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In patients with Huntington's disease (HD), the proteolytic activity of the ubiquitin proteasome system (UPS) is reduced in the brain and other tissues. The pathological hallmark of HD is the intraneuronal nuclear protein aggregates of mutant huntingtin. We determined how to enhance UPS function and influence catalytic protein degradation and cell survival in HD. Proteasome activators involved in either the ubiquitinated or the non-ubiquitinated proteolysis were overexpressed in HD patients' skin fibroblasts or mutant huntingtin-expressing striatal neurons. Following compromise of the UPS, overexpression of the proteasome activator subunit PA28gamma, but not subunit S5a, recovered proteasome function in the HD cells. PA28gamma also improved cell viability in mutant huntingtin-expressing striatal neurons exposed to pathological stressors, such as the excitotoxin quinolinic acid and the reversible proteasome inhibitor MG132. These results demonstrate the specific functional enhancements of the UPS that can provide neuroprotection in HD cells.

Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. Circulating hepcidin levels have been shown to affect iron uptake, release and storage. Hepcidin is mainly liver-derived and regulated, at least in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation each have been shown to influence hepcidin mRNA expression in intact animals. In vitro, regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating the same cell lines with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains unknown. In addition, there is no current technique available that can investigate how iron induces hepcidin expression. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we have established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals' liver after hydrodynamic transfection of hepcidin promoter/luciferase constructs into mice. Transfected hepcidin promoter constructs were shown to respond to both inflammatory and iron stimuli in vivo. This work highlights the ability of this new imaging technique to investigate the key regions of the hepcidin promoter involved in iron induction of hepcidin expression.

The pathogenesis of fibrosis in hepatic cirrhosis remains obscure. This study examines the eventual role of angiogenic factors in the fibrotic process. A series of 55 cirrhotic livers was studied for the proliferation state of fibroblasts, and the expression of vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP) and the basic and acidic fibroblast growth factor (bFGF, aFGF) in both fibroblasts and hepatic cells. The angiogenic and/or fibrogenic factors VEGF, TP, bFGF, and aFGF were clearly expressed in regenerative hepatocytes, but not in fibroblasts of diffuse hepatic fibrosis. The immunohistochemical findings suggest that angiogenic factors and factors promoting oxidative stress (i.e., TP) produced by hepatocytes may contribute to the development of fibrous bands in hepatic cirrhosis.

<h4>Introduction</h4>The causes of coagulation abnormalities and thromboembolic complications during staged Fontan approach in patients with single ventricle remain unclear. This study was designed to evaluate the coagulation profile in the early postoperative period after hemi-Fontan and Fontan procedures with relationship to liver function and hemodynamic variables.<h4>Materials and methods</h4>The prospective study on 43 patients after hemi-Fontan (group 1) and 37 patients after Fontan procedure (group 2) was carried out. Coagulation profile (factor VII, factor VIII, ATIII, fibrinogen, prothrombin), liver function (total serum protein, albumin, AST, ALT, bilirubin), and hemodynamic variables were assessed on postoperative day 1 and 5 and compared to preoperative measures.<h4>Results</h4>Factor VIII concentration was significantly higher on first postoperative day in both groups. On postoperative day 5 the concentration of factor VIII was significantly decreased in group 1 whereas constant in group 2. The concentration of factor VII, ATIII, fibrinogen, and prothrombin was significantly decreased on first and increased on fifth postoperative day after both hemi-Fontan and Fontan procedures. The increase in bilirubin concentration was more distinctive after Fontan operation (p=0.003) with lower AST in this group (p<0.0001). The single ventricle function, pO2 and central venous pressure had significant influence on factor VIII (p=0.034), factor VII (p=0.012), ATIII (p=0.006), and prothrombin (p=0.024) concentrations in group 2 with no significant influence in group 1.<h4>Conclusions</h4>The distinctive causes of coagulation abnormalities during staged Fontan approach are hemodynamic changes and temporary liver dysfunction. Elevated concentration of factor VIII and significant influence of hemodynamics on coagulation profile could contribute to postoperative thromboembolic complications.

Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17bp DNA sequence in the third intron.

<h4>Background & aims</h4>Interferon-induced depression is a major complication in antiviral therapy of chronic hepatitis C. Little is known about underlying mechanisms and reliable predictive factors associated with cytokine-induced depressive symptoms.<h4>Methods</h4>In a cohort of 139 hepatitis C-infected outpatients treated with interferon alfa-2b, we investigated the impact of functional gene variants of the cerebral serotonin (5-HT) signalling pathway previously implicated in depression risk. Depression was monitored using the Hospital Anxiety and Depression Scale (HADS). All patients were genotyped for functional variations in the 5-HT(1A) receptor (HTR1A), 5-HT transporter (SLC6A4, 5-HTT), and tryptophan hydoxylase-2 (TPH2).<h4>Results</h4>Homozygosity for the HTR1A-1019G variant significantly increased both incidence and severity of interferon-induced depression. Maximum increases in HADS depression scores during antiviral therapy correlated with HTR1A variation (P = .011). Clinically relevant depression was significantly associated with the HTR1A-1019G genotype (P = .017; OR, 2.95). 5-HTT and TPH2 variations did not contribute significantly to the prediction of interferon-induced depression by HTR1A (sensitivity, 35.9%; specificity, 84.0%).<h4>Conclusions</h4>Our findings suggest an impact of allelic variation in 5-HT(1A) receptor expression on the development of interferon alfa-induced depression during antiviral treatment of chronic hepatitis C. Prediction models of interferon-induced depressive symptoms based on HTR1A variation offer a perspective for an antidepressant selective serotonin reuptake inhibitor prophylaxis in patients genetically at risk for interferon-induced depression.

There is a need for biomarkers of onset and progression in Huntington's disease (HD), as current outcome measures lack the reliability to enable the efficient conduct of disease-modifying trials. Neurofilament heavy chain (NfH) is a neuron-specific protein for the neuro-axonal compartment that has been proposed as a marker for axonal injury, degeneration and loss and its clinical use as a biomarker has been suggested in several neurodegenerative diseases. We used an enzyme-linked immunosorbent assay to quantify NfH levels in plasma in control subjects, premanifest HD mutation carriers and subjects with early and moderate manifest HD. We found no correlation between plasma NfH level and disease stage, or calculated parameters based on CAG repeat length, the major determinant of disease course in HD, and no evidence that NfH may be a predictor of disease onset. We conclude that plasma NfH concentration is not a useful biomarker of onset or progression in HD.

Prenatal protein malnutrition continues to be a significant problem in the world today. Exposure to prenatal protein malnutrition increases the risk of a number of neuropsychiatric disorders in adulthood including depression, schizophrenia and attentional deficit disorder. In the present experiment, we have examined the effects of stress on extracellular serotonin (5-HT) and dopamine in the medial prefrontal cortex and dorsal hippocampus of rats exposed in utero to protein malnutrition. The medial prefrontal cortex and dorsal hippocampus were chosen as two limbic forebrain regions involved in learning and memory, attention and the stress response. Extracellular 5-HT and dopamine were determined in the medial prefrontal cortex and dorsal hippocampus of adult male Sprague-Dawley rats using dual probe in vivo microdialysis. Basal extracellular 5-HT did not differ between malnourished and well-nourished controls in either the medial prefrontal cortex or the dorsal hippocampus. Basal extracellular dopamine was significantly decreased in the medial prefrontal cortex of malnourished animals. Restraint stress (20 m) produced a significant rise in extracellular dopamine in the medial prefrontal cortex of well-nourished rats but did not alter release in malnourished rats. In malnourished rats, stress produced an increase in 5-HT in the hippocampus, whereas stress produced a decrease in 5-HT in the hippocampus of well-nourished rats. These data demonstrate that prenatal protein malnutrition alters dopaminergic neurotransmission in the medial prefrontal cortex as well as alters the dopaminergic and serotonergic response to stress. These changes may provide part of the bases for alterations in malnourished animals' response to stress.

The phylogeny of placental mammals is a critical framework for choosing future genome sequencing targets and for resolving the ancestral mammalian genome at the nucleotide level. Despite considerable recent progress defining superordinal relationships, several branches remain poorly resolved, including the root of the placental tree. Here we analyzed the genome sequence assemblies of human, armadillo, elephant, and opossum to identify informative coding indels that would serve as rare genomic changes to infer early events in placental mammal phylogeny. We also expanded our species sampling by including sequence data from >30 ongoing genome projects, followed by PCR and sequencing validation of each indel in additional taxa. Our data provide support for a sister-group relationship between Afrotheria and Xenarthra (the Atlantogenata hypothesis), which is in turn the sister-taxon to Boreoeutheria. We failed to recover any indels in support of a basal position for Xenarthra (Epitheria), which is suggested by morphology and a recent retroposon analysis, or a hypothesis with Afrotheria basal (Exafricoplacentalia), which is favored by phylogenetic analysis of large nuclear gene data sets. In addition, we identified two retroposon insertions that also support Atlantogenata and none for the alternative hypotheses. A revised molecular timescale based on these phylogenetic inferences suggests Afrotheria and Xenarthra diverged from other placental mammals approximately 103 (95-114) million years ago. We discuss the impacts of this topology on earlier phylogenetic reconstructions and repeat-based inferences of phylogeny.

Transcriptional dysregulation in Huntington's disease (HD) is a well documented and broadly studied phenomenon. Its basis appears to be in huntingtin's aberrant protein-protein interactions with a variety of transcription factors. The development of therapeutics targeting altered transcription, however, faces serious challenges. No single transcriptional regulator has emerged as a primary actor in HD. The levels of literally hundreds of RNA transcripts are altered in affected cells and it is uncertain which are most relevant. The protein-protein interactions of mutant huntingtin with transcriptional factors do not constitute conventional and easy targets for drug molecules. Nevertheless, potential therapeutic advances, targeting transcriptional deregulation in HD, have been made in recent years. In this chapter we review current progress in this area of therapeutic development. We also discuss possible drug discovery strategies targeting altered transcriptional pathways.

<h4>Background</h4>A tremendous amount of efforts have been devoted to identifying genes for diagnosis and prognosis of diseases using microarray gene expression data. It has been demonstrated that gene expression data have cluster structure, where the clusters consist of co-regulated genes which tend to have coordinated functions. However, most available statistical methods for gene selection do not take into consideration the cluster structure.<h4>Results</h4>We propose a supervised group Lasso approach that takes into account the cluster structure in gene expression data for gene selection and predictive model building. For gene expression data without biological cluster information, we first divide genes into clusters using the K-means approach and determine the optimal number of clusters using the Gap method. The supervised group Lasso consists of two steps. In the first step, we identify important genes within each cluster using the Lasso method. In the second step, we select important clusters using the group Lasso. Tuning parameters are determined using V-fold cross validation at both steps to allow for further flexibility. Prediction performance is evaluated using leave-one-out cross validation. We apply the proposed method to disease classification and survival analysis with microarray data.<h4>Conclusion</h4>We analyze four microarray data sets using the proposed approach: two cancer data sets with binary cancer occurrence as outcomes and two lymphoma data sets with survival outcomes. The results show that the proposed approach is capable of identifying a small number of influential gene clusters and important genes within those clusters, and has better prediction performance than existing methods.

We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5'-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation.

Actin polymerization plays a critical role in clathrin-mediated endocytosis in many cell types, but how polymerization is regulated is not known. Hip1R may negatively regulate actin assembly during endocytosis because its depletion increases actin assembly at endocytic sites. Here, we show that the C-terminal proline-rich domain of Hip1R binds to the SH3 domain of cortactin, a protein that binds to dynamin, actin filaments and the Arp2/3 complex. We demonstrate that Hip1R deleted for the cortactin-binding site loses its ability to rescue fully the formation of abnormal actin structures at endocytic sites induced by Hip1R siRNA. To determine when this complex might function during endocytosis, we performed live cell imaging. The maximum in vivo recruitment of Hip1R, clathrin and cortactin to endocytic sites was coincident, and all three proteins disappeared together upon formation of a clathrin-coated vesicle. Finally, we showed that Hip1R inhibits actin assembly by forming a complex with cortactin that blocks actin filament barbed end elongation.

A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His x 6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His x 6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His x 6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His x 6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His x 6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His x 6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads.

<h4>Background</h4>Heterologous promoter analysis in Plasmodium has revealed the existence of conserved cis regulatory elements as promoters from different species can drive expression of reporter genes in heterologous transfection assays. Here, the functional characterization of different Plasmodium vivax promoters in Plasmodium falciparum using luciferase as the reporter gene is presented.<h4>Methods</h4>Luciferase reporter plasmids harboring the upstream regions of the msp1, dhfr, and vir3 genes as well as the full-length intergenic regions of the vir23/24 and ef-1alpha genes of P. vivax were constructed and transiently transfected in P. falciparum.<h4>Results</h4>Only the constructs with the full-length intergenic regions of the vir23/24 and ef-1alpha genes were recognized by the P. falciparum transcription machinery albeit to values approximately two orders of magnitude lower than those reported by luc plasmids harbouring promoter regions from P. falciparum and Plasmodium berghei. A bioinformatics approach allowed the identification of a motif (GCATAT) in the ef-1alpha intergenic region that is conserved in five Plasmodium species but is degenerate (GCANAN) in P. vivax. Mutations of this motif in the P. berghei ef-1alpha promoter region decreased reporter expression indicating it is active in gene expression in Plasmodium.<h4>Conclusion</h4>Together, this data indicates that promoter regions of P. vivax are poorly or not recognized by the P. falciparum transcription machinery suggesting the existence of P. vivax-specific transcription regulatory elements.

<h4>Aim</h4>To present results from a nested association study of the complement factor H (CFH) gene region using a novel methodology that uses a high-resolution genetic linkage disequilibrium map to estimate a point location for a causal mutation.<h4>Method</h4>Age-related macular degeneration (AMD) case-control data from a genomewide single-nucleotide polymorphism (SNP) panel were used to identify the target interval to be genotyped at higher density in a second independent panel. The pattern of linkage disequilibrium (LD) and segmental duplications across this region are described in detail.<h4>Result</h4>Data were consistent with other studies in that strong association between the Y402H variant and AMD is observed. However, composite likelihood analysis, which combines association data from all SNPs in the region, and uses genetic locations on a high-resolution LD map, gave a point location for a causal variant between exons 1 and 2 of the CFH gene.<h4>Conclusion</h4>The findings are consistent with evidence that, in addition to the widely described Y402H variant, there is at least one and, most probably, several other mutations in the CFH gene which determine disease manifestation in AMD. A genetic model in which multiple mutations contribute to a varying degree to disease aetiology has been previously well described in ophthalmic genetics, and is typified by the COL2A1 and ABCA4 genes.

Previously, several studies have demonstrated changes in the levels of small heat shock proteins (sHSP) in the transgenic mouse models of familial amyotrophic lateral sclerosis (fALS) linked to mutations in Cu/Zn superoxide dismutase. Here, we compared the expression of sHSPs in transgenic mouse models of fALS, Parkinson's disease (PD), dentato-rubral pallido-luysian atrophy (DRPLA) and Huntington's disease (HD); where the expression of mutant cDNA genes was under the transcriptional regulation of the mouse prion protein promoter. These models express G37R mutant Cu/Zn superoxide dismutase (SOD1G37R; fALS), A53T mutant alpha-synuclein (alpha-SynA53T; PD), full-length mutant atrophin-1-65Q, and htt-N171-82Q (huntingtin N-terminal fragment; HD). We found that the levels and solubilities of two sHSPs, Hsp25 and alpha B-crystallin, were differentially regulated in these mice. Levels of both Hsp25 and alpha B-crystallin were markedly increased in subgroups of glias at the affected regions of symptomatic SODG37R and alpha-SynA53T transgenic mice; abnormal deposits or cells intensely positive for alpha B-crystallin were observed in SODG37R mice. By contrast, neither sHSP was induced in spinal cords of htt-N171-82Q or atrophin-1-65Q mice, which do not develop astrocytosis or major motor neuron abnormalities. Interestingly, the levels of insoluble alpha B-crystallin in spinal cords gradually increased as a function of age in nontransgenic animals. In vitro, alpha B-crystallin was capable of suppressing the aggregation of alpha-SynA53T, as previously described for a truncated mutant SOD1. The transgenes in these mice are expressed highly in astrocytes and thus our results suggest a role for small heat shock proteins in protecting activated glial cells such as astrocytes in neurodegenerative diseases.

Ultraconserved elements are stretches of consecutive nucleotides that are perfectly conserved in multiple mammalian genomes. Although these sequences are identical in the reference human, mouse, and rat genomes, we identified numerous polymorphisms within these regions in the human population. To determine whether polymorphisms in ultraconserved elements affect fitness, we genotyped unrelated human DNA samples at loci within these sequences. For all single-nucleotide polymorphisms tested in ultraconserved regions, individuals homozygous for derived alleles (alleles that differ from the rodent reference genomes) were present, viable, and healthy. The distribution of allele frequencies in these samples argues against strong, ongoing selection as the force maintaining the conservation of these sequences. We then used two methods to determine the minimum level of selection required to generate these sequences. Despite the lack of fixed differences in these sequences between humans and rodents, the average level of selection on ultraconserved elements is less than that on essential genes. The strength of selection associated with ultraconserved elements suggests that mutations in these regions may have subtle phenotypic consequences that are not easily detected in the laboratory.

No abstract available.

Autism spectrum disorders (ASDs) are common, heritable neurodevelopmental conditions. The genetic architecture of ASDs is complex, requiring large samples to overcome heterogeneity. Here we broaden coverage and sample size relative to other studies of ASDs by using Affymetrix 10K SNP arrays and 1,181 [corrected] families with at least two affected individuals, performing the largest linkage scan to date while also analyzing copy number variation in these families. Linkage and copy number variation analyses implicate chromosome 11p12-p13 and neurexins, respectively, among other candidate loci. Neurexins team with previously implicated neuroligins for glutamatergic synaptogenesis, highlighting glutamate-related genes as promising candidates for contributing to ASDs.

A rhodopsin-based homology model of the nucleotide-activated human P2Y2 receptor, including loops, termini, and phospholipids, was optimized with the Monte Carlo multiple minimum conformational search routine. Docked uridine 5'-triphosphate (UTP) formed a nucleobase pi-pi complex with conserved Phe3.32. Selectivity-enhancing 2'-amino-2'-deoxy substitution interacted through pi-hydrogen-bonding with aromatic Phe6.51 and Tyr3.33. A "sequential ligand composition" approach for docking the flexible dinucleotide agonist Up4U demonstrated a shift of conserved cationic Arg3.29 from the UTP gamma position to the delta position of Up4U and Up4 ribose. Synthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1 astrocytoma cells. 2'-Amino and 2-thio modifications were synergized to enhance potency and selectivity; compound 8 (EC50 = 8 nM) was 300-fold P2Y2-selective versus P2Y4. 2'-Amine acetylation reduced potency, and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance P2Y2 potency through a predicted hydrophilic interaction possibly because of destabilization of the receptor-favored Northern conformation of ribose. This detailed view of P2Y2 receptor recognition suggests mutations for model validation.

The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Angstrom resolution to R = 0.195 and R(free) = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat.

The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin alpha1LG4-5 domains and provide a mutational analysis of heparin, alpha-dystroglycan, and galactosylsulfatide binding. The two domains of alpha1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin alpha2 LG4-5 but with a substantially different interdomain angle. Recombinant alpha1LG4-5 binding to heparin, alpha-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to alpha-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands.

Due to the increasing power of personal computers, as well as the availability of flexible forward-time simulation programs like simuPOP, it is now possible to simulate the evolution of complex human diseases using a forward-time approach. This approach is potentially more powerful than the coalescent approach since it allows simulations of more than one disease susceptibility locus using almost arbitrary genetic and demographic models. However, the application of such simulations has been deterred by the lack of a suitable simulation framework. For example, it is not clear when and how to introduce disease mutants-especially those under purifying selection-to an evolving population, and how to control the disease allele frequencies at the last generation. In this paper, we introduce a forward-time simulation framework that allows us to generate large multi-generation populations with complex diseases caused by unlinked disease susceptibility loci, according to specified demographic and evolutionary properties. Unrelated individuals, small or large pedigrees can be drawn from the resulting population and provide samples for a wide range of study designs and ascertainment methods. We demonstrate our simulation framework using three examples that map genes associated with affection status, a quantitative trait, and the age of onset of a hypothetical cancer, respectively. Nonadditive fitness models, population structure, and gene-gene interactions are simulated. Case-control, sibpair, and large pedigree samples are drawn from the simulated populations and are examined by a variety of gene-mapping methods.

<h4>Purpose</h4>Glucocorticoids are widely used as adjuvant therapy in hormonal refractory prostate cancer; their therapeutic role, however, remains unclear. Ursolic acid, a natural triterpene, structurally similar to dexamethasone, exhibits antitumor effects in various cell types. Our main objective was to investigate the effects of ursolic acid on cell viability, apoptosis and bcl-2 protein, in human hormone refractory and androgen-sensitive prostate cancer cells.<h4>Methods</h4>The ursolic acid-induced changes in cell viability, apoptosis and bcl-2 protein were examined in human hormone refractory prostate cancer PC-3 cells and androgen-sensitive LNCaP cells, by MTT assay, flow cytometry and western blot analysis, respectively.<h4>Results</h4>Ursolic acid inhibited significantly the cell viability and induced apoptosis in PC-3 cells at 55 microM and in LNCaP cells at 45 microM associated with a downregulation of bcl-2 protein.<h4>Conclusions</h4>The antiproliferative and apoptotic effects of ursolic acid in PC-3 and LNCaP cells implicate its potential therapeutic use for the treatment of hormone refractory and androgen-sensitive prostate cancer. The downregulation of bcl-2 may be one of the molecular mechanisms via which it induces apoptosis in PC-3 and LNCaP cells.

The serotonin (5-hydroxytryptamine) transporter (5-HTT) is considered to affect the pathogenesis of mood disorders. Large number of genetic association studies between 5-HTT functional polymorphisms and vulnerability of mood disorders and therapeutic response to antidepressants has been carried out. We investigated the influence of 5-HTT-linked polymorphic region (5-HTTLPR) and 5-HTT 17 bp variable number of tandem repeat polymorphism (5-HTTVNTR) polymorphisms on concentrations of monoamine metabolites in cerebrospinal fluid (CSF) among treatment-resistant patients with mood disorders. Subjects were 119 Swedish patients with persistent mood disorders and 141 healthy subjects. In 112 of these patients, we measured 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol in CSF. Genotyping for 5-HTT polymorphisms from genomic DNA was carried out by PCR. There was no significant difference in allele/genotype frequency between patients and healthy subjects. In patients with mood disorders, we found significant difference in mean 5-HIAA concentration between 5-HTTLPR genotypes (p=0.03). Although the 5-HIAA concentration showed a tendency to be higher in short (S) carriers than in non-S carriers of the 5-HTTLPR in patients (p=0.06), when considering patients with major depressive disorder (MDD), the 5-HIAA concentration was significantly higher among S carriers than among non-S carriers (p=0.02). Moreover, the 5-HIAA concentration was higher in S/S subjects compared to long (L)/L (p=0.0001) and L/S (p=0.002) subjects in patients with MDD. Similarly, there was higher HVA concentration in S/S subjects compared to L/L (p=0.002) and L/S subjects (p=0.002). There was no effect of 5-HTTVNTR. Our findings show that the 5-HTTLPR polymorphism affects 5-HIAA and HVA concentrations among treatment-resistant patients with mood disorders.

<h4>Background</h4>HCV is one of the major health problems in Egypt, where it is highly prevalent. Genotype 4 is the most common genotype of HCV and its response to treatment is still a controversy.<h4>Methods</h4>HCV genotype 4 quasispecies diversity within the 5' untranslated region (5'UTR) was studied in a series of 22 native Egyptian patients with chronic hepatitis C virus with no previous treatment who satisfied all NIH criteria for combined treatment of pegylated IFN and ribavirine and was correlated with the outcome of treatment. The study also included 7 control patients with no antiviral treatment. HCV sequencing was done using the TRUGENE HCV 5-NC genotyping kit.<h4>Results</h4>At the 48th week of treatment, 15 patients (68%) showed virological response. Whereas HCV-RNA was still detected in 7 patients (32%) in this period; of those, 6 experienced a partial virological response followed by viral breakthrough during treatment. Only one patient did not show any virological or chemical response. The four females included in this study were all responders. There was a significant correlation between the response rate and lower fibrosis (p = 0.026) as well as the total number of mutation spots (including all the insertions, deletions, transitions and transversions) (p = 0.007, p = 0.035).<h4>Conclusion</h4>Patients who responded to interferon treatment had statistically significant less number in both transitions (p = 0.007) and the genetic distances between the quasispecies (p = 0.035). So, viral genetic complexity and variability may play a role in the response to IFN treatment. The consensus alignment of all three groups revealed no characteristic pattern among the three groups. However, the G to A transitions at 160 was observed among non responders who need further study to confirm this observation.

Progressive iron overload is the most salient and ultimately fatal complication of beta-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in beta-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both beta-thalassemia intermedia (th3/+) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/+ and th3/th3 mice, and that transfusion therapy can rescue mice affected by beta-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/+ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/+ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of beta-thalassemia.

<h4>Background/aims</h4>Hyperferritinemia has been associated with cardiovascular mortality in hemodialysis patients. The aim of this study was to evaluate whether serum ferritin was affected by iron and oxidative status and by genetic factors (HFE mutations and the Ala9Val MnSOD polymorphism), and to assess the association between ferritin and cardiovascular damage evaluated by ecocolor-Doppler.<h4>Methods</h4>63 hemodialysis patients were tested for HFE and MnSOD genotype by restriction analysis and oxidative status; vascular damage was assessed by measuring intima-media thickness, and by detecting plaques at carotid and femoral arteries.<h4>Results</h4>Ferritin was correlated with transferrin saturation (p = 0.003), decreased iron-specific serum antioxidant activity (p = 0.01), age (p = 0.03), and C282Y and H63D HFE mutations (p = 0.05), but not with the MnSOD polymorphism. Ferritin was associated with advanced vascular damage, as evaluated by the presence of plaques, both at carotid (p = 0.03) and femoral arteries (p = 0.001), the other risk factors being age and low albumin. Low iron-specific antioxidant activity was associated with carotid plaques (p = 0.03).<h4>Conclusion</h4>In hemodialysis patients, hyperferritinemia reflects a relative increase in iron availability and a decrease in iron-specific antioxidant activity, is favored by HFE mutations, and represents a risk factor for advanced cardiovascular damage.

Diabetes Mellitus is found with increasing frequency in iron overload patients with hemochromatosis. In these conditions, the pancreas shows predominant iron overload in acini but also islet beta-cells. We assess glucose homeostasis status in iron-overloaded hepcidin-deficient mice. These mice presented with heavy pancreatic iron deposits but only in the acini. The beta-cell function was found unaffected with a normal production and secretion of insulin. The mutant mice were not diabetic, responded as the control group to glucose and insulin challenges, with no alteration of insulin signalling in the muscle and the liver. These results indicate that, beta-cells iron deposits-induced decreased insulin secretory capacity might be of primary importance to trigger diabetes in hemochromatosic patients.

Among organisms with chromosome-based mechanisms of sex determination, failure to equalize expression of X-linked genes between the sexes is typically lethal. In C. elegans, XX hermaphrodites halve transcription from each X chromosome to match the output of XO males. Here, we mapped the binding location of the condensin homolog DPY-27 and the zinc finger protein SDC-3, two components of the C. elegans dosage compensation complex (DCC). We observed strong foci of DCC binding on X, surrounded by broader regions of localization. Binding foci, but not adjacent regions of localization, were distinguished by clusters of a 10-bp DNA motif, suggesting a recruitment-and-spreading mechanism for X recognition. The DCC was preferentially bound upstream of genes, suggesting modulation of transcriptional initiation and polymerase-coupled spreading. Stronger DCC binding upstream of genes with high transcriptional activity indicated a mechanism for tuning DCC activity at specific loci. These data aid in understanding how proteins involved in higher-order chromosome dynamics can regulate transcription at individual loci.

Aminoglycoside antibiotics act by binding to 16S rRNA. Resistance to these antibiotics occurs via drug modifications by enzymes such as aminoglycoside 6'-N-acetyltransferases (AAC(6')s). We report here the regioselective and efficient synthesis of N-6'-acylated aminoglycosides and their use as probes to study AAC(6')-Ii and aminoglycoside-RNA complexes. Our results emphasize the central role of N-6' nucleophilicity for transformation by AAC(6')-Ii and the importance of hydrogen bonding between 6'-NH(2) and 16S rRNA for antibacterial activity.

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH(2)) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH(2)) by replacing the His-d-Phe and His-d-Nal(2') fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx=D-Phe/pCl-D-Phe/D-Nal(2')). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists.

Of the three theoretically possible, Bsmoc-related, naphthothiophene sulfone-based amino-protecting groups, the two most readily available derivatives, the alpha- and beta-Nsmoc analogues, have been examined as substitutes for the Bsmoc residue in cases where the latter lead to oily protected amino acids or amino acid fluorides. All of the naphtho systems gave easily handled solid amino acid derivatives. The intermediate sulfone alcohol 11 used as the key reagent for introduction of the alpha-Nsmoc protecting group was readily made from alpha-tetralone (Scheme 1). The corresponding beta-analogue 17 was made similarly on a small scale, but due to the high cost of beta-tetralone, an alternate route involving reaction of rhodanine with alpha-naphthaldehyde was used for large-scale work (Scheme 2). All proteinogenic amino acids were converted to their alpha- and beta-Nsmoc derivatives. Deblocking studies showed that the reactivity toward deblocking by piperidine followed the order alpha-Nsmoc > Bsmoc > beta-Nsmoc. 1H NMR experiments showed that deblocking of the two new systems was mechanistically similar to that previously established for the Bsmoc derivative in that the reaction is initiated by Michael addition to the beta-carbon atom of the alpha,beta-unsaturated sulfone system. Application of alpha- and beta-Nsmoc amino acids to the solid-phase synthesis of two model peptides was examined. An advantage of the alpha-Nsmoc system over the long-known Bsmoc system proved to be the milder conditions needed for the deblocking step relative to the Bsmoc case, which is itself more readily deblocked than the classic Fmoc analogue.

<h4>Background</h4>Human stem cells are viewed as a possible source of neurons for a cell-based therapy of neurodegenerative disorders, such as Parkinson's disease. Several protocols that generate different types of neurons from human stem cells (hSCs) have been developed. Nevertheless, the cellular mechanisms that underlie the development of neurons in vitro as they are subjected to the specific differentiation protocols are often poorly understood.<h4>Results</h4>We have designed a focused DNA (oligonucleotide-based) large-scale microarray platform (named "NeuroStem Chip") and used it to study gene expression patterns in hSCs as they differentiate into neurons. We have selected genes that are relevant to cells (i) being stem cells, (ii) becoming neurons, and (iii) being neurons. The NeuroStem Chip has over 1,300 pre-selected gene targets and multiple controls spotted in quadruplicates (approximately 46,000 spots total). In this study, we present the NeuroStem Chip in detail and describe the special advantages it offers to the fields of experimental neurology and stem cell biology. To illustrate the utility of NeuroStem Chip platform, we have characterized an undifferentiated population of pluripotent human embryonic stem cells (hESCs, cell line SA02). In addition, we have performed a comparative gene expression analysis of those cells versus a heterogeneous population of hESC-derived cells committed towards neuronal/dopaminergic differentiation pathway by co-culturing with PA6 stromal cells for 16 days and containing a few tyrosine hydroxylase-positive dopaminergic neurons.<h4>Conclusion</h4>We characterized the gene expression profiles of undifferentiated and dopaminergic lineage-committed hESC-derived cells using a highly focused custom microarray platform (NeuroStem Chip) that can become an important research tool in human stem cell biology. We propose that the areas of application for NeuroStem microarray platform could be the following: (i) characterization of the expression of established, pre-selected gene targets in hSC lines, including newly derived ones, (ii) longitudinal quality control for maintained hSC populations, (iii) following gene expression changes during differentiation under defined cell culture conditions, and (iv) confirming the success of differentiation into specific neuronal subtypes.

Ferroportin disease is caused by mutation of one allele of the iron exporter ferroportin (Fpn/IREG1/Slc40a1/MTP1). All reported human mutations are missense mutations and heterozygous null mutations in mouse Fpn do not recapitulate the human disease. Here we describe the flatiron (ffe) mouse with a missense mutation (H32R) in Fpn that affects its localization and iron export activity. Similar to human patients with classic ferroportin disease, heterozygous ffe/+ mice present with iron loading of Kupffer cells, high serum ferritin, and low transferrin saturation. In macrophages isolated from ffe/+ heterozygous mice and through the use of Fpn plasmids with the ffe mutation, we show that Fpn(ffe) acts as a dominant negative, preventing wild-type Fpn from localizing on the cell surface and transporting iron. These results demonstrate that mutations in Fpn resulting in protein mislocalization act in a dominant-negative fashion to cause disease, and the Fpn(ffe) mouse represents the first mouse model of ferroportin disease.

A susceptibility locus for coronary artery disease (CAD) has been mapped to chromosome 3q13-21 in a linkage study of early-onset CAD. We completed an association-mapping study across the 1-LOD-unit-down supporting interval, using two independent white case-control data sets (CATHGEN, initial and validation) to evaluate association under the peak. Single-nucleotide polymorphisms (SNPs) evenly spaced at 100-kb intervals were screened in the initial data set (N=468). Promising SNPs (P<.1) were then examined in the validation data set (N=514). Significant findings (P<.05) in the combined initial and validation data sets were further evaluated in multiple independent data sets, including a family-based data set (N=2,954), an African American case-control data set (N=190), and an additional white control data set (N=255). The association between genotype and aortic atherosclerosis was examined in 145 human aortas. The peakwide survey found evidence of association in SNPs from multiple genes. The strongest associations were found in three SNPs from the kalirin (KALRN) gene, especially in patients with early-onset CAD (P=.00001-00028 in the combined CATHGEN data sets). In-depth investigation of the gene found that an intronic SNP, rs9289231, was associated with early-onset CAD in all white data sets examined (P<.05). In the joint analysis of all white early-onset CAD cases (N=332) and controls (N=546), rs9289231 was highly significant (P=.00008), with an odds-ratio estimate of 2.1. Furthermore, the risk allele of this SNP was associated with atherosclerosis burden (P=.03) in 145 human aortas. KALRN is a protein with many functions, including the inhibition of inducible nitric oxide synthase and guanine-exchange-factor activity. KALRN and two other associated genes identified in this study (CDGAP and MYLK) belong to the Rho GTPase-signaling pathway. Our data suggest the importance of the KALRN gene and the Rho GTPase-signaling pathway in the pathogenesis of CAD.

The histological hallmark feature of Huntington's disease (HD) and other polyglutamine repeat diseases is the presence of intracellular inclusions. Much work has been devoted to trying to determine the relationship between inclusion formation and neuronal injury. However, little attention has been paid to the variability and characteristics of inclusions themselves. Here, we characterize the morphological and biochemical composition of inclusions in both a transgenic mouse model (R6/2 line) and an inducible cell culture model of HD (iPC12Q74). We identified several morphologically distinct kinds of inclusions in different locations (nuclei, cytoplasm and cellular processes). Ubiquitin colocalized completely with all of these inclusions in both the iPC12Q72 and R6/2 models. In the inclusions in iPC12Q74 cells, the 20S and 11S proteasome subunits colocalized variably, and the 19S subunit did not colocalize at all. In inclusions in R6/2 mouse neurons, the 20S subunit colocalized completely, but neither the 11S nor the 19S subunits colocalized at all. While the role of inclusions in the pathogenesis of HD continues to be debated, we suggest that the content and structure of inclusions vary considerably, not only from cell to cell but even within individual cells. Their role in the pathogenesis of HD is likely to depend on their location as well as their composition.

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100-300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons.

Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.

FR901464 is a potent anticancer natural product that lowers the mRNA levels of oncogenes and tumor suppressor genes. In this article, we report a convergent enantioselective synthesis of FR901464, which was accomplished in 13 linear steps. Central to the synthetic approach was the diene-ene cross olefin metathesis reaction to generate the C6-C7 olefin without the use of protecting groups as the final step. Additional key reactions include a Zr/Ag-promoted alkynylation to set the C4 stereocenter, a mild and chemoselective Red-Al reduction, a reagent-controlled stereoselective Mislow-Evans-type [2,3]-sigmatropic rearrangement to install the C5 stereocenter, a Carreira asymmetric alkynylation to generate the C4' stereocenter, and a highly efficient ring-closing metathesis-allylic oxidation sequence to form an unsaturated lactone. The decomposition pathways of FR901464's right fragment were studied under physiologically relevant conditions. Facile epoxide opening by beta-elimination gave two enones, one of which could undergo dehydration via its hemiketal to form a furan. To prevent this decomposition pathway, a right fragment was rationally designed and synthesized. This analogue was 12 times more stable than the right fragment of the natural product. Using this more stable right fragment analogue, an FR901464 analogue, meayamycin, was prepared in 13 linear steps. The inhibitions of human breast cancer MCF-7 cell proliferation by synthetic FR901464 and meayamycin were studied, and the GI50 values for these compounds were determined to be 1.1 nM and 10 pM, respectively. Thus, meayamycin is among the most potent anticancer small molecules that do not bind to either DNA or microtubule.

<h4>Background</h4>Polymorphisms in GSTT1, GSTM1 and GSTP1 impact detoxification of carcinogens by GSTs and have been reported to increase susceptibility to environmentally related health outcomes. Individual factors in arsenic biotransformation may influence disease susceptibility. GST activity is involved in the metabolism of endogenous and exogenous compounds, including catalyzing the formation of arsenic-GSH conjugates.<h4>Methods</h4>We investigated whether polymorphisms in GSTT1, GSTP1 and GSTM1 were associated with risk of skin lesions and whether these polymorphisms modify the relationship between drinking water arsenic exposure and skin lesions in a case control study of 1200 subjects frequency matched on age and gender in community clinics in Pabna, Bangladesh in 2001-2002.<h4>Results and discussion</h4>GSTT1 homozygous wildtype status was associated with increased odds of skin lesions compared to the null status (OR1.56 95% CI 1.10-2.19). The GSTP1 GG polymorphism was associated with greater odds of skin lesions compared to GSTP1 AA, (OR 1.86 (95%CI 1.15-3.00). No evidence of effect modification by GSTT1, GSTM1 or GSTP1 polymorphisms on the association between arsenic exposure and skin lesions was detected.<h4>Conclusion</h4>GSTT1 wildtype and GSTP1 GG are associated with increased risk of skin lesions.

<h4>Objectives</h4>To investigate the relation between the HFE C282Y and H63D variants with arthralgia and joint pathology in the population-based Rotterdam Study.<h4>Methods</h4>From a cohort of 7983 people aged 55 years and over, 2095 randomly drawn subjects were genotyped for C282Y and H63D variants. We compared the frequency of arthralgia, and the presence of chondrocalcinosis, osteophytes, joint space narrowing and radiographic osteoarthritis in hand, hip and knee joints, and Heberden's nodes in carriers of HFE variants with that in non-carriers.<h4>Results</h4>Overall, there was a significantly higher frequency of arthralgia (odds ratio 1.6; 95% CI 1.0 to 2.6), oligoarthralgia (2.3; 1.2 to 4.4) and Heberden's nodes (2.0; 1.1 to 3.8) in H63D homozygotes compared with non-carriers. In subjects aged 65 years or younger, H63D homozygotes had significantly more often polyarthralgia (3.1; 1.3 to 7.4), chondrocalcinosis in hip or knee joints (4.7; 1.2 to 18.5), and more hand joints with osteophytes (6.1+/-1.0 vs 4.4+/-0.3), space narrowing (2.8+/-0.5 vs 1.0+/-0.1), radiographic osteoarthritis (4.4+/-0.7 vs 2.0+/-0.2) and Heberden's nodes (3.1; 1.3 to 12.8) than non-carriers. We found no relation of arthralgia or joint pathology to C282Y, but compound heterozygotes had a significantly higher frequency of arthralgia (2.9; 1.0 to 9.3), chondrocalcinosis in hip joints (6.5; 1.8 to 22.3), and an increased number of osteophytes in knee (6.9+/-1.2, n = 5 vs 2.4+/-0.1) joints at a later age (>65 years).<h4>Conclusions</h4>The HFE H63D variant may explain, at least in part, the prevalence of arthralgia in multiple joints sites, chondrocalcinosis, and hand osteoarthritis in the general population.

Achieving greater understanding of the genomic influence on milk synthesis in dairy cows represents a daunting challenge. Bovine-specific microarrays have allowed for high-throughput gene expression analysis of the mammary transcriptome. However, real-time PCR (qPCR) still represents the method of choice for accurate expression profiling of small numbers of genes and verification of key microarray relationships. This method is extremely sensitive but requires data normalization to account for analytical errors. Ideally, expression of genes used as internal controls should not be affected by specific treatments or physiological state. Mammary biopsies were collected from five cows each at -15, 1, 15, 30, 60, 120, and 240 days relative to parturition for gene expression profiling. We evaluated expression of nine genes (RPS9, ACTB, GAPD, GTP, ITGB4BP, MRPL39, RPS23, RPS15, and UXT) that could serve as internal controls in mammary tissue using qPCR. Due to gradual increases in mammary RNA concentration (mug/mg tissue) over lactation, all genes investigated experienced a dilution effect. We used pairwise comparison of expression ratios to analyze the reliability of these genes as internal controls. UXT, RPS9, and RPS15 had the most stable expression ratios across cow and time. We also assessed co-regulation among genes through network analysis. Network analysis suggested co-regulation among most of the genes examined, with MYC playing a central role. Pairwise comparison was suitable for finding appropriate internal controls in mammary gland tissue. Results showed that the geometrical average of UXT, RPS9, and RPS15 expression could be used as internal control for longitudinal mammary gene expression profiling.

Serotonin transporter (5-HTT) is a key component of the serotonergic neurotransmitter system. Few studies have focused on polymorphisms of the serotonin transporter and antipsychotic response and, in particular, there have so far been no published studies on the association between the serotonin transporter and response to risperidone. This study examined the relationship between two polymorphisms of the serotonin transporter and the efficacy of risperidone treatment in 129 patients with schizophrenia. Our results revealed that patients with l allele of HTTRLP showed a greater improvement than those without l allele on the overall brief psychiatric rating scale (BPRS) (P=0.025). But no such relationship was found for the HTTVNTR. In haplotype analysis, the frequency of L-12 haplotype showed a significant difference between the responder group and the non-responder group (P=0.005). Our study has, for the first time, produced evidence that the potential for therapy in patients with schizophrenia is related to the HTTRLP polymorphism in the HTT gene and haplotype L-12 may help to predict risperidone treatment efficiency.

Lung cancer is the most common cause of cancer mortality in male and female patients in the US. The etiology of non-small cell lung cancer (NSCLC) is not fully defined, but new data suggest that estrogens and growth factors promote tumor progression. In this work, we confirm that estrogen receptors (ER), both ERalpha and ERbeta, occur in significant proportions of archival NSCLC specimens from the clinic, with receptor expression in tumor cell nuclei and in extranuclear sites. Further, ERalpha in tumor nuclei was present in activated forms as assessed by detection of ER phosphorylation at serines-118 and -167, residues commonly modulated by growth factor receptor as well as steroid signaling. In experiments using small interfering RNA (siRNA) constructs, we find that suppressing expression of either ERalpha or ERbeta elicits a significant reduction in NSCLC cell proliferation in vitro. Estrogen signaling in NSCLC cells may also include steroid receptor coactivators (SRC), as SRC-3 and MNAR/PELP1 are both expressed in several lung cell lines, and both EGF and estradiol elicit serine phosphorylation of SRC-3 in vitro. EGFR and ER also cooperate in promoting early activation of p42/p44 MAP kinase in NSCLC cells. To assess new strategies to block NSCLC growth, we used Faslodex alone and with erlotinib, an EGFR kinase inhibitor. The drug tandem elicited enhanced blockade of the growth of NSCLC xenografts in vivo, and antitumor activity exceeded that of either agent given alone. The potential for use of antiestrogens alone and with growth factor receptor antagonists is now being pursued further in clinical trials.

The ferroportin (FPN1) Q248H polymorphism has been associated with increased serum ferritin (SF) levels in sub-Saharan Africans and in African Americans (AA). AA participants of the HEIRS Study who did not have HFE C282Y or H63D who had elevated initial screening SF (> or =300 microg/L in men and >= or =200 microg/L in women) (defined as cases) were frequency-matched to AA participants with normal SF (defined as controls) to investigate the association of the Q248H with elevated SF. 10.4% of cases and 6.7% of controls were Q248H heterozygotes (P=0.257). Q248H homozygosity was observed in 0.5% of the cases and none of the controls. The frequency of Q248H was higher among men with elevated SF than among control men (P=0.047); corresponding differences were not observed among women. This appeared to be unrelated to self-reports of a previous diagnosis of liver disease. Men with elevated SF were three times more likely than women with elevated SF to have Q248H (P=0.012). There were no significant differences in Q248H frequencies in men and women control participants. We conclude that the frequency of the FPN1 Q248H polymorphism is greater in AA men with elevated SF than in those with normal SF.

In mammals, growth hormone-releasing hormone (GHRH) is the most important neuroendocrine factor that stimulates the release of growth hormone (GH) from the anterior pituitary. In nonmammalian vertebrates, however, the previously named GHRH-like peptides were unable to demonstrate robust GH-releasing activities. In this article, we provide evidence that these GHRH-like peptides are homologues of mammalian PACAP-related peptides (PRP). Instead, GHRH peptides encoded in cDNAs isolated from goldfish, zebrafish, and African clawed frog were identified. Moreover, receptors specific for these GHRHs were characterized from goldfish and zebrafish. These GHRHs and GHRH receptors (GHRH-Rs) are phylogenetically and structurally more similar to their mammalian counterparts than the previously named GHRH-like peptides and GHRH-like receptors. Information regarding their chromosomal locations and organization of neighboring genes confirmed that they share the same origins as the mammalian genes. Functionally, the goldfish GHRH dose-dependently activates cAMP production in receptor-transfected CHO cells as well as GH release from goldfish pituitary cells. Tissue distribution studies showed that the goldfish GHRH is expressed almost exclusively in the brain, whereas the goldfish GHRH-R is actively expressed in brain and pituitary. Taken together, these results provide evidence for a previously uncharacterized GHRH-GHRH-R axis in nonmammalian vertebrates. Based on these data, a comprehensive evolutionary scheme for GHRH, PRP-PACAP, and PHI-VIP genes in relation to three rounds of genome duplication early on in vertebrate evolution is proposed. These GHRHs, also found in flounder, Fugu, medaka, stickleback, Tetraodon, and rainbow trout, provide research directions regarding the neuroendocrine control of growth in vertebrates.

The adaptive immune response requires waves of T-cell clonal expansion on contact with altered self and contraction after elimination of antigen. In the case of persisting antigen, as occurs for example in cytomegalovirus or Epstein-Barr virus infection, this critical process can become dysregulated and responding T-cells enter into a dysfunctional senescent state. Longitudinal studies suggest that the presence of increased numbers of such T-cells is a poor prognostic factor for survival in the very elderly. Understanding the nature of the defects in these T-cells might facilitate intervention to improve immunity in the elderly. The process of clonal expansion under chronic antigenic stress can be modelled in vitro using continuously cultured T-cells. Here, we have used cDNA array technology to investigate differences in gene expression in a set of five different T-cell clones at early, middle and late passage in culture. Differentially expressed genes were confirmed by real-time polymerase chain reaction, and relationships between these assessed using Ingenuity Systems evidence-based association analysis. Several genes and chemokines related to induction of apoptosis and signal transduction pathways regulated by transforming growth factor beta (TGFbeta), epidermal growth factor (EGF), fos and beta-catenin were altered in late compared to early passage cells. These pathways and affected genes may play a significant role in driving the cellular senescent phenotype and warrant further investigation as potential biomarkers of aging and senescence. These genes may additionally provide targets for intervention.

<h4>Background</h4>Inhalation of diesel exhaust particles (DEPs) is characterized by lung injury and inflammation, with significant increases in the numbers of polymorphonuclear leukocytes and alveolar macrophages. This influx of cellular infiltrates is associated with the activation of multiple genes, including cytokines and chemokines, and the production of reactive oxygen species.<h4>Objective</h4>The pathogenesis of the lung injury is not fully understood, but alterations in the presence or abundance of a number of proteins in the lung have been observed. Our objective in this study was to further characterize these changes and to ask whether additional changes could be discerned using modern proteomic techniques.<h4>Methods</h4>The present study investigates global alterations in the proteome of bronchoalveolar lavage fluid taken from rats 1, 7, or 30 days after exposure to 5, 35, or 50 mg/kg of animal weight of DEPs.<h4>Results</h4>Analysis by surface-enhanced laser desorption/ionization-time of flight mass spectrometry identified two distinct peaks that appeared as an acute response postexposure at all doses in all animals. We identified these two peaks, with mass to charge ratios (m/z) of 9,100 and 10,100, as anaphylatoxin C3a and calgranulin A by additional mass spectral investigation using liquid chromatography coupled to mass spectrometry.<h4>Conclusions</h4>With this approach, we found a number of inflammatory response proteins that may be associated with the early phases of inflammation in response to DEP exposure. Further studies are warranted to determine whether serum levels of these proteins could be markers of diesel exhaust exposure in workers.

The iron that is required to meet the metabolic needs of cells and tissues is derived from the plasma. Plasma iron in turn reflects the release of iron from various body cells, principally the macrophages of the reticuloendothelial system, and the absorption of dietary iron by the proximal small intestine. This iron donation is highly regulated and the liver-derived peptide hepcidin has emerged as the key modulator of cellular iron export. Following its synthesis and secretion from the liver, circulating hepcidin reduces iron export into the plasma by binding to the iron efflux protein ferroportin1 on the surface of enterocytes, macrophages and other cell types and causing its internalization. The level of hepatic hepcidin expression is influenced by HFE, transferrin receptor 2 and hemojuvelin, and the signal transduction pathway(s) linking these proteins to hepcidin are only beginning to be revealed. Hemojuvelin has recently been shown to signal through the bone morphogenetic protein pathway, ultimately activating receptor SMAD/SMAD4 complexes to alter hepcidin transcription. Circulating differic transferrin has emerged as a possible upstream regulator of the liver-based hepcidin regulatory pathway. In addition to being regulated by body iron requirements, hepcidin expression can be modulated by pro-inflammatory cytokines such as interleukin-6. The continuing analysis of inherited disorders of iron metabolism combined with biochemical analysis of signal transduction pathways is essential to fully define this important regulatory system.

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

Expansion of polyglutamine (polyQ) tracts within proteins underlies a number of neurodegenerative diseases, such as Huntington disease, Kennedy disease, and spinocerebellar ataxias. The resulting mutant proteins are unstable, forming insoluble aggregates that are associated with components of the ubiquitin system, including ubiquitin, ubiquitin-like proteins, and proteins that bind to ubiquitin. Given the presence of these ubiquitin-binding proteins in the insoluble aggregates, we examined whether heterologous expression of short motifs that bind ubiquitin, termed ubiquitin-interacting motifs (UIMs), altered the aggregation of polyQ-expanded huntingtin (Htt), the protein product of the Huntington disease gene. We found that a subset of UIMs associated with mutant Htt. The ability to interact with ubiquitin was necessary, but not sufficient, for interaction with mutant Htt. Furthermore, we found that expression of single, isolated UIMs inhibited aggregation of mutant Htt. These data suggest that isolated UIMs might serve as potential inhibitors of polyQ-aggregation in vivo.

The initial step of retinal ganglion cell (RGC) axon pathfinding involves directed growth of RGC axons toward the center of the retina, the optic disc, a process termed "intraretinal guidance". Due to the accessibility of the system, and with various embryological, molecular and genetic approaches, significant progress has been made in recent years toward understanding the mechanisms involved in the precise guidance of the RGC axons. As axons are extending from RGCs located throughout the retina, a multitude of factors expressed along with the differentiation wave are important for the guidance of the RGC axons. To ensure that the RGC axons are oriented correctly, restricted to the optic fiber layer (OFL) of the retina, and exit the eye properly, different sets of positive and negative factors cooperate in the process. Fasciculation mediated by a number of cell adhesion molecules (CAMs) and modulation of axonal response to guidance factors provide additional mechanisms to ensure proper guidance of the RGC axons. The intraretinal axon guidance thus serves as an excellent model system for studying how different signals are regulated, modulated and integrated for guiding a large number of axons in three-dimensional space.

<h4>Background and aim</h4>The development of autoantibodies against antigens of the pancreatic islet cells is a typical phenomenon in patients with type 1 diabetes. The expression of densin, recently shown to be present in kidney podocytes, was explored in the pancreas. Additionally, we studied whether densin and filtrin, another molecule shared between the kidney podocytes and pancreatic islet cells, can act as autoantigens and whether autoantibodies against these can be detected in patients with type 1 diabetes.<h4>Methods</h4>Expression of pancreatic densin was studied with reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence. Children and adolescents (n = 66) with type 1 diabetes and control subjects were analysed for densin autoantibodies (DAA) and filtrin autoantibodies (FAA) using radioimmunoprecipitation assay. The serum samples were obtained at the time of diagnosis and after a duration of 2, 5 and 10 years.<h4>Results</h4>Densin expression was observed in the pancreas, localising to the beta cells. DAA were detected in 33% of the patients and the positivity was typically seen already at diagnosis. FAA were observed in 11% of the patients. The proportion of islet cell antibody (ICA) positive, GADA positive and protein tyrosine phosphatase-related islet antigen 2 antibody (IA-2A)-positive patients decreased during the follow-up period, and a similar trend was seen for DAA but not for FAA. Among the 14 patients with signs of renal injury, four tested positive for DAA and two for FAA.<h4>Conclusions</h4>Densin is a novel molecule shared by the kidney glomerular podocytes and pancreatic islet cells. Densin and filtrin can act as autoantigens, and autoantibodies against these can be detected in patients with type 1 diabetes.

Migraine is a complex familial condition that imparts a significant burden on society. There is evidence for a role of genetic factors in migraine, and elucidating the genetic basis of this disabling condition remains the focus of much research. In this review we discuss results of genetic studies to date, from the discovery of the role of neural ion channel gene mutations in familial hemiplegic migraine (FHM) to linkage analyses and candidate gene studies in the more common forms of migraine. The success of FHM regarding discovery of genetic defects associated with the disorder remains elusive in common migraine, and causative genes have not yet been identified. Thus we suggest additional approaches for analysing the genetic basis of this disorder. The continuing search for migraine genes may aid in a greater understanding of the mechanisms that underlie the disorder and potentially lead to significant diagnostic and therapeutic applications.

Depression is the most common psychiatric comorbidity in epilepsy. To better understand the contribution of seizures versus environment to depression in epilepsy, we investigated differential gene expression using microarray and quantitative RT-PCR, and depressive behavior, in the Porsolt forced swim test in juvenile rats reared in different environments after kainic acid (KA)-induced seizures. We selected for genes significantly down-regulated by KA seizures and upregulated by environmental enrichment. This common gene selection process yielded one known gene involved in mood and affect: serotonin receptor 5B. The changes in serotonin receptor gene expression were paralleled by decreased mobility in the forced swim tests; depressive behavior exhibited after seizures was no longer evident in rats reared in environmental enrichment. Our results suggest that seizures lead to increased susceptibility to depression through transcriptional regulation while environment, in turn, can interact with gene expression to influence the behavioral outcome of epilepsy.

<h4>Purpose</h4>We determined the effects of selenomethionine, the major organic selenium containing compound found in the diet and the form of selenium being used in the Selenium and Vitamin E Cancer Prevention Trial, on prostate cancer cells.<h4>Materials and methods</h4>We assessed global transcript profiles of selenomethionine treated LNCaP using cDNA microarrays and compared them to those of cells treated with methylselenic acid, a direct precursor of methylselenol, which is the active form of selenium in vivo.<h4>Results</h4>After treatment with selenomethionine 2,336 unique genes showed expression changes of at least 1.5-fold in at least 3 time points during 48 hours and 366 unique transcripts differed significantly between selenomethionine and methylselenic acid treated LNCaP. Approximately half of the 76 cell cycle regulated genes affected by selenomethionine were down-regulated and enriched for genes associated with the G2/M phase. Flow cytometry analysis showed that selenomethionine induced G2/M arrest in LNCaP at low concentrations. Selenomethionine also affected expression levels of 35 known androgen responsive genes and 18 of these transcripts showed changes that were the inverse of those seen after androgen stimulation. At high concentrations selenomethionine decreased prostate specific antigen promoter driven luciferase expression.<h4>Conclusions</h4>Selenomethionine modulates transcript levels of genes involved in a number of biological processes, including cell cycle/apoptosis androgen signaling, signal transduction and transcriptional regulation. Although the pathways affected paralleled in many ways those that are modulated by methylselenic acid, distinct differences in transcript patterns and effects on cell cycle regulation suggest that different selenium compounds could exert unique effects in prostate cells.

Iron status and dietary correlates of iron status have not been well described in Hispanic older adults of Caribbean origin. The aim of this study was to evaluate iron status and describe dietary components and correlates of iron status in Hispanic older adults and in a neighborhood-based comparison group of non-Hispanic white older adults. Six hundred four Hispanic and non-Hispanic white adults (59-91 y of age) from the Massachusetts Hispanic Elders Study were included in the analysis. We examined physiological markers of iron status as well as dietary factors in relation to iron status. Dietary intake was assessed by FFQ. Our results revealed that Hispanics had significantly lower geometric mean serum ferritin (74.1 microg/L vs. 100 microg/L; P<0.001), lower hemoglobin concentrations (137+/-13 vs. 140+/-12 g/L; P<0.01), higher prevalence of anemia (11.5 vs. 7.3%; P<0.05), and suboptimal hemoglobin concentrations (<125 g/L) for this age group (21.4 vs. 13.3%; P<0.05). Iron deficiency anemia was higher (7.2% vs. 2.3%; P<0.05) in Hispanic women. Hispanics had lower mean intakes of total iron, vitamin C, supplemental vitamin C, and total calcium than did non-Hispanic whites. After adjusting for age, sex, BMI, alcohol use, smoking, total energy intake, inflammation, diabetes, and liver disease, intake of heme iron from red meat was positively associated and dietary calcium was negatively associated with serum ferritin. This population of Hispanic older adults was significantly more likely than their non-Hispanic white neighbors to suffer from anemia and poor iron status, particularly among women. Cultural variation in dietary patterns may influence iron availability and body iron stores and contribute to an increased risk for iron deficiency anemia among some Hispanic older adults.

Homozygosity for a novel hemochromatosis (HFE) gene splice site mutation (IVS5+1 G/A) was previously reported in a 48-year-old Vietnamese man residing in Germany who had an elevated serum ferritin (SF) and transferrin saturation (TS) and severe iron overload on liver biopsy. This mutation was not found in 222 controls of central European origin but has been found in Southeast Asians living in Vietnam without evidence of iron overload. Hemochromatosis and iron overload screening (HEIRS) Study is an ongoing, multiethnic, primary care-based study of 101,168 North American adults, including 12,772 Asians, a group that the HEIRS Study found has a significantly higher than expected prevalence of elevated serum TS and SF but very low prevalence of the common C282Y and H63D HFE alleles usually associated with hereditary hemochromatosis. It was hypothesized that the IVS5+1 G/A splice site mutation might explain some elevated biochemical iron measures in North American Asians. Overall, 200 Vietnamese subjects from the Los Angeles Field Center who had TS and SF values greater than the 75th percentile of all HEIRS Study participants after adjusting for covariates and 149 controls randomly selected to represent this Vietnamese population were genotyped. Among cases, 1 homozygous mutant and 7 heterozygotes were found; among controls, 1 homozygous mutant and 4 heterozygotes were found yielding an allele frequency of 2.32% for cases and 2.04% for controls (P>0.5). This finding suggests that the HFE IVS5+1 G/A splice site mutation is not the major explanation for unexpectedly high prevalence of TS and SF in North American Asians.

To translate basic research findings into clinical practice, it is essential that information about mutations and variations in the human genome are communicated easily and unequivocally. Unfortunately, there has been much confusion regarding the description of genetic sequence variants. This is largely because research articles that first report novel sequence variants do not often use standard nomenclature, and the final genomic sequence is compiled over many separate entries. In this article, we discuss issues crucial to clear communication, using examples of genes that are commonly assayed in clinical laboratories. Although molecular diagnostics is a dynamic field, this should not inhibit the need for and movement toward consensus nomenclature for accurate reporting among laboratories. Our aim is to alert laboratory scientists and other health care professionals to the important issues and provide a foundation for further discussions that will ultimately lead to solutions.

Genetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. Hepcidin, a negative regulator of intestinal iron absorption, is found to be inappropriately low in both patients and in animal models, indicating that proper control of basal hepcidin levels requires both hemojuvelin and HFE. In mice, repulsive guidance molecule c (Rgmc, the hemojuvelin mouse ortholog) and hepcidin levels are transcriptionally regulated during inflammation. Here, we report that basal Rgmc levels in Hfe-deficient mice are normal and that these mice retain the ability to suppress Rgmc expression after lipopolysaccharide (LPS) challenge. Thus, Rgmc regulation by LPS is Hfe-independent. The response of Rgmc to LPS involves signaling through toll-like receptor 4 (Tlr4), because Tlr4-deficient mice do not show altered Rgmc expression after LPS administration. We further show that tumor necrosis factor-alpha, but not interleukin-6, is sufficient to cause Rgmc down-regulation by LPS. These results contrast with previous data demonstrating that hepcidin levels are directly regulated by interleukin-6 but not by tumor necrosis factor-alpha. The regulation of iron-related genes by different cytokines may allow for time-dependent control of iron metabolism changes during inflammation and may be relevant to chronic inflammation, infections, and cancer settings, leading to the development of anemia of chronic disease.

<h4>Background</h4>In order to recapitulate tumor progression pathways using epigenetic data, we developed novel clustering and pathway reconstruction algorithms, collectively referred to as heritable clustering. This approach generates a progression model of altered DNA methylation from tumor tissues diagnosed at different developmental stages. The samples act as surrogates for natural progression in breast cancer and allow the algorithm to uncover distinct epigenotypes that describe the molecular events underlying this process. Furthermore, our likelihood-based clustering algorithm has great flexibility, allowing for incomplete epigenotype or clinical phenotype data and also permitting dependencies among variables.<h4>Results</h4>Using this heritable clustering approach, we analyzed methylation data obtained from 86 primary breast cancers to recapitulate pathways of breast tumor progression. Detailed annotation and interpretation are provided to the optimal pathway recapitulated. The result confirms the previous observation that aggressive tumors tend to exhibit higher levels of promoter hypermethylation.<h4>Conclusion</h4>Our results indicate that the proposed heritable clustering algorithms are a useful tool for stratifying both methylation and clinical variables of breast cancer. The application to the breast tumor data illustrates that this approach can select meaningful progression models which may aid the interpretation of pathways having biological and clinical significance. Furthermore, the framework allows for other types of biological data, such as microarray gene expression or array CGH data, to be integrated.

Autism is a dynamic neurodevelopmental syndrome in which disabilities emerge during the first three postnatal years and continue to evolve with ongoing development. We briefly review research in autism describing subtle changes in molecules important in brain development and neurotransmission, in morphology of specific neurons, brain connections, and in brain size. We then provide a general schema of how these processes may interact with particular emphasis on neurotransmission. In this context, we present a rationale for utilizing pharmacologic treatments aimed at modifying key neurodevelopmental processes in young children with autism. Early treatment with selective serotonin reuptake inhibitors (SSRIs) is presented as a model for pharmacologic interventions because there is evidence in autistic children for reduced brain serotonin synthesis during periods of peak synaptogenesis; serotonin is known to enhance synapse refinement; and exploratory studies with these agents in autistic children exist. Additional hypothetical developmental interventions and relevant published clinical data are described. Finally, we discuss the importance of exploring early pharmacologic interventions within multiple experimental settings in order to develop effective treatments as quickly as possible while minimizing risks.

<h4>Aim</h4>To examine the clinical characteristics of a subgroup of patients with hepatocellular carcinoma (HCC) and compare them to those with known risk factors.<h4>Methods</h4>We used the HCC database of 306 patients seen at our institution from January 1, 1995 to December 31, 2001. Of the 306 patients, 63 (20%, group 1) had no known risk factors (hepatitis C virus, hepatitis B virus, alcohol, hemochromatosis or cirrhosis from any cause) and 243 (group 2) had one or more risk factors.<h4>Results</h4>The median age was similar in both groups, but there were disproportionate numbers of younger (< 30 years old), older (> 80 years) patients, women (33% vs 18%), and Caucasians (81% vs 52%) in group 1 as compared to group 2. There were fewer Asians (2% vs 11%) and African Americans (13% vs 27%) in group 1. Abdominal pain (70% vs 37%) was more common while gastrointestinal bleeding (0% vs 11%) and ascites (4% vs 17%) were less common in group 1 compared to group 2. Group 1 had larger tumor burden (median size 9.4 cm vs 5.7 cm) at the time of presentation, but there were no differences in the site (right, left or bilateral lesions), or number of tumors between the two groups.<h4>Conclusion</h4>HCC patients without identifiable risk factors have different characteristics and clinical presentation compared to those with known risk factors. Absence of cirrhosis and larger tumor burden may explain the differences in the presenting symptoms.

<h4>Context</h4>Depression in bipolar disorder is clinically indistinguishable from that observed in major depressive disorder. As in major depression, selective serotonin reuptake inhibitors targeting brain serotonin transporters are first-line treatments for bipolar depression. Associations of serotonin transporter promoter polymorphisms and bipolarity have been reported; however, research on alterations in serotonergic neurotransmission in bipolar depression remains scant.<h4>Objectives</h4>To assess in vivo brain serotonin transporter binding potential (BP(1), proportional to serotonin transporter number) in patients with bipolar depression and controls and to examine the relationship between serotonin transporter binding and genotype.<h4>Design</h4>Case-control study.<h4>Setting</h4>University hospital.<h4>Participants</h4>A sample of 18 medication-free patients with bipolar depression and 41 controls.<h4>Main outcome measures</h4>In vivo brain serotonin transporter binding was measured using positron emission tomography and radiolabeled trans-1,2,3,5,6,10-beta-hexahydro-6-[4-(methylthio)phenyl]pyrrolo-[2,1-a]-isoquinoline ([(11)C](+)-McNeil 5652). Participants were genotyped assessing biallelic and triallelic 5-HTTLPR polymorphisms.<h4>Results</h4>Patients with bipolar disorder had 16% to 26% lower serotonin transporter BP(1) in the midbrain, amygdala, hippocampus, thalamus, putamen, and anterior cingulate cortex. Triallelic 5-HTTLPR genotypes were unrelated to serotonin transporter BP(1).<h4>Conclusions</h4>Lower serotonin transporter BP(1) in bipolar depression overlaps with that observed in major depression and suggests that serotonergic dysfunction is common to depressive conditions.

Neurotransmitters have emerged as important players in the control of programmed cell death in the cerebral cortex. We report that genetic depletion of serotonin, dopamine, and norepinephrine in mice lacking the vesicular monoamine transporter (VMAT2 KO mice) causes an increase in cell death in the superficial layers of the cingulate and retrosplenial cortices during early postnatal life (postnatal days 0-4). Electron microscopy and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling indicated that this represents a form of apoptosis. Caspase-3 and -9 are over activated in the VMAT2 KO cortex and Bcl-X(L) is downregulated, whereas the apoptosis-inducing factor caspase-8 and FasL/FasR pathway are not involved. Partial inhibition of serotonin or/and catecholamine synthesis by pharmacological treatments or genetic reduction of serotonin neuron number in mice lacking the transcription factor Pet-1 (pheochromocytoma 12 E26 transformation-specific) did not modify the cell death ratios in the cerebral cortex. However, when monoamine oxidase type A was invalidated in the VMAT2 KO background (VMAT2-MAOA DKO mice), increases in 5-HT levels coincided with a reduction of cell death and a normalization of Bcl-X(L) expression. trkB signaling is not implicated in the anti-apoptotic effects of MAOA inhibition because BDNF mRNA levels were unchanged in VMAT2-MAOA DKO mice and because the massive cell death in the cerebral cortex of trkB KO mice is also reverted by genetic invalidation of the MAOA gene. Finally the broad 5-HT2 receptor agonist (-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride prevented the increase in cell death of VMAT2 KO mice. Altogether, these results suggest that high levels of serotonin, acting through 5-HT2 receptors, have neuroprotective action on cortical neurons by controlling Bcl-X(L) mRNA levels and that this action is independent of trkB signaling.

Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation.

Obesity and hypertension are increasing medical problems in adolescents. Serotonin transporter (5-HTT) is involved in mood and eating disturbances. Encoded by the gene SLC6A4, the promoter shows functional insertion/deletion alleles: long (L) and short (S). Because individuals who are carriers for the short version are known to be at risk for higher levels of anxiety, we hypothesized that this variant may be associated with overweight. Data and blood samples were collected from 172 adolescents out of a cross-sectional, population-based study of 934 high school students. To replicate the findings, we also included 119 outpatients from the Nutrition and Diabetes Section of the Children's County Hospital. We found that the S allele was associated with overweight (BMI > 85th percentile), being a risk factor for overweight independently of sex, age, and hypertension [odds ratio (OR): 1.85; 95% confidence interval (CI): 1.13, 3.05; p < 0.02]. Additionally, in the outpatient study, compared with the homozygous LL subjects, S allele carriers showed a higher BMI z-score (1.47 +/- 1.09 vs. 0.51 +/- 1.4; p < 0.002) and were more frequent in overweight children. In conclusion, the S allele of the SLC6A4 promoter variant is associated with overweight being an independent genetic risk factor for obesity.

The subunit composition of GABA(A) receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of gamma2 subunit-containing GABA(A) receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA(A) receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of gamma2 subunit-containing GABA(A) receptors. The surface numbers of diazepam binding sites (alpha/gamma2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (alpha/beta subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA(A) receptors and GABA(A) receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA(A) receptor beta subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of gamma2 subunit-containing GABA(A) receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.

An established model for mechanistic analysis of lung carcinogenesis involves administration of 3-methylcholanthrene to mice followed by several weekly injections of the tumor promoter 2,6-di-tert-butyl-4-methylphenol (BHT). BHT is metabolized to quinone methides (QMs) responsible for promoting tumor formation. QMs are strongly electrophilic and readily form adducts with proteins. The goal of the present study was to identify adducted proteins in the lungs of mice injected with BHT and to assess the potential impact of these modifications on tumorigenesis. Cytosolic proteins from treated mouse lungs were separated by two-dimensional electrophoresis, adducts detected by immunoblotting, and proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eight adducts were detected in the lungs of most, or all, of six experimental groups of BALB mice. Of these adducts, several were structural proteins, but others, namely, peroxiredoxin 6 (Prx6), Cu,Zn-superoxide dismutase (SOD1), carbonyl reductase, and selenium-binding protein 1, have direct or indirect antioxidant functions. When the 9000g supernatant fraction of mouse lung was treated with BHT-QM (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone), substantial lipid peroxidation and increases in hydrogen peroxide and superoxide formation were observed. Studies with human Prx6 and bovine SOD1 demonstrated inhibition of enzyme activity concomitant with adduct formation. LC-MS/MS analysis of digests of adducted Prx6 demonstrated adduction of both Cys 91 and Cys 47; the latter residue is essential for peroxidatic activity. Analysis of QM-treated bovine SOD1 by matrix-assisted laser desorption/ionization time-of-flight MS demonstrated the predominance of a monoadduct at His 78. This study provides evidence that indicates Prx6, SOD1, and possibly other antioxidant enzymes in mouse lung are inhibited by BHT-derived QMs leading to enhanced levels of reactive oxygen species and inflammation and providing a mechanistic basis for the effects of BHT on lung tumorigenesis.

The human major histocompatibility complex HLA is located on the short arm of chromosome 6. It is known to be the most polymorphic genetic system in humans. The biological role of the HLA class I and class II molecules is to present processed peptide antigens. The HLA system is clinically important as transplantation antigens. Molecular HLA allele typing is routinely performed to provide HLA class I and class II allele matching in unrelated donor hematopoietic stem cell transplantation. Prospective lymphocyte crossmatching is critical in solid organ transplantation to prevent allograft rejection. HLA alloimmunization causes various problems in transfusion therapy. The HLA system is associated with certain diseases, but its underlying mechanisms are not yet fully explained.

Huntingtin-interacting protein 1 (HIP1) is an endocytic adaptor protein that plays a role in clathrin-mediated endocytosis and the ligand-induced internalization of AMPA receptors (AMPARs) (Metzler et al., 2003). In the present study, we investigated the role of HIP1 in NMDA receptor (NMDAR) function by analyzing NMDA-dependent transport and NMDA-induced excitotoxicity in neurons from HIP1-/- mice. HIP1 colocalizes with NMDARs in hippocampal and cortical neurons and affinity purifies with NMDARs by GST (glutathione S-transferase) pull down and coimmunoprecipitation. A profound decrease in NMDA-induced AMPAR internalization of 75% occurs in neurons from HIP1-/- mice compared with wild type, using a quantitative single-cell-based internalization assay. This defect in NMDA-dependent removal of surface AMPARs is in agreement with the observed defect in long-term depression induction in hippocampal brain slices of HIP1-/- mice and supports a role of HIP1 in AMPAR internalization in vivo. HIP1-/- neurons are partially protected from NMDA-induced excitotoxicity as assessed by LDH (lactate dehydrogenase) release, TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling) and caspase-3 activation assays, which points to a role of HIP1 in NMDA-induced cell death. Interestingly, phosphorylation of Akt and its substrate huntingtin (htt) decreases during NMDA-induced excitotoxicity by 48 and 31%, respectively. This decrease is significantly modulated by HIP1, resulting in 94 and 48% changes in P-Akt and P-htt levels in HIP1-/- neurons, respectively. In summary, we have shown that HIP1 influences important NMDAR functions and that both HIP1 and htt participate in NMDA-induced cell death. These findings may provide novel insights into the cellular mechanisms underlying enhanced NMDA-induced excitotoxicity in Huntington's disease.

Anticoagulation treatment can prevent systemic embolism in patients with mitral stenosis (MS) and atrial fibrillation (AF), but this treatment is under debate if patients are in sinus rhythm. The authors aimed to determine the hemostatic changes in patients with MS and sinus rhythm. Forty-six patients (28 in sinus rhythm and 18 in AF) with mitral stenosis were enrolled in this study. They studied systemic venous fibrinogen, D-dimer, antithrombin-III, tissue plasminogen activator (tPA), plasminogen activator inhibitor-I (PAI-I), von Willebrand factor (vWF), and platelet factor 4 (PF 4) in these patients. The patients were first classified according to their rhythm as sinusal and AF, and then according to the presence of left atrial spontaneous echo contrast (LASEC). Fibrinogen, D-dimer, antithrombin-III, vWF, and PF 4 levels were significantly greater in patients with MS and sinus rhythm or atrial fibrillation compared to the control group (p < 0.05). Whether the rhythm was sinus or AF, fibrinogen, D-dimer, antithrombin-III, vWF, and PF 4 levels were significantly higher in patients with LASEC than in the control group (p < 0.05). Only PF 4 was higher in the AF group than in those with sinus rhythm (p < 0.05). As to plasminogen activator and PAI-I levels, only tissue plasminogen activator levels were found to be higher in the AF group than in those with sinus rhythm and the control group (p < 0.05). In patients with mitral stenosis and sinus rhythm, if LASEC is present, coagulation activation, platelet activation, and endothelial dysfunction are similar in patients with AF, and anticoagulation should be considered in these patients.

<h4>Aim</h4>To investigate whether serum levels of two soluble forms of extracellular cytokeratin 18 (M30-antigen and M65-antigen) may differentiate nonalcoholic steatohepatitis (NASH) from simple steatosis in patients with nonalcoholic fatty liver disease (NAFLD).<h4>Methods</h4>A total of 83 patients with suspected NAFLD and 49 healthy volunteers were investigated. Patients with suspected NAFLD were classified according to their liver histology into four groups: definitive NASH (n=45), borderline NASH (n=24), simple fatty liver (n=9), and normal tissue (n=5). Serum levels of caspase-3 generated cytokeratin-18 fragments (M30-antigen) and total cytokeratin-18 (M65-antigen) were determined by ELISA.<h4>Results</h4>Levels of M30-antigen and M65-antigen were significantly higher in patients with definitive NASH compared to the other groups. An abnormal value (> 121.60 IU/L) of M30-antigen yielded a 60.0% sensitivity and a 97.4% specificity for the diagnosis of NASH. Sensitivity and specificity of an abnormal M65-antigen level (> 243.82 IU/L) for the diagnosis of NASH were 68.9% and 81.6%, respectively. Among patients with NAFLD, M30-antigen and M65-antigen levels distinguished between advanced fibrosis and early-stage fibrosis with a sensitivity of 64.7% and 70.6%, and a specificity of 77.3% and 71.2%, respectively.<h4>Conclusion</h4>Serum levels of M30-antigen and M65-antigen may be of clinical usefulness to identify patients with NASH. Further studies are mandatory to better assess the role of these apoptonecrotic biomarkers in NAFLD pathophysiology.

In this follow-up of a randomized placebo-controlled clinical trial of nicotine replacement transdermal patch for smoking cessation, 741 smokers of European ancestry who were randomized to receive active patch or placebo patch were genotyped for the serotonin transporter gene-linked polymorphic region. The study setting was a primary care research network in Oxfordshire, United Kingdom. The primary outcome measures were biochemically verified sustained abstinence from cigarette smoking at end of treatment and 24-week follow-up. The main effect of genotype was not associated with sustained abstinence from smoking at either end of treatment (SL: p=.33; SS: p=.81) or 24-week follow-up (SL: p=.05; SS: p=.21), and we found no evidence for a genotypextreatment interaction effect. In summary, despite the theoretically important contribution of serotonin neurotransmission to smoking cessation, the serotonin transporter gene was not associated with treatment response to nicotine patch for smoking cessation in this primary care-based trial.

<h4>Aim</h4>To elucidate the sequential transfer of iron amongst ferritin, transferrin and transferrin receptor under various iron status conditions.<h4>Methods</h4>Incorporation of 59Fe into mucosal and luminal proteins was carried out in control WKY rats. The sequential transfer of iron amongst ferritin, transferrin and transferrin receptor was carried out in iron deficient, control and iron overloaded rats. The duodenal proteins were subjected to immunoprecipitation and quantitation by specific ELISA and in situ localization by microautoradiography and immunohistochemistry in tandem duodenal sections. Human duodenal biopsy (n = 36) collected from subjects with differing iron status were also stained for these proteins.<h4>Results</h4>Ferritin was identified as the major protein that incorporated iron in a time-dependent manner in the duodenal mucosa. The concentration of mucosal ferritin was significantly higher in the iron excess group compared to control, iron deficient groups (731.5 +/- 191.96 vs 308.3 +/- 123.36, 731.5 +/- 191.96 vs 256.0 +/- 1.19, P < 0.005), while that of luminal transferrin which was significantly higher than the mucosal did not differ among the groups (10.9 +/- 7.6 vs 0.87 +/- 0.79, 11.1 +/- 10.3 vs 0.80 +/- 1.20, 6.8 +/- 4.7 vs 0.61 +/- 0.63, P < 0.001). In situ grading of proteins and iron, and their superimposition, suggested the occurrence of a sequential transfer of iron. This was demonstrated to occur through the initial binding of iron to luminal transferrin then to absorptive cell surface transferrin receptors. The staining intensity of these proteins varied according to the iron nutrition in humans, with intense staining of transferrin receptor observed in iron deficient subjects.<h4>Conclusion</h4>It is concluded that the intestine takes up iron through a sequential transfer involving interaction of luminal transferrin, transferrin-transferrin receptor and ferritin.

Iron can accumulate in the liver in a variety of conditions, including congenital, systemic iron-loading conditions (hereditary hemochromatosis), conditions associated with systemic macrophage iron accumulation (transfusions, hemolytic conditions, anemia of chronic disease, etc), in some hepatitidies (hepatitis C, alcoholic liver disease, porphyria cutanea tarda), and liver-specific iron accumulation of uncertain pathogenesis in cirrhosis. The anatomic pathologist will be faced with the task of determining whether iron accumulation in the liver is significant and, if so, the nature of the disease that lead to the accumulation (ie diagnosis). The tools available to the pathologist include (most importantly) histologic examination with iron stain, quantitative iron analysis, clinical history, laboratory iron tests (serum iron and iron-binding capacity, serum ferritin) and germline genetic analysis for mutations in genes known to be associated with hemochromatosis (HFE, ferroportin, hepcidin, hemojuvelin, transferrin receptor-2). This article provides an overview of the above.

The aim of the study was to verify the presence of mutated tumor suppresser gene p53 in intestinal mucosa with histologically confirmed premalignant lesions and gastric carcinoma, and assess its prognostic value. The paper presents prospective study that included 50 patients with gastric adeno-carcinoma of intestinal type that were treated at Gastroenterohepatology Clinic, and 50 patients with histologically confirmed chronic atrophic H. pylori positive gastritis. In the mucosa biopsy samples, we analyzed presence, frequency and severity of inflammatory-regenerative, metaplastic and dysplastic changes. We typed intestinal metaplasia immunohistochemically and confirmed the presence of p53 onco-protein in antigen positive gastric carcinoma cells, and evaluated its prognostic value. Our results suggest that H. pylori acts as an initiator of inflammatory processes in gastric mucosa, which are followed by emergence of precancerous lesions. p53 is expressed late in carcinogenesis (14%) and as such, may be considered as an indicator of transformation of premalignant into malignant lesion.

The effect of crystallization of hydroxyapatite on the secondary structure of bovine serum albumin (BSA) was studied by circular dichroism spectrum, Fourier transform infrared spectroscopy, derivative, deconvolution and curve-fitting techniques in the present paper. The CD results show that pure bovine serum albumin is composed of 56.8% alpha-helices, 5.8% beta-sheets, 14.1% beta-turns and 23.9% random structures, while the bovine serum albumin in the Ca10(PO4)6(OH)2/bovine serum albumin solution is composed of 25.4% alpha-helices, 25.0% beta-sheets, 20.0% beta-turns and 29.7% random structures. The results of Fourier transform infrared spectroscopy are in good agreement with those from the CD spectra. From these results it can be seen that the percentage of alpha-helix decreased, while that of the beta-sheet increased with the formation of the crystal of hydroxyapatite, and with the reaction time increasing, the percentages of alpha-helix obviously dropped and those of beta-sheet markedly rose. These results showed that alpha-helix transformed into beta-sheet. Furthermore the essence of these changes is discussed.

We review association studies that have examined the genetic basis of eating disorders. Overall, findings suggest that serotonin, brain-derived neurotrophic factor, and estrogen genes may be important for the development of the disorders. These neuronal systems influence behavioral and personality characteristics (e.g., anxiety, food intake) that are disrupted in eating disorders. Future studies would benefit from larger sample sizes and inclusion of behavioral and personality covariates in analyses. Consideration of the mechanisms of genetic effects and interactions between genes and environment is also needed to extend conceptualizations of the genetic basis of these disorders.

The structural protein group-specific antigen (Gag) is the only viral product required for retrovirus assembly and will form virus-like particles (VLPs) in vitro in the presence of nucleic acids. The nature of the contribution of nucleic acid to particle assembly and structure is not fully understood. Further, interactions of Gag protein with nucleic acids have not been characterized in any detail. We have utilized a quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate the possible assembly of Gag particles in the presence of three oligonucleotides—TGx2.5, TGx10, and TGx20 (a 5-mer, 20-mer, and 40-mer, respectively). QCM-D provides information on both mass changes, measured as a change in frequency, and structural changes measured as changes in energy dissipation. TGx2.5, TGx10, and TGx20 were immobilized, via a biotin linker, to an amine-coupled NeutraAvidin (Pierce Chemical Co., Rockford, IL) surface. HIV Gag protein was then passed over the surfaces via a peristaltic pump. QCM-D data are summarized as follows: The frequency values decreased with time when Gag was flowed over the oligo surfaces, indicating binding of protein. A continuous decrease in dissipation and frequency values was observed during the binding of Gag protein with the longest oligonucleotide, TGx20, indicating increase in the rigidity of the oligo-protein film. This suggests the formation of intermolecular complex(es) between the bound oligo and the protein. Binding of Gag with TGx10 and TGx2.5 showed varying dissipation values with time. This indicates oligo-protein assemblies go through several structural changes during the course of binding. The control NeutraAvidin surface exhibited an increase in dissipation on binding with Gag that is typical of mass adsorption, indicating an increase in the viscoelasticity of the adsorbed film.

In addition to the general secretion pathway, Mycobacterium tuberculosis utilizes an alternative secretion apparatus called ESX-1, which is essential for control of host-cell responses to infection. Despite its importance in virulence, this system is poorly characterized and has few known substrates. There are five duplicate loci of the ESX-1 system in the genome, further increasing the difficulty in defining the nature of substrate secretion. Previous efforts to map the secreted proteome of this bacterium have been less successful because of the inability to differentiate true secreted substrates from those that are present due to lysis or sample preparation. We established and quantified secreted substrates from M. tuberculosis using a novel application of a known mass-spectrometric quantitative technique employing isobaric mass tags and applied it to culture supernatants from wild-type M. tuberculosis and mutants deficient for components of ESX-1. Using this technique, we sought to identify ESX-1 substrates and determine whether duplicate Cfp-10 paralogs are secreted in an ESX-1-dependent manner. This has enabled us to definitively illustrate that most of the Cfp-10 paralogs are secreted in the absence of functional ESX-1, uncovering the presence of alternative secretion systems that have not been characterized. Also, we paradoxically found a quantitative increase in the secretion of substrates dependent upon Sec in ESX-1 mutants, suggesting cross-talk between general and alternative secretion systems. Further characterization of these substrates by mass-spectrometric quantification of dominant-negative mutants gave further insight into the specificity and mechanism of ESX-1 substrate secretion.

Selenomethionine substitution is the preferred method for preparing heavy-atom derivatives of proteins for crystal structure determination using the multi-wavelength anomalous diffraction phasing method. This approach allows researchers to take advantage of the anomalous signal from a number of diverse atoms. We recently published a protocol describing a number of variables that play a role in determining incorporation efficiency of selenomethionine into mammalian expression systems.1 Here we describe, in detail, a simple method for assessing selenomethionine substitution by replacement of methionine in homogeneous protein preparations. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) technology following trypsin proteolysis of the recombinant protein, we are able to evaluate variables that play roles in affecting selenomethionine incorporation. Examples will illustrate (a) the ease of identification of modified peptides containing the selenomethione and (b) relative quantitation of such peptides when compared with the control, unmodified peptides.

Based on specially treated large-pore silica and enhanced with a proprietary bonding process, Vydac MS reversed-phase (RP) high-performance liquid chromatography columns offer superior performance for peptides and proteins. The deamidation of human growth hormone (HGH) has been monitored for many years by RP using Vydac columns. The Vydac MS C4 column provides the best overall performance characteristics (recovery, resolution, and peak symmetry) for the common important assay of HGH and desamido HGH. Although hydrophobic membrane proteins are particularly difficult to separate, the Vydac MS C4 column provides better separation and recovery (up to 86% higher than other leading columns) for a reptilian reovirus p14 protein and myristolyated form, a component of a potentially new vaccine-delivery system. Separation of the trypsin digest of fetuin, a glycoprotein, exhibits improved selectivity for peptide mapping on a Vydac MS C18 column compared to other C18 columns, revealing some peaks otherwise not seen. The improved selectivity for peptides on the Vydac MS columns results in better primary structure definition and easier identification of degradation products and other protein characteristics.

Objective: To apply advanced statistical model-building procedures to derive proteomic “signatures” from 2D gels and validate the approach by predicting double-blinded samples. Methods: A large experiment was used to explore the power of the predictive modeling process (340 samples, 18 groups, seven double-blinded and three unknown). The images were geometrically corrected and then analyzed at a pixel level. On completion of this procedure, areas important for obtaining good group discrimination were automatically identified. The areas were ranked and visually examined. Up to 10 per group were selected for the next stage of analysis. The 117 resultant spots were then used to build predictive models. The models were explored in the context of the experiment and also for their prediction performance. This process enables the selection of candidate spots that may be below standard univariate thresholds (such as p < 0.05, 1.5-fold change). Results: Models were successfully built that gave perfect performance on the training sets. The blind samples were successfully predicted and interesting information on the unknown samples was produced and is the subject of further experimentation. The effective “systems” dimension for the 18 group sets was estimated to be 12, which suggests we may have more groups than is supported by the data. A “minimal spot set” was calculated and showed a saturation in prediction performance at around 60 spots. A follow-on procedure was employed to choose the best spots for group discrimination and also to specify the spot number vs. performance relationship. Conclusion: Proteomics data provide a rich source for advanced statistical modeling techniques, and using standard double-blind procedures can add an intuitive confidence to the experimental results. The techniques are very powerful in assisting in the exploration of the complex relationships intrinsic to the data.

A non-isobaric label chemistry is introduced that allows the triplexed quantification of proteomic samples based on the isotope-coded protein-labeling technology (ICPL). Amino groups of intact proteins were derivatized with three isotopically different nicotinoyl reagents (L: 12C6H4; M: 12C6D4; and H: 13C6H4) prior to protein separation and digestion. Here, we describe different approaches in respect of MS instruments and protein separation. Protein samples with different complexity were labeled with ICPL. For one sample, the proteins were digested, separated by CAP LC, and spotted on disposable targets for subsequent MALDI measurements. The other sample was separated on a 1D gel, excised, digested, and supplied to LC-MS/MS using a Qq orthogonal time-of-flight instrument. Excellent results were obtained from the LC-MALDI and the Qq TOF approach with the three protein samples labeled differentially and measured together. The multiplexed labeling allows us to reduce the experimental effort when proteomics experiments are run as replicates and when more than two different states are to be compared. A unique property of ICPL compared to other label chemistries is its compatibility with protein pre-fractionation, as the labeling reaction works on the undigested protein.

The reversible phophorylation of serine, threonine, and tyrosine is one of the most important post-translational modifications involved in various cellular functions. Identification of phophorylation sites by mass spectrometry is challenging due to the low abundance of phosphopeptides and their limited ionization efficiency. Therefore, it is critical to selectively enrich the phosphopeptides prior to MS analysis. In this study we investigated the affinity extraction of phosphopeptides using a metal oxide–based solid-phase extraction (SPE) microscale device. This sorbent shows high affinity towards phosphopeptides; acidic peptide adsorption is greatly minimized. When dealing with highly complex samples (e.g., cell lysate), the selectivity of phosphopeptides can be further improved by mixing the sample with additive, aromatic carboxylic acids prior to loading onto the SPE. The metal oxide SPE is compared with other methods such as immobilized metal affinity chromatography and titanium dioxide–based phosphopeptide enrichment. Enhanced performance was observed in terms of the selectivity. MALDI-TOF and nanoLC/MS/ MS were used to study the phosphopeptide recovery and selectivity. Phosphopeptide standards, alpha-casein tryptic digest, and yeast cell lysate were used to evaluate the phosphopeptide enrichment method’s performance.

The up- and down-regulation of protein expression has long been a focus of proteomics researchers and has given rise to several mass spectrometry–based quantitation techniques. In the past decade, a great deal of attention has turned toward the subproteomes, particularly those representing post-translational modifications (PTMs), such as phosphorylation. Those PTMs often play key roles in signaling cascades, and affect a number of cellular processes; their study offers insight into intracellular function. We illustrate here phosphoproteome quantitation through the pairing of immobilized metal-affinity chromatography (IMAC) and 18O labeling, which allows for the determination of modulated phosphorylated species in response to external stressors. Following application of varying biological stressors and isolation of each resultant proteome, the samples were proteolytically cleaved using immobilized trypsin. IMAC enrichment of phosphorylated peptides was accomplished using a nitrilotriacetic acid (NTA) analog bonded to silica, presenting Ga3+ as the chelated metal species. Immobilized trypsin was also employed for incorporation of 18O into the sample for quantitative global labeling. It was determined that IMAC enrichment utilizing this combination of chelate, substrate, and metal provided significant enrichment of phosphopeptides that afforded signal levels, which allowed for quantitative analysis of the phosphoproteome. The use of 18O labeling of the samples permitted a global approach to targeting the phosphoproteome concurrent with the non-phosphorylated component. Using MS analysis before and after IMAC-based phosphopeptide enrichment revealed a distinction in regulation of expression and modulation through phosphorylation of the proteins. The coupling of phosphopeptide enrichment with global isotopic labeling provides a valuable and enabling resource for the vastly under-explored area of quantitative phosphoproteomics.

Available peptide fragmentation interpretation software is focused on sequence database–driven protein identification, rather than on primary structure elucidation. Searching for posttranslational modification (PTM) or sequence errors currently needs time-critical manual intervention and evaluation. Here we describe the results and performance of a novel interpretation software, which was used to characterize more than 50 recombinant serine/threonine kinases, receptor tyrosine kinases, or cytoplasmatic tyrosine kinases. LC-MS/MS data was acquired after tryptic digestion of the recombinantly produced kinases. The datasets have been imported to the proteome bioinformatics platform ProteinScape. The spectra were screened for a set of modifications, amino acid substitutions, unsuspected large measurement errors, enzyme no-specificity, and unknown mass shifts. The software restricts the search space by testing only sequences of interest. In widely used sequence database searches, testing all modifications and possible non-specific cleavages is not feasable. Besides the increase in sequence coverage basically caused by detection of one side non-specifically cleaved peptides, numerous modifications were found—namely, phosphorylation. methylation, pyroglutamate formation, methinonine oxidation, and N-terminal acetylation. As spectra of phosphorylated peptides are almost always in the minority compared to their unmodified counterparts, their detection is a challenge, but internal significance analysis revealed a substantial amount of phosphorylation.The phenomenon of auto-phosphorylation of kinase proteins was successfully monitored. The phosphorylation sites are categorized according to their sequence motive, and additionally their distribution is compared to phosphoryation sites described in public databases. Using this software triggered by the proteome database software proteinscape, searches were performed in a highly automated manner. Manual analysis could be reduced to minutes for the LC-MS/MS datasets containing more than 1000 spectra. Integrated result presentation strategies, which use clustering of spectra results on the amino acid level to annotate the protein sequence of interest, avoided the the possibility of seeing excess PTM contained in the large amount of acquired spectra.

Previously we reported on a protocol for the complete de novo sequencing of a therapeutic monoclonal antibody. We are continuously evaluating biochemical, analytical, and bioinformatics methods to add to our toolbox for increased throughput. Microwave-assisted proteomics has recently emerged as a way to decrease biochemical and proteolytic reaction time. Here, we describe an investigation into microwave-assisted proteolytic cleavage using trypsin, Lys-C, and Asp-N. Microwave irradiation was also applied to the digestion of proteins for removal of N-linked sugars using PNGase-F and removal of pyroglutamyl group employing pyroglutateamino peptidase (PGAP). Method: Proteins were digested with trypsin, Lys-C, Asp-N in the thermocycler at 37°C for 5, 10, and 30 min, or in the CEM microwave at 37°C, 2 W, at the same time points. Generated peptides were then analyzed by SDS-PAGE for quantitation or via LC/MS/MS for sequence coverage. Microwave-assisted deglycosylation of proteins was performed at 37–60°C for 2 min to 1 h or in the thermo-cylcer at 37°C for 2 min to overnight, and molecular weight analysis was performed. PGAP digestion was performed in the thermocycler or microwave at 90°C at different time points, and proteins were sequenced by Edman degradation analysis to assess de-blocking of the N-terminus. Preliminary Results: For proteolytic digestion, a significant reduction in incubation time was observed for trypsin when microwave irradiation was applied. However, no benefit was observed with microwave-assisted Lys-C or Asp-N digestion. Deglycosylation of proteins using microwave irradiation markedly decreased the incubation time from several hours to 10 min for most proteins. The initial deblocking using PGAP occurred more rapidly in the microwave versus thermocycler, but overall incubation times were not significantly reduced.

Objective: To develop a procedure that can serve to identify phosphopeptides in complex mixtures. The identification and characterization of phosphopeptides by mass spectrometry is an active area of investigation due to the importance of the modification in a variety of regulatory processes. Phosphopeptides exhibit high binding affinity for a number of metal ions, including iron(III) and gallium(III). We have investigated whether MALDI-MS allows the detection of cationized phosphopeptides. A number of metal ions, including Fe(III), Ga(III) and Ca(II), were tested in a variety of matrices. It was found that Fe(III) binds preferentially to phosphopeptides in the presence of non-phosphorylated peptides under acidic conditions. Preliminary results: When incubating phosphopeptides with millimolar concentrations of metal salts (FeCl3, Ga(NO3)3, CaCl2), cationized species were observed in MALDI-MS spectra with all matrices tested. The major cationized species carried a single positive charge, e.g., [M –2H +Fe]+. The cationized species typically exhibited a signal intensity 10–20% that of the protonated one ([M+H]+). To test the specificity of the metal ion binding, a model phosphopeptide (beta-casein 48-63) was mixed with a BSA tryptic digest and incubated with metal salts on the MALDI target in the presence of matrix. Cationization was observed to a varying extent in all cases, but only one metal ion/matrix combination exhibited specificity for the phosphopeptide. Iron(III) bound to the phosphopeptide exclusively when employing ACHC as the matrix. A variety of phosphopeptides containing phosphorylated serine, threonine, and tyrosine residues were analyzed in the same manner with similar results. We were able to obtain TOF/ TOF fragmentation spectra when selecting the [M+H]+ ion corresponding with the phosphopeptides but not for the cationized precursor. Currently, conditions are being tested that increase the specificity of Fe(III) for phosphopeptides as opposed to other acidic and potentially chelating peptides.

Western blotting systems are most commonly used after separation of proteins on one-dimensional (1D) SDS-PAGE gels. The fluorescent-based ECL Plex Western Blotting Detection System (GE Healthcare, Piscataway, NJ) provides sensitivity, linearity, and a dynamic range of nearly four orders of magnitude, as well as the possibility of multiplex analysis-detection of two protein epitopes on the same membrane simultaneously. Multiplexing is commonly used for quantitation of one protein relative to a protein of known abundance (housekeeping protein). It can also be used for the detection and quantitation of post-translational modifications such as phosphorylation, provided that there are antibodies available. In this study, a two-dimensional Western blotting approach was evaluated in addition to the traditional 1D workflow, using fluorescent secondary antibodies, namely the ECL Plex Western Blotting Detection System. Human PC-3U cell lysate was separated by 1D and 2D SDS-PAGE, followed by blotting and incubation with primary antibodies against non-phosphorylated and Ser 9-phosphorylated forms of glycogen synthase kinase-3 beta (GSK3β). ECL Plex CyDye Cy3- and Cy5-conjugated secondary antibodies were used for simultaneous detection of the non-phosphorylated and phosphorylated forms of GSK3β. The 1D Western blotting experiment showed that both the non-phosphorylated and phosphorylated forms of GSK3β could be detected in PC-3U cells without transforming growth factor beta (TGF-β) activation. The 2D Western blotting experiment gave information on phosphorylation states that could not be obtained by 1D Western blotting. A 48-kDa band corresponding to phosphorylated GSK3β in the 1D experiments was resolved into at least five distinct protein isoforms, two of which were phosphorylated at serine 9.

The generation of peptide mass fingerprints followed by a database search is a common tool for the mass spectrometric identification of proteins. To provide a high sensitivity, proteins must be efficiently purified and concentrated prior to enzymatic digestion. Common desalting procedures, like ultrafiltration or dialysis, are very time consuming and work best for high protein concentrations. Another critical point is the proteolysis of the investigated protein, which works efficiently only with concentrated protein solutions. Therefore, efficient concentration and simultaneous purification using solid phase extraction (SPE) will be the method of choice to receive pure and highly concentrated protein solutions prior to enzymatic digestion. In this work, we manufactured magnetic reversed phase particles for the efficient purification and simultaneous concentration of protein samples with volumes up to several millilitres. The SPE procedure was compared with dialysis using commercial available microconcentrators with a cut-off membrane. Due to the magnetic core, each washing and elution step could be performed within 15 minutes. Then, the bound protein was digested directly on the beads, resulting in a remarkable increase of protein detection and better mass structural analysis. Useful MOWSE scores were achieved using bovine serum albumin as a model protein with concentrations as low as 50 ng/ml (720 pM). Compared to the dialysis procedure, which needs several hours, the isolation and purification of protein can be performed in minutes with the reversed phase particles.

Protein fractionation is often necessary in mass spectrometry (MS) proteomic studies to help detect low-abundance proteins. Protein Forest has developed the digital ProteomeChip (dPC) technology, which is a rapid, reproducible, and easy to use protein MS pre-fractionation tool. The dPC consists of discrete pH gel features with pH intervals of 0.1 or smaller, which, when placed in an electric field for 30 min, will each trap and collect proteins simultaneously from a complex mixture according to the proteins’ respective isoelectric points (pI). The resultant protein fractions can then be tryptically digested for subsequent MS identification analysis, transferred to traditional SDS-PAGE, or blotted to a membrane for antibody staining. The individual pH features allow researchers to reproducibly extract protein fractions from a complex mixture for detailed analysis, such as in biomarker discovery and clinical assay applications. We evaluated the isoelectric fractionation reproducibility of the dPC technology using I125-labeled human growth hormone (hGH) as radioactive tracers spiked into a model complex protein mixture (E. coli lysate). We collected focused I125-hGH predominantly in the pH 5.2 to 5.4 fractions, and the average radioactive counts recovered were very reproducible, with a coefficient of variance of 16%. We also demonstrated the utility of a dPC in fractionating E. coli lysates when coupled to subsequent LC/ESI-MS, SDS-PAGE, and immunoblotting analyses. The dPC is a novel fractionation technology that provides improved throughput and reproducibility compared to existing protein fractionation techniques. Our technology can be easily coupled to most existing downstream analysis techniques, allowing great research flexibility. Finally, the robustness of the chip format, along with the digital nature, also makes the dPC amenable to customization and automation.

With the advent of nucleic acid silencing technologies and the need for high purity diagnostic and therapeutic oligonucleotides, there is a need for high-capacity chromatographic supports that can deliver economic purification processes. A new, 30 micron, mono-sized, polymeric resin has been recently developed that provides high resolution and high capacity for synthetic oligonucleotides. Physical properties of this new resin will be described, including particle size uniformity, ion exchange capacity, pressure stability, and chemical stability. The purification of two synthetic 12mer DNA oligonucleotides (AAA CCT GAT GTG and GGT GGT GGT GGT) will be demonstrated, and will compare yields and purities to those from commercially available anion exchange resins.

Electrospray ionization (ESI) is a very gentle form of ionization, which enables the intact transfer into the gas phase and detection of large multi-protein structures with little or no fragmentation; when ESI is coupled to mass spectrometry, non-covalently assembled macromolecular protein complexes can be detected and accurately mass measured. The transfer of non-covalently associated protein-protein complexes from solution to the gas phase generally results in the formation of ions possessing relatively few charges; the m/z values are often above 10,000. In some of the data presented in this poster, MS/MS activation of such biomolecular complexes can produce ions with m/z values in excess of 20,000. By coupling an ion mobility separator (IMS) with a time-of-flight (ToF) mass spectrometer, one can not only accurately mass measure intact biomolecular complexes, but also one can measure their collisional cross-sections and differences in cross-section produced upon activation, and detect subtle conformation differences, which are not evident from spectral data alone. Here, we show the analysis of several different, non-covalently associated protein-protein complexes, which differ vastly in mass and collisional cross-section, by IMS-ToF-MS and IMS-ToF-MS/MS.

The conventional MS “bottom up” strategy for protein identification is the tandem MS/MS analysis of tryptic digests of complex proteins mixtures. Via enzymatic digestion, even larger proteins are cut into smaller peptides. In order to reduce the resulting complexity of the tryptic digest, additional chromatographic separation is used in front of the tandem MS/MS analysis. The bottom-up strategy, however, has shown its limitation when the characterization of multiply post-translational modified protein is of interest. The unique identification of several individual peptides from a tryptic digest does not provide the needed information if the interaction of the different modifications of the intact protein is of primary interest. Electron transfer dissociation (ETD) is particularly dedicated for the sequence analysis of larger peptides as well as for the identification of PTMs, as weakly bonded PTMs survive the prompt odd-electron-driven fragmentation of the electron transfer. However, if a multiply charged protein (z > 10) is analyzed via tandem ETD MS/MS, the resulting fragmentation spectrum shows a highly complex mixture of multiply charged fragment ions (zfragment ion = 1–9). We have modified the nCI source as well as the acquisition software of the HCTultra PTM Discovery System to allow the selective and consecutive ion accumulation of different types of reagent anion. For the initial ETD step, odd-electron reagent anions are selectively accumulated, and for the subsequent proton-transfer reaction, even-electron reagent anions revealing a strong proton affinity are accumulated. The second PTR step is necessary to significantly reduce the complexity of the above-mentioned ETD MS/MS-data, particularly when highly multiply charged proteins are investigated. The final charge stripping step produce a mixture of 1+, 2+, 3+, and 4+ fragment ions. With the present setup we were able to characterize the amino acid sequence of the intact proteins (e.g., ubiquitine, cytochrome C).

The increasing number of MS/MS spectra, generated by new mass spectrometers with fast scanning (LTQ, ThermoElectron, etc.), requires better and more robust spectra filtering. Retention time is one of the important peptide characteristics, but it is not used very often with the aim of peptide sequence validation. Here, we present an approach to filter Mascot (Matrixscience) search results on the basis of comparison of experimental retention times with predicted ones. Proteins were digested in-gel or in-solution with trypsin according to conventional protocols. Resulting peptide mixtures were analyzed on line with an LC-MS system (HP 1100 Nanoflow HPLC [Agilent] and LTQ Mass Spectrometer [ThermoElectron]). Peptides were separated on custom-packed 75-μm i.d., 10-cm-long PicoTip columns (New Objective) packed with 3.5-μm C18 beads (Pursuit, Varian). The column effluent was sprayed directly into the transfer tube of the mass spectrometer. The Sequence Specific Retention Calculator (http://hs2.proteome.ca/SSRCalc/SSRCalc.html) was used for calculation of peptide relative hydrophobicities. This program uses a refined model for determination of hydrophobicity, presented recently by Oleg Krokhin (University of Manitoba). Two columns are automatically added to the standard Mascot peptide summary page: scan number (reflecting retention time) and relative hydrophobicity. The plot of scan number vs. hydrophobicity is also created. The linear regression could be calculated with a subset of “good peptides” (having high Mascot score, e.g., >50). Based on this regression, predicted scan numbers could be identified. The rest of the peptides (with score <50) are tested, and sequences with scan numbers too far from predicted are rejected as false-positive hits. The algorithm was trained on standard digests such as BSA, and is now applicable to any LC-MS Mascot search results. Rejected hits were confirmed by manual inspection of MS/MS files. It appeared that this script is especially useful for validation of singly charged peptides, which often give fragmentation spectra of poor quality.

Cells are organized spatially and functionally into subcellular organelles, and a protein’s subcellular localization can provide a useful clue to its function. Additionally, changes in protein subcellular localization are involved in regulation of interactions, stability, and activity. For this reason, studying global changes in protein subcellular localization can provide useful insights into cellular functions. We had previously developed localization of organelle proteins by isotope tagging (LOPIT), a high-throughput technique for protein localization to subcellular organelles. Organelles are partially separated by density gradient centrifugation. Gradient fractions are labeled with isotopic iTRAQ tags for quantitation by mass spectrometry. Proteins from the same organelle co-sediment exhibit similar distributions in the density gradient. By comparing distributions of unknown proteins to those of known organelle markers, subcellular localization can be assigned. In this study, we extend the technique to look at protein localization dynamics in the Drosophila melanogaster embryo during Wnt/Wingless signaling. This signaling pathway is involved in proliferation and differentiation, and several components are known to alter their localization in response to activation. We have used LOPIT to simultaneously localize 851 Drosophila proteins to multiple organelles, including the plasma membrane, endoplasmic reticulum, and mitochondria. The localizations were reproducible in independent experiments. Known organelle markers localized as expected, and novel proteins with previously unknown localizations were assigned putative localizations. Repeating the analysis using embryos in which the Wingless pathway has been hyper-activated, we have identified proteins that move in response to Wingless signaling, and are in the process of carrying out functional assays to dissect their role in the pathway.

Proteomics requires large amounts of highly pure and correctly folded protein. This need is often met using heterologous expression systems such as Escherichia coli. Over-expression of foreign proteins in E. coli commonly results in the formation of inclusion bodies—dense, insoluble aggregates of misfolded protein. Though commonly viewed as detrimental, inclusion bodies are easily purified, resistant to proteolysis, and can be solubilized with chaotropic agents. Defining conditions that promote refolding of a chemically solubilized target protein into its native conformation, however, is largely empirical. The chances of identifying an optimal refolding condition can be increased by simultaneously and systematically evaluating a large number of refolding conditions. To meet this need, we have developed the iFOLD Protein Refolding systems, a collection of 96-well plate-based refolding screens. System 1 uses N-lauroylsarcosine, a chaotropic anionic detergent, to denature the inclusion bodies, while System 2 uses either guanidine hydrochloride or urea as the denaturant. With both systems, proteins are refolded by rapid dilution of the denatured inclusion bodies into the 96-well refolding plate. The plate contains 92 (System 1) or 95 (System 2) unique protein refolding solutions, each comprised of buffers, salts, redox couples, and protein refolding additives. Using the systems, we have successfully identified high-yielding refolding conditions for a green fluorescent fusion protein, a mammalian endopeptidase, a matrix metalloprotease, and a viral protease. Refolding was measured using spectrophotometric, fluorescent, and target-specific activity assays. High-yielding refolding conditions identified by the screens have been scaled 10,000-fold from 50 μg to 500 mg. Significantly, all steps of the iFOLD systems are equally compatible with manual use and high-throughput automated liquid-handling systems.

Peroxisome proliferator-activated receptor (PPAR)γ ligands decrease early atherosclerosis lesions in experimental animal models, but their effects on advanced atherosclerosis are not known. A unique process in advanced atherosclerosis is the accumulation of free cholesterol (FC) by macrophages (Mfs), which, according to previous in-vivo studies, can result in Mf death, lesional necrosis, inflammation, and plaque susceptibility to rupture. Mf apoptosis occurs through all stages of atherosclerosis, and inefficient phagocytic clearance of dying Mfs may lead to cellular necrosis and release of plaque-destabilizing factors. Here we sought to determine how PPARy agonists would affect phagocytosis of FC-induced apoptotic cells. We found that with the use of these PPARγ agonists, phagocytosis of these apoptotic cells increased significantly. This enhanced phagocytic uptake was associated with increased accumulation of filamentous actin at the interface between phagocyte and apoptotic cell, suggesting an effect of PPARy ligands on intracellular actin signaling. Indeed, macrophages treated with the PPARy ligand rosiglitazone had decreased levels of the actin-modulating protein RhoA. Thus, compounds designed to activate PPARy, inducing those in clinical use such as rosiglitazone and pioglitazone may reduce atherosclerotic maturation by enhancing phagocytic clearance of dying macrophages. Future derivations of PPARγligands may be selected to further exploit this phagocytic property.

The development of new methodology for the direct analysis of protein-protein interactions is of high interest, as protein complexes are involved in all cellular processes. If mass spectrometry is routinely used for protein analysis, it is still challenging to use this analytical tool for the analysis of protein-protein interactions. Most of the succesful studies in this field have been done using electrospray ionization, requiring time-consuming optimizations both on sample preparation and instrumental settings. MALDI mass spectrometry is a faster method for the analysis of proteins but is rarely used for the direct analysis of protein-protein interactions. The principal reason is the high tendency of non-covalent interactions to dissociate during the ionization step. Another issue is the detection of the intact complexes as single-charge pseudomolecular ions. Here, we present the analysis of intact protein complexes by MALDI mass spectrometry using the combination of chemical cross-linking and high-mass MALDI mass spectrometry. To circumvent the dissociation problem, a specific chemical cross-linking reaction is performed prior to the MS analysis, maintaining the integrity of the intact complex. To allow the detection of the complex, a new high-mass detection system is used, allowing sensitive analysis (nM range) in the 10–1200 kDa range. Different applications will be presented: Immunochemistry: Direct analysis of immuno-complexes between prion/antiprion; HAtag/antiHA; GSTtag/antiGST; BSA/AntiBSA in the 150–400 kDa range; analysis of epitope by competition assays, sandwich assays. Protein pathways: Direct analysis of complex stoichiometry for the complexes: AMPk, CDC42-SopE, thymidine K, ATP synthase, ferritin complex. Supershifting: The supershifting application consists in determining the presence of a subunit in a complex by MALDI high-mass mass spectrometry using monoclonal antibodies. An example of a supershifting experiment will be presented with the complexes formed by the fusion proteins GST-SopE and GST-CDC42 using a monoclonal antibody anti-GST.

Imaging the spatial distribution of molecules in tissue using MALDI mass spectrometers is a rapidly developing technique. The acquisition of accurate mass data in this type of experiment can be hampered in axial MALDI Tof systems. Even small changes in sample position and laser energy in the source region of this type of mass spectrometer affect mass measurement accuracy and mass spectral resolution. Here, we show how the use of an orthogonal Tof MALDI mass spectrometer circumvents these problems by decoupling the MALDI source from the mass analyser. Imaging data were acquired on a MALDI Q-Tof mass spectrometer. The tissue sections were mounted on a target plate and moved in a raster pattern relative to the laser. To reduce interference from the biological matrix and enhance specificity the instrument was operated in MS/MS mode, a quadrupole was used for precise precursor ion selection. The sensitivity of specific ions was further enhanced by synchronising the high voltage push of the Tof mass analyser with the arrival of ions of appropriate m/z in the acceleration region. MALDI imaging information has been obtained from thin sections of rat tissue from animals doped with drugs, e.g., the well studied D2/D3 dopamine receptor antagonist Raclopride as well as from untreated animals. Data obtained on the spatial distribution of drugs/drug metabolites and endogenous species will be presented. Challenges and future directions with MALDI imaging sample preparation are discussed.

Proteomic workflow frequently involves gel separations. Poor peptide recovery from in-gel digestion procedures is at large influenced by the properties of polyacrylamide gel, which restricts access of proteolytic enzymes to the protein substrate as well as prevents complete extraction of certain peptides. We have designed an optimized centrifugal device which allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, roughly 100 μm, prior to the enzymatic digestion. Such treatment increases the surface area of gel pieces and allows the enzymes and solvent to better penetrate the gel lattice. The performance of such novel device has been evaluated using several gel-separated model proteins pre-labeled with thiol-specific multiplexed stable isotope tags, followed by relative quantification by a MALDI-TOF/TOF mass spectrometer. The performance of the new device, called the Gel Shredder, has been compared to standard in-gel digestion protocols. It has been shown that an improvement in peptide recovery can be reproducibly obtained by using the Gel Shredder devices. It has been determined that the application of the Gel Shredder is particularly useful for recoveries of high molecular weight hydrophobic peptides, presumably due to their higher affinity to the polyacrylamide gel lattice. Gel Shredder also allows to save time usually spent on gel band manipulation during in-gel digestion experiments.

DNA microarrays allow many simultaneous parallel measurements; and transcriptional profiling has provided scientists with a wealth of information. The same technology can be used to build protein arrays which hold similar promise. We describe here an approach using protein arrays to screen cells for gene knockdowns. Small interfering RNAs (siRNA) can be used to degrade mRNA levels of a specific gene, thereby reducing the corresponding protein levels within cells. The use of siRNAs has many implications in functional genomics and proteomics as well as therapeutics, as researchers use this tool to identify proteins involved in specific pathways. We demonstrate the use of microarrays as a screening method to identify cells that have been treated successfully with a beta-actin siRNA. We have silenced the β-actin gene in HeLa cells using siLentMer Dicer-Substrate siRNA Duplexes, and confirmed the knockdown with a combination of 2-D gel analysis, western blotting and quantitative PCR (qPCR). An increase in the level of phosphorylated cofilin was also detected (Liu, N., et al. 2006 in press). We now demonstrate the use of reverse phase protein microarrays to screen for these proteins. These arrays enable higher throughput than traditional protein detection, and add multiplexing capabilities. Arrays were produced on the benchtop with the BioOdyssey Calligrapher miniarrayer. Antibodies against β-actin and phosphocofilin were tested for specificity by western blots. In one experiment, arrays were processed to monitor the concentration of β-actin in cells, with a standard curve of purified human actin printed on the grids. In another experiment, changes in phosphorylation levels of cofilin with an antibody specific for phosphocofilin were detected. We also demonstrate that these printed arrays can be screened using antibodies either singly or in pairs. Finally, the microarray results were validated using qPCR.

Due to their low abundance and limited number of trypsin cleavage sites, integral membrane proteins are notoriously difficult to identify and to analyze by mass spectrometry. Our approach to this problem is based on a novel technology for peptide sample preparation that uses plates with pre-deposited matrix spots of sub-micron size CHCA crystals prepared by vacuum sublimation onto an ultraphobic surface (Mass·Spec·Turbo Chips, QIAGEN). Such a design enables highest sensitivity analysis and reproducible and automatable sample preparation, suitable for both gel- and LC-based MALDI-MS approaches. In order to compare the chips’ performance with that of standard stainless steel target plates, different sample amounts of two model membrane proteins—bacteriorhodopsin and subunit A of ATP synthase—were separated by SDS-PAGE and in-gel digested with trypsin. After digestion, aliquots were spotted either as classical dried droplet preparations on stainless steel plates using CHCA as matrix, or applied directly onto the matrix spots pre-deposited on the chip surfaces. Spectra were acquired on Voyager DE-Pro or 4800 MALDI-TOF instruments (Applied Biosystems) and protein identification was performed by peptide mass fingerprint or MS/MS ion search using the Mascot algorithm (Matrix Science). The results were evaluated by comparing S/N ratios, sequence coverage, Mascot scores and the total ratio of protein identifications vs. unsuccessful attempts. Analyses of the spectra obtained revealed a significantly larger number of peptide signals using the chip approach, which resulted in increased sequence coverage and more reliable protein identifications.

Quantitative real-time reverse-transcription PCR (RT-qPCR) is presently the method of choice for validating gene expression results from high-density microarrays. However, the low throughput of traditional gene-by-gene RT-qPCR makes this process labor intensive and time consuming. To accelerate this laborious task, the SuperArray RT²Profiler PCR Array combines SYBR Green–based real-time RT-qPCR technology with a multi-gene array plate format to simultaneously analyze a panel of genes related to a specific disease or biological pathway. Each assay on the PCR array plate has been experimentally validated to insure gene-specific amplification. The reliability and reproducibility of the RT²Profiler PCR Array have been demonstrated by DNA sequencing and intra/inter-laboratory reproducibility comparisons. The DNA sequencing demonstrated 100% of the PCR products amplified from the correct target genes. In one laboratory, individual PCR assays produced a standard deviation of 0.24 cycles and a coefficient of variance of 0.92% in technical replicates. The correlation coefficient for Ct values between replicate runs was 0.997 and for fold changes (ΔΔCt) across thermocyclers was 0.976. Comparisons between two different laboratories using different thermocyclers showed correlation coefficients of 0.972 and 0.976 for ΔCt and ΔΔCt, respectively. Each PCR array also includes stringent controls to monitor RNA quality by assessing reverse transcription efficiency and genomic DNA contamination to ensure the reliability of the PCR array data. A practical application for the PCR array was demonstrated by identifying human pancreatic tumor–associated genes using the Cancer PathwayFinder RT²Profiler PCR Array. Results showed 23 genes exhibiting a statistically significant threefold or greater change in expression between a human pancreatic tumor and normal pancreas, including many genes previously linked to pancreatic cancer. Hence, the RT2Profiler PCR Array system offers a simple, reliable, and convenient tool for multi-gene profiling and microarray data validation.

We analyzed mRNAs regulated during differentiation of rat neural stem cells using the ABI1700 microarray platform. This microarray, while technically advanced, suffers from the difficulty of integrating hybridization results into public databases for systems-level analysis. This is particularly true for the rat array, since many of the probes were designed for transcripts based on predicted human and mouse homologs. Using several strategies, we increased the public annotation of the 27,531 probes from 43% to over 65%. To increase the dynamic range of annotation, probes were mapped to numerous public keys from several data sources. Consensus annotation from multiple sources was determined for well-scoring alignments, and a confidence-based ranking system established for probes with less agreement across multiple data sources. Previous attempts at genomic interpretation using the Celera annotation model resulted in poor overlap with expected genomic sequences. Since the public keys are more precisely mapped to the genome, we could now analyze the relationships between predicted transcription factor binding sites and expression clusters. Results collected from a differentiation time course of two neural stem cell clones were clustered using a model-based algorithm. Transcription factor binding sites were predicted from upstream regions of mapped transcripts using position-weight matrices from either JAS-PAR or TRANSFAC, and the resulting scores were used to discriminate between observed expression clusters. A classification and regression tree analysis was conducted using cluster numbers as gene identifiers and TFBS scores as predictors, pruning back to obtain a tree with the lowest gene class prediction error rate. Results identify several transcription factors, the presence or absence of which are sufficient to differentiate clusters of mRNAs changing over time from those that are static, as well as clusters describing cell-line differences. Public annotation of the ABI1700 rat genome array will be valuable for integrating results into future systems-level analyses.

No abstract available.

MicroRNAs (miRNAs) are short 22–24 nucleotide RNAs that play a vital roles in regulating gene expression. These single stranded RNA molecules are incorporated into the RISC (RNA-inducing silencing complex) and regulate gene expression through various methods including translational inhibition, transcriptional cleavage and transcriptional gene silencing. Recent interest in these non-coding RNAs has been explosive because of its implication in cellular differentiation and disease genesis. Therefore, there is an urgent need to identify and validate miRNA expression in cell lines, tissues, and organs. qRT-PCR has become the standard for microarray data validation, as well as an invaluable tool for quantifying individual or subsets of miRNAs with the greatest sensitivity and accuracy. Most commercially available miRNA qRT-PCR systems employ proprietary, pre-designed miRNA-specific primers for cDNA synthesis by reverse transcription. Unfortunately, this approach requires that the sequence of the miRNA is publicly available and a commercial qRT-PCR assay has been developed for that specific sequence, limiting the availability of qRT-PCR assays for many model organisms as well as recently discovered miRNAs or proprietary miRNAs. Here, we describe a novel, universal, and user-friendly qRT-PCR method, that was developed to measure and characterize miRNA expression in almost all organisms, starting from either total RNA and/or enriched miRNA. The NCode miRNA SYBR Green qRT-PCR protocol is based on carefully optimized polyadenylation reaction with the reverse transcriptase, SuperScript III RT, in a “universal” 1st strand cDNA synthesis reaction.. The miRNA specific amplification occurs during the PCR reaction where the sequence of the miRNA of interest is used as the target-specific PCR primer. Platinum SYBR qPCR Supermix combines Platinum Taq DNA polymerase with SYBR Green fluorescent dye, delivers excellent sensitivity in the quantification of target sequences. This protocol offers 7 logs of dynamic range for maximum sensitivity and single nucleotide discrimination for distinguishing between closely related miRNA families.

Living organisms translate genetic information in many ways, occasionally altering transcription signals and changing reading frames. A consequence of these post-transcriptional actions (recoding) is that a single coding sequence can produce multiple gene products, or a single product from two overlapping open reading frames (ORFs). Although examples of recoding have been uncovered in all well-studied organisms from viruses to bacteria to humans, we know little about the global contribution recoding makes to proteomic complexity. Proteomic methods tailored to expose recoding events will have enormous impact. In examining the proteome of Syntrophus aciditrophicus, a gram-negative bacterium that grows syntrophically (cooperatively) with methanogens or sulfate reducers, we found several examples of translational frameshifting, apparently arising from particularly “slippery” mRNA sequences (XXXY YYZ). De novo sequencing of LC-MS/MS spectra obtained from trypsin-digested 2D gel spots reveals multiple peptide sequences arising from a single stretch of mRNA sequence, providing a rare opportunity to view proteins generated in vivo by recoding. Linking sequenced peptides to 2D gel spots presents the extraordinary opportunity to monitor ratios of recoding products with respect to changes in culture conditions and even whether S. aciditrophicus is cultivated syntrophically or in isolation. Our studies revealed that predicted 4- and 16-kDa ORFs actually correspond to a 30-kDa product with at least two frameshifts (peptides identified in all 3 reading frames). More than ten tryptic peptides are observed that do not correspond to the primary sequence in any single reading frame. Instead, they define the actual recoding site. These data extend the questions about small ORFs beyond “real or unreal?” to “small or enlarged with frame-shift?” They also promise to elucidate factors regulating frameshifts, translational bypassing, and readthrough. Finally, they raise important questions about present capabilities to predict and quantify gene products, and future capabilities needed to address these issues in high-throughput.

Proteomic analyses typically produce massive amounts of mass spectrometric data, which are analyzed in an automated way by database search engines for retrieval of peptide sequences and subsequent inference on the corresponding protein sequences. However, this process turned out to be error prone, producing false positives and multiple hits for the same proteins for various reasons. In this study we analyzed the human serum glycosubproteome. For this, glycoproteins were produced in five separate extractions by affinity interaction chromatography using magnetic beads coated with the lectins ConA, WGA, LCA, and AIA, as well as with boronic acid. The eluates from the beads were digested using trypsin and subsequently analyzed by LC-MALDI-MS/MS as well as LC-ESI-MS/MS. The analyses were carried out as up to triple replicates. All the data were submitted to the ProteinScape database system. Within ProteinScape, the datasets were searched against Mascot and Phenyx. The resulting peptide identifications were analyzed by the ProteinExtractor tool to reduce the list by the false positives using a decoy strategy, and subsequently merged into a combined list of identified proteins for the respective sample preparations. This allows the combination of data from different search engines as well as from different experiments (ESI and MALDI). The use of decoy strategies as well as application of the ProteinExtractor to overcome the protein inference problem minimizes the need for manual validation (which is nevertheless easily possible using raw spectra information). In parallel, the use of a single data repository allows for easy access to the combined information from different work-flows and links to external tools complement the system for project-spanning comparisons of datasets.

The measurement of gene expression from formalin-fixed paraffin-embedded (FFPE) tissue has proven to be problematic. Consequently, archives of FFPE samples remain unexploited in the quest to elucidate and validate the molecular mechanisms of diseases, cellular processes, and drug activity/safety. We validated the measurement of gene expression from FFPE tissue by a new multiplexed assay, the quantitative nuclease protection assay (qNPA). qNPA measures the RNA cross-linked to tissue without its having to be solubilized, plus the soluble RNA pool—i.e., the total RNA contained in the FFPE sample. This is likely one explanation of qNPA success where methods that measure only soluble RNA have failed. Cross-linked RNA is the major pool in FPE samples, and the fraction it makes up varies from sample to sample, presumably due to differences in fixation time or sample age. Consistent with the observation that qNPA measures the total RNA in fixed tissue, and the fact that it measures the total RNA in fresh samples, is the result that identical quantitative levels of gene expression are measured from fresh fixed and 18-y-old FFPE tissue and from matched fresh or fixed tissue. The expression level for a set of low to moderately expressed genes from fresh vs. FFPE tissue correlated with an R2 = 0.99, slope = 1. Gene expression measurements in FFPE tissue provided average CVs <10%. A retrospective study using clinical diffuse large B-cell lymphoma samples was carried out, validating prognostic biomarkers of disease, disease subtype, and survival. The levels of gene expression measured by qNPA correlated with protein product levels measured by IHC. These results validate that qNPA provides a high-quality gene expression assay of FFPE tissue, enabling research and clinical assays not previously possible.

In order to characterize in vivo interactions in Drosophila melanogaster, and further our understanding of developmental processes, hybrid PiggyBac/P-element YFP traps with Strep Tag affinity tags were generated to isolate multi-protein complexes. The incorporated Strep affinity tag allows the target protein to be isolated from its native environment, by tandem affinity purification, along with any associating proteins. Purification efficiency can be followed visually by the YFP marker. Complex proteins are then identified by tandem mass-spectrometry against the Drosophila melanogaster database using the Mascot search engine. Due to the vast number of putative proteins, a high throughput purification and identification system has to be adopted to maximize coverage. Protein extraction techniques have been optimized to include isolation of tagged membrane proteins whilst maintaining their native structure and complexes. Strep-Tactin columns have proven to be inefficient at eluting high enough yields of Strep-tagged purified protein thus making the purification time consuming and tedious. However, modifying elution protocols has enabled us to load eluates directly onto the Mass Spec and thus identify all proteins in a single run. The addition of a FLAG tag will also greatly improve the purification efficiency. Vectors have been designed and new fly lines are underway. Mass Spec results were confirmed by visualizing the corresponding sized proteins on SDS-PAGE gels and western blotting using a GFP antibody to identify the tagged bait. Data from this analysis will be uploaded onto a worldwide accessible database and supersede data generated from the yeast two-hybrid interaction screens.

No abstract available.

The Fms-interacting protein (FMIP, MW 78 kDa) acts as a shuttling protein during macrophage differentiation in the human immune system.1 Previous work on an enzymatic digest of this protein has revealed a peptide with a precursor mass corresponding to the FMIP peptide YTCQELQR with the addition of 80Da. This mass difference may be attributable to either phosphorylation or sulfonation. The type of post-translational modification (PTM) could not be determined by detailed analysis of the resulting MS/MS spectrum obtained under typical collision-induced dissociation conditions. In order to distinguish the PTM, we analyzed the sample on a Thermo Fisher LTQ-FT Ultra mass spectrometer making use of its very high and reliable mass accuracy. In addition, electron capture dissociation (ECD) was performed on the peptide in an attempt to assign unambiguously the site of modification. The sample was separated using a nanoLC setup (ThermoFisher Scientific Micro Autosampler and Surveyor MS Plus pump) equipped with a C18 trapping column and a C18 100 × 0.075 mm analytical column (both: www.nanoseparations.com). Standard HPLC solvents (MS grade) were used for the analysis: water/acetonitrile (98:2 v/v, 0.1% formic acid) as solvent A and acetonitrile/water (80:20 v/v, 0.1% formic acid) as solvent B. The applied method used a FTMS full scan and three data-dependent ECD scans per cycle, picking the top three signals from the full scan and subsequently excluding these for 20 sec. The analysis clearly reveals the type of modification as well as the site. The poster will present the data as well as their interpretation.

MALDI-TOF MS is used successfully in investigating in vitro glycation of normal and variant hemoglobins (Hbs). Singly glycated, doubly glycated, and/or multiply glycated glycoisoforms of the alpha-globin, beta-globin, and gamma-globin of Hbs are observed. Different glycation rates are observed for normal and variant Hbs, with the normal Hb A having the slowest rate. The normal Hb A is more stable than the Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego upon condensation with glucose at 37°C. Data reveal that with longer incubation time (up to 5 d), higher glucose concentration (up to 1 M), and higher temperature (up to 37°C), the number of glycated amino acid residues of Hbs increase. The extent of the glycation of both Hb A and Hb F increases upon changing the solvent from PBS (pH 7.4) to carbonate buffer (pH 10). However, this pH change has a lesser effect on the glycation of the Hb C, Hb E, or Hb Leiden. In this study, higher concentration of the glucose is used to increase the reaction rate of the less reactive residues, and higher pH is used to alter the structures of proteins and consequently expose the reactive amino acid residues. However, incubation time is kept relatively short. Results obtained in this study could lead to the inference that effects of the incubation time, glucose concentration, temperature, and pH on the linkage of Hbs with glucose would be similar under the diabetes conditions in vivo (~37°C, ~neutral pH, ~0.007 M glucose concentration). This conclusion agrees with our previous report on glycation of all the peptides/proteins that we have studied with glucose.

The Mouse Genome Informatics (MGI; http://www.informatics.jax.org/) database integrates genetic and genomic data with the primary mission of facilitating the use of the mouse as a model system for understanding human biology and disease processes. MGI is the authoritative source of official mouse genetic nomenclature, gene ontology annotations, mammalian phenotype annotations, and mouse anatomy terms. MGI staff enforce the use of standardized genetic nomenclature, ontologies, and controlled vocabularies to describe mouse sequence data, genes, strains, expression data, alleles, and phenotypes. Extensive links between gene-centric information in MGI and other informatics resources (e.g., OMIM, Ensembl, UCSC, NCBI, UniProt) are maintained and updated on a regular basis. Using the Web-based query interfaces for MGI, users can query for a mouse gene or genes according to diverse biological attributes of those genes, including phenotype associations, gene expression, functional annotation, and genome location. The MGI MouseBLAST server allows users to interrogate the MGI database using nucleotide and/or protein sequences. Functional and phenotypic data from MGI can be viewed in a broader genomic context using an interactive genome browser called Mouse GBrowse. The power of the MGI database as a research tool for biomedicine stems from the degree to which data from diverse sources are integrated. Integration, in turn, allows the data to be evaluated in new contexts. For example, integration makes possible such complex queries as “Find all genes from Chromosome 1 where the function is annotated as transcription factor and there is a knockout allele that results in eye dysmorphology.”

Introduction: Protein phosphorylation is one of the most important post-translational modifications that regulate the function, localization, and binding specificity of target proteins. Specifically, protein phosphorylation works as a switch to turn “on” and “off” protein activity or a cellular pathway. Protein phosphorylation is also known to be both spatial and dynamic in its distribution. Therefore, a work-flow that provides the detailed information of phosphorylation site location, site occupancy under specific conditions, etc., is necessary. Current strategies such as ELISA, radio isotope workflow, and Western blotting lack the ability to ascertain independent information of each phosphorylation site. Our mass spectrometry–based method using a chemical labeling strategy provides dynamic information about phosphorylation on each phosphorylation site. Method: A model system using JNK1 kinase and the substrate protein myelin basic protein (MBP) was selected. JNK1 phosphorylates serine and threonine residues just before the proline residue, typical of this MAP kinase family. The MBP substrate was incubated with JNK1 in vitro, and samples were removed at four different time points (0, 5, 30, and 60 min). These samples were digested and labeled with iTRAQ reagent in accordance with the standard procedure. A nano-LC-MS/MS experiment was performed using the 4000 Q TRAP System, and the data were analyzed by ProteinPilot Software. Results: All of the peptides possessing a possible phosphorylation site motif in MBP were detected. For each identified peptide, the iTRAQ reagent reporter ions clearly showed the change in abundance of specific MBP peptides as they become phosphorylated by JNK1 over the time course of the assay. These results indicate that this workflow is a robust strategy for routinely characterizing the variation of phosphorylation through the interaction between protein substrate and kinase.

In today’s proteomics research, various techniques and instrumentation bioinformatics tools are necessary to manage the large amount of heterogeneous data with an automatic quality control to produce reliable and comparable results. Therefore a data-processing pipeline is mandatory for data validation and comparison in a data-warehousing system. The proteome bioinformatics platform ProteinScape has been proven to cover these needs. The reprocessing of HUPO BPP participants’ MS data was done within ProteinScape. The reprocessed information was transferred into the global data repository PRIDE. ProteinScape as a data-warehousing system covers two main aspects: archiving relevant data of the proteomics workflow and information extraction functionality (protein identification, quantification and generation of biological knowledge). As a strategy for automatic data validation, different protein search engines are integrated. Result analysis is performed using a decoy database search strategy, which allows the measurement of the false-positive identification rate. Peptide identifications across different workflows, different MS techniques, and different search engines are merged to obtain a quality-controlled protein list. The proteomics identifications database (PRIDE), as a public data repository, is an archiving system where data are finally stored and no longer changed by further processing steps. Data submission to PRIDE is open to proteomics laboratories generating protein and peptide identifications. An export tool has been developed for transferring all relevant HUPO BPP data from ProteinScape into PRIDE using the PRIDE.xml format. The EU-funded ProDac project will coordinate the development of software tools covering international standards for the representation of proteomics data. The implementation of data submission pipelines and systematic data collection in public standards–compliant repositories will cover all aspects, from the generation of MS data in each laboratory to the conversion of all the annotating information and identifications to a standardized format. Such datasets can be used in the course of publishing in scientific journals.

Peptide arrays are useful tools to characterize antibodies, enzyme substrates or sequence specificities of interaction partners with given peptide sequences (e.g., SH2, SH3, MH2 and other domains). Here we present a new method1 that allows production of hundreds of identical peptide arrays from a single synthesis run on modified, individual cellulose-disks. The disks are dissolved in the acid cleavage-mixture after synthesis and the resulting solutions of peptide-cellulose-conjugates are then spotted onto multiple slides by conventional spotting techniques. As application example we show results obtained with arrays of kinase substrate libraries and various consensus sequences of known kinase targets. These arrays can be used with different detection methods to profile known and unknown kinases for their substrate specificity. The new arrays are derived from the the well known SPOT method2 but offer several major improvements: A smaller volume of sample (only 100 μL) is needed for incubation, and a high number of identical copies of the arrays enables large scale, parallel screening experiments. The cost of an individual array is considerably lower than that of a SPOT membrane. Unlike DNA hybridization, protein-protein interactions frequently suffer from low binding affinities. The new cellulose substrate with peptides linked to it generates a three dimensional scaffold on the array support with a peptide loading exceeding that of a monolayer by a factor of 100. The high peptide density of the spots should be advantageous to identify protein-interaction sites, even if their binding constants are low. Figure 1

2D-PAGE experiments can provide a powerful means of investigating protein expression behavior in a cell or tissue across different disease states or other experimental conditions. Traditional analysis of 2D experiments typically requires a large amount of post-detection editing in order to prepare the data for statistical research. With the application of SameSpots, all protein spots are fully matched and thus no missing values exist. This enables a more robust statistical exploration of the data with a reduction in subjective editing. The SameSpots workflow has a semi-automatic gel alignment step and uses the arcsinh transform introduced by Huber et al.1 for inter-gel calibration and variance stabilization (VSN). To validate these techniques we repeated the work of Nishihara and Champion2 and Karp and Lilley.3 The results of this study demonstrate that gel alignment has no adverse affect on spot volume quantitation, and we also show that VSN outperforms traditional normalization and variance stabilizing methods used in 2D gel analysis.

Protein phosphorylation is crucial to the global regulation of the intricate biochemical networks of cell signaling pathways, allowing protein functional activity, stability, association into multi-subunit complexes, and subcellular localization to be integrated and tuned for generation of highly specific biological responses crucial to fundamental cellular functions. An antibody-free process workflow is described involving orthogonal phosphomonoester selective binding strategies. First, complex protein samples, such as rat liver cytosol proteins, are separated by conventional gel electrophoresis and a fluorophore-conjugated alkoxide-bridged dinuclear zinc complex is employed to selectively highlight phosphoproteins via binding to the phosphomonoester dianion moieties of serine, threonine, and tyrosine residues at neutral pH. Interaction with other anionic residues, including carboxylate residues on proteins, is insignificant. As little as 1 ng of phosphoprotein is detectable by this method using standard charge-coupled device camera- or laser-scanner-based imaging systems. Then, phosphoprotein bands are excised and subjected to proteolytic digestion. Constituent phosphopeptides are subsequently purified using titanium dioxide thin-film-coated magnetic beads at acidic pH. Phosphopeptides are eluted at alkaline pH and directly characterized by MALDI-TOF or tandem mass spectrometry, without chemical modification by methyl esterification. Phosphopeptides can readily be identified from as little as 78 fmol of starting material, with minimal contamination of samples by acidic peptides, as is often encountered using conventional trivalent ferric- or gallium-based metal ion affinity approaches. The high affinity and capacity titanium dioxide-based purification method is also suitable for the direct enrichment of phosphopeptides from human serum, which could lead to new approaches for biomarker discovery from biological fluids.

Reversible phosphorylation of proteins plays an important role in many cellular processes. To investigate the functions of kinases and their substrates in vivo and in vitro—for example, production of phospho-specific antibodies or generation of phospho-site mutants—the precise identification of phosphorylation sites is mandatory. A rapid and efficient workflow has been elaborated using prespotted 384 alpha-cyano-4-hydroxycinnamic acid MALDI targets as fractionation units; these are ideally suited to visualize 32P-labeled phosphopeptides. Ingel-tryptic digests of phosphorylated AMP-activated protein kinase (either by protein kinase B or autophosphorylation in the presence of γ32P-ATP) were separated on an Agilent 1100 capillary LC system coupled to a microfractionation unit. The tryptic peptides were deposited in 1-μL portions onto a PAC-MALDI target with 384 prespotted matrix preparations as well as calibrant spots. In order to visualize 32P-labeled peptides, the PAC plate was then exposed to a Kodak MR autoradiography film. Accordingly, the 32P-positive spots were screened for phosphorylation sites by performing MS and MSMS with the Ultraflex II TOF/ TOF. This new workflow for phosphorylation site identification eliminates tedious manual handling of digested samples, thus reducing the initial sample material dramatically. Furthermore, we observe that the separated peptides can be stored on the PAC plate for several weeks without any significant loss of resolution and signal intensity.

We have developed a sandwich immunoassay method for quantitatively measuring site-specific phosphorylation of multiple proteins and at multiple sites within a protein simultaneously, without the need for multiple, phospho-site-specific detection antibodies. This approach utilizes antibodies to capture a unique peptide sequence (EpiTag) proximal to the phosphorylation site of interest. The captured peptides containing the phosphorylation site(s) of interest are then interrogated with a commercially available, pan-anti-phospho antibody to determine whether the site is indeed phosphorylated. Unlike traditional immunoassays, select epitopes are made available to the antibodies by a unique sample-processing procedure. The procedure involves proteolytic fragmentation of proteins in order to segregate multiple phosphorylation sites within a given protein. Since proteolytic cleavage is predictable, quantitation is achieved by interpolating protein concentrations from standard curves generated with synthetic phosphopeptide standards. This approach obviates the need for phosphoproteins, which can be difficult to generate and qualify. The sandwich immunoassays demonstrate titration over a large dynamic range (≥3 logs) and achieve sensitivity at or below picomolar concentrations of standard. In contrast, no sandwich formation is observed with standards that have been dephosphorylated with lambda phosphatase. While the approach is limited to measuring previously discovered phosphorylation sites and does not distinguish between multiple sites that lie within the same proteolytic fragment, this proprietary approach is directly applicable to both bead-based and planar arrays, and has been multiplexed and used to measure >30 phosphotyrosine sites on various targets across multiple signaling pathways. In addition, the approach is currently being evaluated for multiplexed measurements of other phosphoresidues, and we believe this technology can be applied specifically, to study and quantify particular signaling events, as well as on a more global level, to identify and “fingerprint” activated signaling pathways during different disease states or in response to therapeutic strategies.

It now appears that many of the health effects of exercise are influenced by the balance of stress mediators and growth factors. Related to these agents are the soluble adhesion molecules (sAM: ICAM and VCAM) and soluble selectins (sS: E, L, and P selectins) which have been linked to such illnesses as asthma and cardiovascular disease. We hypothesized that brief exercise would alter circulating levels of sAM and sS, and that the response would be modified by the child’s pubertal status. Thirty healthy males (14 early pubertal, EP; 16 late pubertal, LP) performed 10 2-min bouts of exercise on a cycle ergometer. Blood was sampled at pre-exercise (PE) and end-exercise (EE). Levels of sAM and sS were analyzed using commercially available ELISAs. Mean PE levels were significantly different between groups for ICAM (p < 0.005) and VCAM (p < 0.05), and at EE for ICAM (p < 0.005). VCAM significantly increased in response to exercise in LP males (p < 0.05). A significant change between groups was demonstrated for the selectins at PE (E and P p < 0.005; L p < 0.05) and EE (all sS p < 0.05). Only LP males demonstrated a significant change from PE to EE (all sS p < 0.05). Baseline levels of circulating vascular adhesion molecules are significantly different between healthy EP and LP males. Moreover, exercise in LP males caused a significant increase in these vascular mediators, possibly preparing the organism to adapt to stressors imposed by exercise and the vascular demands needed for muscle growth. NIH- P01HD048721, RO1HL080947, K23 ES014923-02.

Detergents are commonly used in hydrophobic cell membrane protein purification and viral removal/inactivation. These detergents must eventually be removed during the purification process. A rapid detergent removal protocol using short chromatographic columns has been developed. Polystyrene divinylbenzene polymers, packed in chromatography columns, were employed for the removal of non-ionic, zwitterionic, and anionic detergents from protein solutions. The results demonstrate that this resin exhibited high capacity and was effective in removing a wide range of detergents at high linear velocities (>600cm/hr) with minimal loss in protein yield.

Already before the occurrence of pathological symptoms in the development of severe diseases changes in the proteome pattern in the plasma of patients can be observed. Therefore, to follow these pathological changes, plasma samples from several time points should be analysed before clinical symptoms arise. Future patients can be statistically expected within the pool of periodical blood donors. From the biobank (blood donor centre of the Bavarian red cross) colon cancer patients can be derived out of a pool of nearly 300,000 donors every year. Within this project (20 individuals) plasma samples of five different time points, collected each from the same individual were depleted from 12 high abundant proteins, labelled with ICPL and separated by 2D-gelelektrophoresis. Proteins were stained with Sypro-Ruby, picked and enzymatically cleaved with Trypsin. Peptides and corresponding proteins were identified by MALDI-MS and MS/MS analysis. From several gene products up to 30 different isoforms could be found, many of them differentially regulated. These isoforms, usually derived from one single gene, are produced due to splicing, degradation and posttranslational events. Importantly, they would not have been discriminated if GIST or if shotgun proteomics approaches would have been used. Applying these methods, the proteome would have been cleaved enzymatically before labelling. After enzymatic cleavage of similar protein species their peptides could not be reliably used for quantification since they may be obtained from differently regulated proteins. Only a few proteotypic peptides (which are specific for a single protein species) are suited for accurate quantification. However, with usual sequence coverage of far less than 50% chances are quite high to miss these peptides. During the ICPL approach the stable isotopic label is already introduced on protein level, thereby bypassing the disadvantages of GIST approaches and maintaining the benefits of correlating protein modifications with their regulation.

In the early 1990s the discovery that Ethidium bromide (EtBr) could be used to monitor PCR cycles through binding double stranded DNA (dsDNA), resulting in a fluorescence increase, ushered in a new age in PCR. By the end of the decade new detection methods using probes (i.e. TaqMan) and instrumentation supported the launch of quantitative real time PCR (qPCR) technology. Since then many instruments and detection chemistries (FRET Hybrids, Molecular Beacons, SYBR Green and others) have been developed. Among these chemistries, the intercalating chemical SYBR Green is the most similar to EtBr and widely used. It is a fluorophore that binds dsDNA in the minor groove in a non-sequence-dependent manor. Upon binding, its fluorescence increases to ~10 times that of EtBr. This detection method is simple, sensitive and relatively inexpensive to other methods currently available. Since SYBR Green detects all dsDNA, primer dimers form during PCR will also be detected and quantified. These primer dimers and non-specific amplification can be minimized by using a “hot start” polymerase which prevents reaction initiation. To help alleviate detection of primer-dimers and other non specific amplification Takara has created SYBR Premix Ex Taq (Perfect Real Time). A convenient premix consisting of Takara’s high sensitivity-high performance Ex Taq Hot Start DNA Polymerase and SYBR Green I. Here we investigate this SYBR Green/ Ex Taq Hot Start combination and its compatibility with several qPCR instruments.

Introduction: Nuclear receptor transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) are the molecular targets of drugs for the treatment of a wide range of diseases. Ligand-induced changes in structure/dynamics of the receptor drive dissociation of co-repressors, heterodimer formation, co-activator binding, and subsequent transcriptional activity. Characterization of the dynamics of the receptor is critical to understanding the mechanism of activation. Methods: Hydrogen/deuterium (H/D) exchange is a technique for measuring protein dynamics. Here we describe a two-part approach to generate a high-throughput (<1 h/compound) H/D method to measure changes in the dynamics of the receptor ligand-binding domain (LBD). First, a comprehensive set of differential H/D data are acquired with a number of well-characterized ligands (~24 h/compound). From these data, a single period of H/D on-exchange is chosen that provides the maximal distance between dynamics of ligand-bound and apo-receptor. Next, the chromatography is optimized for the rapid elution of a subset of peptides that span the LBD. To aid this step, we incorporated 1.9-μm stationary phase HPLC columns into our H/D exchange platform. Single time point H/D exchange experiments are then used to probe the dynamics of the receptor. Results: Comprehensive H/D exchange experiments were performed with five ligands that induce varying degrees of PPARγ activity (rosiglitazone, MRL-20, MRL-24, nTZDpa, BVT.13). For all regions of the protein, 60 sec on-exchange provided the maximum difference between the apo- and ligand-bound receptor. Fifteen peptides were selected that span the LBD. A chromatography method was developed that allowed for the separation of the peptides of interest in <10 min. We then proceeded to characterize 27 ligands in a 24-h time period. Conclusions: We have developed an H/D exchange method for rapid characterization of ligand binding to PPARγ LBD. The approach is suited to the analysis of other nuclear receptors.

Current protocols for protein quantitation using iTRAQ reagents utilize a tryptic peptide–labeling strategy. While there are many advantages to this workflow, it precludes the effective use of protein-level separation techniques. We compare two protein-level labeling workflows. The first is in-gel digestion after SDS-PAGE, and the second is solution-phase digestion after size-exclusion chromatography. Chromatography offers increased sample load relative to electrophoresis and thus potentially increased access to proteins of lower abundance. The study was conducted on a human cell line (U937) infected with dengue virus, a mosquito-borne flavivirus of the Flaviviridae family. Intact protein samples were reduced, alkylated, and labeled at the lysine residues with one of the isobaric iTRAQ reagent tags, pooled, and then separated by size-exclusion chromatography. After separation, the fractions were digested with trypsin. These workflows resulted in larger peptides and reduced complexity, as the proteins are digested only at the arginine residues. The digests were then analyzed by LC-MALDI on a 4800 MALDI TOF/TOF Analyzer. Initial data show approximately 750 proteins identified using the gel-based workflow, with a similar number of identifications from the size-exclusion data. The analysis will be focused on global protein expression changes as a result of infection in order to elicit information on how the virus competes with normal cellular mRNAs for the translational machinery of the cell.

The most common form of dementia is Alzheimer’s disease. According to the amyloid hypothesis, the disease is preceded by an accumulation of the amyloid-β (Aβ) protein, which leads to downstream events including activation of microglia, inflammation, synaptic dysfunction, and neuronal loss. The objective of this research is to address the physiology of Aβ in humans by measuring its in vivo metabolic rates. A method was previous developed in our laboratory for measuring the in vivo synthesis and clearance rates of total Aβ within the cerebrospinal fluid (CSF) of normal healthy volunteers using a metabolic label of 13C6 leucine. After a spinal tap, the total Aβ peptide pool was isolated from the CSF by immunoprecipitation, followed by mass spectrometry. As in selected reaction monitoring experiments, we use signals obtained from fragmentation of precursor peptide ions containing unlabeled and labeled leucine, but with a full scan tandem MS, instead of using precursor ions for quantitation as done in isotope-labeling experiments. Total Aβ in human CSF was measured by ELISA methods to be about 5000–20,000 pg/mL (~2 pmol total and 20 fmol 1% labeled). The metabolism of Aβ was measured by determining the percent 13C6 leucine labeling in Aβ in CSF samples taken every hour over a 36-h time course. The incorporated label was not detected until the fifth hour of label infusion, followed by an increase to a steady state at 20 to 24 h, then a decrease over the last 12 h. In vivo metabolic protein labeling can be measured with high sensivtivity (low fmol labeled peptides), accuracy (R2 = 0.99), and reproducibility. These technical advances using common commercially available instruments may be applied to other proteins of interest and adapted to measuring total amounts of proteins or percent labeled proteins, for absolute and relative quantitation respectively.

Recombinant monoclonal antibodies (MAb) comprise a significant proportion of biopharmaceuticals used in diagnostic and therapeutic applications. Although the general structural features of monoclonal antibody such as disulfide bond patterns have been known for decades, the intrinsic heterogeneity of such molecules has imposed the needs for thorough analytical characterizations so the structural details of each pharmaceutical MAb are determined, and safe, effective, and reproducible products can be generated. The analysis of intact MAb molecules is challenging due to their high molecular mass, hydrophobic nature, and presence of sugar moieties. Conversely, the analysis of intact protein is also advantageous because it reduces dramatically the time for sample preparation and data interpretation compared with peptide mapping and sequencing. Furthermore, it also minimizes the chance of introducing putative modifications, which are often observed during peptide mapping. The aim of this study is to perform structural characterization of a recombinant monoclonal antibody (IgG1) by a newly developed electrospray ion-mobility time-of-flight mass spectrometer (ESI-IM-TOF MS) using a top-down approach. In this approach, the molecular masses of the intact and the deglycosylated antibodies as well as the light and heavy chains of the antibody were measured by direct infusion to MS. The mass difference between intact and the deglycosylated antibody establishes the glycosylation profile of the antibody. For the reduced antibody, the ligh and heavy chains are separated in the gas phase by the ion mobility functionality of the instrument. Thus, the masses of light and heavy chains are accurately measured to yield further evidence for other post-translational modifications. Furthermore, the fragmentation of the intact light chain subunit and subsequent separation of fragment ions by ion mobility allows us to quickly obtain partial sequence of the light chain. All of the results demonstrate that ESI-IM-TOF-MS is a superior tool to characterize MAbs and other complex protein pharmaceuticals.

Many of the available methods for the analysis of hemoglobin using mass spectrometry rely upon mathematical reconstruction of the data to generate information about the different isoforms. Although these produce the ability to speciate fine mass differences between various isoforms, they are computationally intensive and time consuming. These forms of mathematical reconstruction can lead to artifacts in the reconstructed data, which may interfere with the accurate quantification of the different forms. This poster presents data evaluating a number of different methods for the determination of levels of modified hemoglobin. These include protein reconstruction, selected reaction monitoring (SRM) of protein charge states, and multireaction monitoring (MRM) evaluation of the specific modified peptides. Using SRM screening, a number of different charge states have been monitored, which allows for shifts in the protein envelope due to differences in buffer composition or due to variations in the protein concentration. This showed a low degree of specificity when compared to the use of protein reconstruction. The use of peptide MRM with subsequent MS/MS provided the best specificity, removing any ambiguity in the data. This also provided the best route for automation using a liquid chromatography system and the Analyst Software 1.4.2 quantification module. This allows a fully automated workflow utilizing tools that provide features that assist users with good laboratory practices compliance.

Protein phosphorylation is a critical signal transduction event in many areas of therapeutic interest, including cancer and inflammation. Understanding of these signaling events, their role in disease, or their modulation by therapy, necessitates identification and characterization of phosphoproteins regulated by hormones, circulating factors, or pharmacological agents. Identification of regulated phosphoproteins requires sensitive and robust methods of phosphoprotein enrichment, profiling, identification, and characterization. In this study we utilized a well-characterized cellular inflammation model, LPS-stimulated and drug treated THP-1 cells, to quantitatively assess the enrichment of known and total phosphoproteins with a commercial phosphoprotein enrichment kit. We measured the enrichment of Hsp27 with pan- and phospho-specific antibodies using flourescence based assays and 1D/2D Western blots, and measured total phosphoprotein enrichment with two phosphoprotein quantitation kits. These enriched fractions were then processed and profiled by 2D-DIGE to identify known and unique regulated phosphoproteins. Several phosphoprotein candidates were characterized by 1D- and 2D-Western blotting to verify regulation. Our results indicate that current phosphoprotein enrichment reagents are: 1) subject to poor recovery and preferential enrichment of multiply phosphorylated proteins; 2) that sample processing (desalting) of enriched intact proteins for 2D gel analysis can lead to significant sample loss and artifactual regulation, and 3) that verification of regulation by 1D and 2D Western blotting is an essential step in the identification of phosphoprotein markers of interest.

Proliferating cell nuclear antigen (PCNA) is a small nuclear protein that plays a central role in DNA replication. Although it has been intensely studied, there are strangely few reports of post-translational modifications to this protein. A site of ubiquitination has been described, and some have claimed the protein is not phosphorylated. A recent publication proposed partial methyl esterification of a number of Glu and Asp residues. Very recently, we found a phosphorylated Tyr (211) and showed it regulates stability of the protein. Increased phosphorylation at this site coincides with pronounced cell proliferation, and also correlates better with poor survival of breast cancer patients than total PCNA level. We describe here the strategy and experimental details of how we found Tyr211 to be phosphorylated.

Enhanced heat shock protein (HSP) expression protects cells and tissues from injury and HSP expression can improve survival in experimental illness models. Our laboratory has shown that treatment with glutamine (GLN) can enhance HSP expression in tissues of stressed animals. An important part of GLN’s protective effect may be via rapid O-glycosylation of key cellular proteins, potentially via enhanced activity of the hexosamine pathway following stress. This is known to occur in seconds, prior to the expression of stress proteins, and can prevent proteosomal degradation of these vital proteins. The activation of the O-GlcNAc pathway is known to induce a cellular protective response, including enhanced HSP expression which may serve as a nutritional sensor of the cell’s environment. In this study, we pre-treated HSF1 wild-type cells with DMEM + 10% FBS for 21 h and 20 min. Cells were treated with either 50 mM glutamine or 5 mM glucosamine 15 min prior to heat shock. After 4 h, cells were fractionated into nuclear and cytoplasmic fractions and lysed. Anti-O-GlcNAc was used to immunoprecipitate modified proteins from the lysates, which were then separated using 1D gel electrophoresis. Bands showing changes were excised, digested and identified by nanoLC/MS/MS and database searching. The majority of protein changes were observed in the nuclear fraction, where treatment with glutamine, and to a lesser extent glucosamine, rescued some heat shock proteins to near control levels of expression. As expected, in our preliminary work, the expression of O-GlcNAc modified HSP-70 was at similar levels for control and glutamine-treated heat shocked cells, while expression was reduced for untreated heat shocked cells. Other modified proteins detected include myosin, plectin 1, alpha filamin, vimentin and actin. Future efforts will include replicating these experiments in cell and mouse models to confirm these results.

As the study of protein biomarkers increases in importance, technical limitations to the detection of low-abundance proteins and high-throughput, high-precision quantitation remain to be overcome. The complexity and dynamic range of the plasma proteome makes the task of specific, quantitative detection even more challenging. Multiple reaction monitoring (MRM) capabilities of triple quadrupole MS systems have been explored as solutions to this challenge due to their well-known sensitivity and selectivity for components in complex matrices such as plasma. Recently, a suite of >100 MRMs representing ~50 plasma protein markers were monitored quantitatively in a single assay using the MRM-based technique showing detection of proteins down to the level of L-selectin (~1μg/mL) with minimal sample preparation and no peptide or protein standards for most of the plasma protein markers.1 As more extensive candidate biomarker panels are being identified, MRM assays will need to be more rapidly developed to verify the expression changes of these proteins across larger clinical sample sets. To do this, the unique combination of triple-quadrupole and ion-trapping capabilities of the hybrid triple quadrupole–linear ion trap mass spectrometer have been utilized. A strategy for rapid MRM assay development for larger-scale profiling and qualification of biomarker candidates without having to first prepare synthetic peptide standards is currently being investigated and involves a chemical labeling strategy to create global reference standards to enable quantitative comparisons between clinical samples. Single assays consisting of ~500s of MRM transitions have been developed for this rapid qualification phase, facilitated by intelligent use of retention time windows during an LC analysis, while maintaining an optimum number of data points for improved precision of peak area and quantitative profiling. This presentation will demonstrate the details of this workflow with human plasma examples.

We developed a new strategy and computer algorithm for high-throughput analysis of protein-protein or intra-protein interaction sites from chemically cross-linked protein(s). In this strategy, we directly analyze LC-MS/MS data obtained with LTQ-FTMS without any comparison with control sample or isotopic labeling. We developed a computer algorithm, X!Link, to find cross-links of two peptides, which takes only ~10 seconds to analyze ~5000 MS/MS spectra. It is very sensitive and has low false-positive rate. We applied this method for the cross-linking site analysis of cytochrome c and Dnm1 G385D homodimer chemically cross-linked by BS3. We also adopted X!Tandem and Sequest for intra-peptide cross-linking and monolink modification analysis. Total of twenty one cross-links are identified in a single LC-MS/MS data of cytochrome c, in which thirteen is inter-peptide cross-links in 42 MS/MS spectra. High coverage of BS3 modified Lys (84%, 16/19) is owing to the high sensitivity of the present method. Monomer and dimer SDS-PAGE bands of Dnm1 G385D were studied to investigate inter-protein interaction sites of this homodimer. Total of forty five cross-links including thirty seven inter-peptide cross-links from 243 MS/MS spectra were identified in four LC-MS/MS datasets, which is the most cross-links identified so far in a single protein. Closer look of the data demonstrates importance of careful manual inspection for the correct assignment of cross-linking sites when multiple sites exist in a peptide or other similar sequences exist. Two such cases are shown in Figure 1. FIGURE 1 Top:Two internal lys residues in a peptide. Bottom: exactly the same parent mass between cross-linked peptides of two subsequent peptide sequences and a single peptide with monolink modification.

After separating the highly abundant proteins (HAP) by IgY affinity column, the next layer of abundant protein, moderately abundant proteins (MAP), becomes an obstacle to access the low abundant proteins (LAP), where the majority of biologically relevant and clinically important biomarkers reside. Therefore, isolation of MAP is a new challenge for effective detection and analysis of LAPs. To tackle this challenge, we further developed the IgY-microbead system by immunizing chickens with a flow-through fraction of IgY12 column and constructing the column with affinity-purified IgY antibodies against the flow-through proteins of IgY12 column. The column developed, called SuperMix, was applied for further partitioning of the flow-through fraction of IgY12, which resulted in a bound/eluted fraction (designated as MAP fraction) and the flow-through fraction (designated as LAP fraction). Unfractionated and serial-fractionated samples using IgY12 and SuperMix columns were analyzed by SDS-PAGE and 2DE. Our data demonstrate that SuperMix columns specifically and reproducibly remove the post-IgY12 layer of the abundant proteins. A case study using SDS-PAGE coupled with LC/MS/MS demonstrates that the SuperMix column enabled specific capturing of 207 MAP, with 77 proteins being uniquely identified in high confidence (≥95%). This novel approach enables deeper and more effective access into the population of LAPs. In addition to digging deeper with the SuperMix column, we also have progressed in the direction of digging faster. One of the present challenges of plasma biomarker discovery is sample throughput limitations. In collaboration with PSS Bio Instruments, GenWay Biotech has developed a novel multiplex automated system (SepproTip) for high-throughput plasma sample processing. It permits processing of 12 samples at a time. This Seppro-Tip system can process plasma samples using both IgY12 and SuperMix tips. The turnaround time of 12 samples per 65 min allows a large number of samples to be processed without decrease in sample preparation quality.

Rationale: Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of the recognized contributors in the pathogenesis of lung diseases. It evokes a dramatic increase of phosphorylation events in the inflammatory cells after stimulation. Lipoxin A4 (LxA4) is a novel lipid mediator with putative proresolution properties that appear to function as inhibitory signal during the time course of inflammation. We investigated the influence of LxA4 on intracellular events during stimulation with GM-CSF. Methods: 2D electrophoresis was used to resolve whole-cell proteome, subcellular compartments, and GM-CSF signaling-specific proteome, using biotinylated ligand immunoprecipitation and GM-CSF receptor immunoprecipitation as well. Overall protein phosphorylation was assessed on the gels with phospho-specific stain Pro-Q-Diamond and on the Western blot using anti-phosphotyrosine immunostaining. Phosphorylation of ERK, SHP-2, and STAT-5 was revealed with specific antibodies. Protein interactions were assessed by co-immunoprecipitation. Cytokine profile was measured with multiplex assay. Results: Pretreatment of the cells with LxA4 before GM-CSF stimulation led to the decrease of cytokine production as well as to changes in overall protein phosphorylation and tyrosine phosphorylation. Significant changes were also observed in phosphorylation of key kinases of GM-CSF signaling. Among signaling molecules affected by LxA4, STAT-5 transcription factor and SHP-2 phosphatase were detected. 2D electrophoresis showed that LxA4 pretreatment changed GM-CSF signaling in the proteome. Conclusions: The inhibitory effect of Lipoxin A4 on GM-CSF-induced cellular function may indicate its potential for downregulation of GM-CSF-driven inflammatory processes. We propose mechanisms of inhibition of GM-CSF action by Lipoxin A4 involving different cornerstones of GM-CSF signaling, including protein phosphatases and transcription factor STAT-5.

The age of proteomics and resulting advances in technology have resulted in an increase in the number of requests for recombinant protein construction, production, and purification projects. One of the mainstays of the Molecular Biology Core Facility (MBCF) at Trudeau Institute is the production of Major Histocompatibility (MHC) Class I and Class II multimeric fluorescently labeled protein-peptide complexes, which allow the tracking of antigen-specific CD8+ and CD4+ T-cells using fluorescence-activated cell sorting (FACS) analysis. In addition, the MBCF has generated fusion proteins to be utilized for immunization strategies and viral coat proteins from influenza and murine gamma herpes virus 68 for detecting epitope-specific antibodies. Another simple but extremely useful and money-saving protein production project is the production and purification of Taq enzyme for a laboratory that uses large quantities of Taq enzyme in a limiting dilution assay for latent viral detection. Requests for milligram amounts of native, endotoxin-free proteins to be used for in vivo vaccination are increasing. The MBCF utilizes a variety of expression systems, but primarily bacterial (using T7 promoter–based plasmids) and the insect cell–based Drosophila Expression System (DES) (Invitrogen). Recently, the Gateway system (Invitrogen) has been introduced into the MBCF to facilitate the whole procedure. Purification schemes are mainly based on size exclusion or affinity chromatography using either protein A, Ni-NTA, or glutathione sepharose.

The use of ion-pairing reagents for reversed phase peptide separations has been investigated in numerous publications. Reversed phase oligonucleotide separations typically employ TEAA as an ion-pairing reagent. However, other ion-pair reagents may provide better separation results. We investigated several different ion-pairing reagents using a ten micron, mono-sized, reversed phase polymeric resin, Amberchrom HPR10. DMT-off purifications of two synthetic 12mer DNA oligonucleotides (AAA CCT GAT GTG and GGT GGT GGT GGT) were performed, and yields and purities from the different purifications were compared in order to determine the most favorable ion-pairing conditions.

In a Top-Down workflow (ECD, ETD, ISD) the whole protein molecule is subjected to fragmentation and proteins are not digested (as in the Bottom-Up approach). This allows the detection of signal peptides, modifications, sequence variations and mutations. Top-Down analyses can be done using FTMS/ECD/ESI or MALDI-ISD spectra. For the analysis of Top-Down datasets flexible algorithms are required that allow the creation of sequence tags based on peak mass differences and several ways to process the sequence tags in a subsequent step. Sequence tags can be created that can include various modifications, user-defined mass range, inclusion of selected peaks and exclusion of peaks matched by accepted tags. The resulting sequence tags are scored and can be used to perform MS-BLAST homology searches or Mascot sequence tag searches to identify unknown proteins. An alternative workflow that was developed involves a known protein sequence and the identification of protein modifications or mutations. Using these algorithms it is possible to automatically suggest the signal peptide structure from the mass offset between the experimental tag and the theoretical value that is based on the protein sequence database entry, modifications or a mutations. We used the algorithms that are included in the BioTools 3.1 software package to analyze reISD-MALDI-TOF and ECD-FTICR spectra from undigested proteins in the molecular weight range 6–70 kDa and could automatically detect, e.g., the length of the pre-pro peptide of bovine serum albumin (67 kDa). Other examples involved proteins with N/D and Q/E ambiguities in their database record such as carbonic anhydrase. An interesting field of application is the localization of protein phosphorylation sites based on the undigested protein as in the top-down analysis suppression of the phosphorylated peptides is not observed.

The microbial mats growing in the runoff channels of the hot springs of Yellowstone National Park (YNP) are a rich mix of bacterial, archaeal, and eukaryotic species. Mat samples were gathered from Octopus Hot Spring in 2005 and 2006.The samples were subjected to labeling with iTRAQ reagents followed by shotgun proteomics. Mascot was used to query an in-house YNP database derived from the microbial portion of the NCBInr. It was expected that the majority of the proteins mapping to species with high temperature optima would be associated with the sample taken at a higher temperature, while those proteins from species with lower optima would be associated with the sample from lower temperature. Although Synechococcus is the most abundant microorganism in the mat community when abundance is measured by the percentage of DNA in a metagenomics sample, more peptides were identified from Roseiflexus sp. RS-1 than from any other organism. This discrepancy is most likely due to the sample being a mix of the large red and small green layers of the mat. Synechococcus resides only in the green layer. The large number of distinct peptides from the Roseiflexus extracellular binding protein resulted in the highest Mowse score of the proteins identified. Eighty percent of the peptides associated with the extracellular binding protein were isolated from the 58°C sample, while only 20% of this protein came from the 71°C sample. This trend continues throughout the housekeeping proteins quantified from Roseiflexus, as might be expected of a thermophile with a lower temperature optimum. The remainder of the identified proteins showed the association with collection temperature that would be expected from the temperature optimums of the mciroorganisms from which they were extracted. The majority of the proteins identified in this experiment were housekeeping and structural proteins.

Biacore’s surface plasmon resonance technology is widely used in the study of affinity and kinetics of interactions between two or more biomolecules, receptor-ligand interactions, and in understanding the kinetics of biomaterials. The analyte protein bound to the immobilized ligand on the sensor chip can be recovered for subsequent identification by mass spectrometry. Earlier studies have demonstrated that Biacore can be used to fish out proteins from crude mixtures in a highly purified state, and the recovered protein can be identified by upstream mass spectrometry. However, earlier studies have been done mainly for the known target protein. The study of RNA-binding proteins is very important for understanding the assembly and function of RNA-protein complexes in the splice-osome. In the current study, a biotinylated short single hairpin loop RNA was immobilized on the streptavidin sensor chip, and the HeLa cell nuclear extract was passed over it to identify the proteins bound to the biotinylated RNA stem-loop. The results of recovered analytes identified by mass spectrometry show that the Biacore 3000 could be a useful technique to identify previously unknown RNA-binding protein/s from nuclear extracts.

As altered epigenetic regulation is a common feature of cancer, a greater understanding is required of the role of histone post-translational modifications as epigenetic regulators of changes in chromatin structure. Chromatin remodeling may also be important in the cellular response to DNA damage induced by anti-cancer drugs. A greater understanding of the nature of histone PTMs and resulting DNA damage in normal and cancerous tissues might indicate differences in the activity of enzymes involved in modifying histones, which could yield potential targets for cancer-specific therapy. Trichostatin A (TSA), a histone deacetylase inhibitor, promotes acetylation of histones and subsequently increases the accessibility to cellular machinery by the unraveling of DNA. Using high-resolution 9.4T and 12T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers, the aim of this work was to characterise, in an unbiased fashion, the histone PTMs induced in histone H4 in response to treatment with TSA and certain environmental stimuli. FT-ICR mass spectrometry is not well suited to online analysis, owing to the extended scan time required to achieve optimum resolving power. The TriVersa Nanomate enables fraction collection of the HPLC eluent allowing FT acquisition time to be decoupled from separation time. Following further sample cleanup, it proved possible to infuse unfractionated complex histone samples. The most abundant classes of histone present in the cell (H2A, H2B, H3, H4) were observed The resolving power of FT-ICR permits the post-translational modification of H4 to be observed directly. It can be seen that in the case of cells treated with TSA, there was considerable variation in the acetylation state of histone H4. It was also possible to affect the acetylation state using certain environmental stimuli.

Heat lysis is a very reliable method for high-throughput labs to decrease costs and increase throughput with little sacrifice in quality of sequence data compared to typically used two-plate or magnetic-bead DNA purification methods. Laboratories employing this procedure on high-copy plasmids, using a sequencing reaction with a 1/8th Big Dye (Applied Biosystems) dilution, have reported generating data with 85% of all wells having read lengths of at least 600 bases with quality value (QV) 20 or above.1 We have adopted this procedure in our laboratory and optimized it for sequencing from various in-house cDNA and genomic shotgun libraries cloned into high-copy plasmids, as well as for libraries constructed outside of our sequencing center. To fully develop the procedure, we utilized a number of culture plates, testing different plate volumes, well shapes, and growth times. Also, we tested various volumes of resuspension buffer in order to generate the highest sequencing success rates and read lengths. To further cut costs, we optimized the sequencing reactions by testing various dilutions of Big Dye and template amounts. Based upon over 55,000 reads, we have been able to consistently generate sequencing results with average success rates of 90–95% and read lengths of over 700 bases with QV 20 or above. Our past protocol employed a standard two-plate DNA preparation method with a 1/16th Big Dye dilution in the sequencing reaction. In contrast, we now use the optimized heat lysis protocol combined with a 1/32nd Big Dye dilution. These changes have increased throughput and produced the high-quality sequencing results stated above, yet reduced our consumables cost by over 55%.

Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins and automated analysis procedures for the detection, identification, and structural characterization of the corresponding peptide modification. Selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids). Isolated proteins were released from the beads under acidic conditions, dried, and subsequently re-dissolved and digested with trypsin. The resulting complex mixture of peptides was subjected to LC-MALDI analysis. The respective glycoproteins were identified by direct MS/MS analysis and subsequent database searching. Intact glyco-peptides enriched by a second magnetic-bead purification on peptide level were directly subjected to LC-MALDI analysis to get structural information about the glycan and peptide parts. A precondition to this novel approach was the discovery of certain consensus peak patterns in the MALDI-MS/MS spectra, allowing the automatic determination of the peptide part and the glycosidic information of the glycopeptides supported by bioinformatics tools. Applying this fast and simple approach, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from experiments using magnetic ConA, LCA, WGA, jacalin, and boronic acid beads, respectively. The use of jacalin and boronic acid beads facilitates the enrichment of O-glycosidically modified proteins. In contrast, ConA, WGA, and LCA specifically bind N-glycosylated peptides and proteins. Few non-glycosylated proteins were identified, probably due to co-precipitation with glycosylated proteins.

Characterization of glycoproteins by electrospray ionization tandem mass spectrometry of glycosylated peptides using collisionally induced dissociation (CID) results in the preferential fragmentation of the glycosidic bonds, while the peptide bonds are more stable. As low-energy CID of glycopeptides mainly provides information on the glycan moiety, a selective analysis of the peptide chain itself usually requires enzymatic removal of the carbohydrate part. The recently introduced electron transfer dissociation (ETD) option for ion-trap mass spectrometers shows preferred fragmentation of the peptide backbone, leaving posttranslational modifications widely intact. Thus, a combination of ETD and CID is a highly promising tool for the analysis of glycopeptides. A high-capacity three-dimensional quadrupole ion-trap MS with ETD option enables the application of the two complementary fragmentation techniques. For fragmentation via ETD, radical anions of fluoranthene are generated in a negative chemical ionization source and added to previously isolated multiply charged peptide cations inside the ion trap. Different glycosylated proteins were analyzed either offline or with on-line nano-LC-MS/MS in the positive-ion mode. Enriched glycopeptides from a tryptic digest of horseradish peroxidase were analyzed offline using nanospray. For an on-line nano LC approach, digests of human IgG3 were chosen. With ETD and CID, widely complementary data from these glycopeptides were obtained: CID MS/MS spectra showed mainly sugar fragmentation, while ETD MS/MS allowed for selective fragmentation of the peptide backbones with retention of the intact glycan moiety, thereby allowing the facile deduction of peptide sequence and glycan attachment site. In conclusion, glycopeptides were characterized in detail with regard to carbohydrate composition and structure as well as peptide sequence and modification sites. Importantly, this approach can be incorporated into a conventional glycoproteomics work flow, as sensitivity and speed of data acquisition allow the combined use of ETD and CID with direct LC coupling.

Equine protozoal myeloencephalitis (EPM) is a common and costly neurologic disease of horses caused by Sarcocystis neurona, protozoa that parasitize the nervous system. Confirmation of the clinical diagnosis is a challenge for veterinarians because the current immunologic tests lack specificity. Multiplexing strategy approaches including two-dimensional fluorescence difference gel electrophoresis (2D DIGE) and mass spectrometry were employed to identify differential protein expression associated with EPM. Cerebrospinal fluid (CSF) samples were collected from normal and EPM horses. EPM was diagnosed if neurological signs were present, Western blot against S. neurona on the CSF was positive, and other neurological diseases were excluded by ancillary tests and/or necropsy. CSF was collected under general anesthesia, at the atlanto-occipital site to avoid blood contamination. The CSF proteins were precipitated, labeled with CyDye DIGE fluor dyes, and separated on 2D gels. The 2D gels were scanned with Typhoon, and the digitized images were analyzed with DeCyder software. The differentially expressed proteins were excised with a ProPic II spot picker, in-gel trypsin digested, and the tryptic peptides were analyzed with the electrospray ionization mass spectrometer. A number of differentially expressed proteins were identified in the various horse CSF, including serum albumin, haptoglobin, prostaglandin-D synthase, and apolipo-proteins E and A-I. Various isoforms of serum albumin were up-regulated with EPM compared to the normal horse CSF. The above proteins, including the isoforms, may be associated with EPM, and the characterization of differentially expressed additional proteins could give insight into the pathogenesis and diagnosis of EPM.

The standard mass spectrometric technique for the analysis of phosphopeptides on a linear ion trap mass spectrometer is to perform MS/MS followed by MS3 of the putative neutral loss peak observed by MS/MS. This is usually termed data-dependent neutral loss MS3 (DDN-LMS3). The strength of this approach is the sensitivity in full scan MSn of linear ion trap technology and the characteristically strong neutral loss peak, which flags the MS/ MS spectrum as potentially belonging to a phosphopeptide. This approach has been much applied to phospho-proteomic analyses typically involving up-front phosphopeptide enrichment, such as TiO2 chromatography. Indeed, a recent article from Mann and co-workers identified 6600 phosphorylation sites utilizing a hybrid linear ion trap Fourier transform mass spectrometer. However, there are also weaknesses, such as the fact that phosphotyrosine residues rarely produce a neutral loss from the precursor during MS/MS. Recently, a new dissociation technique, electron transfer dissociation (ETD), has been invented in the lab of Don Hunt. ETD has different but complementary characteristics compared to CID. For example, (1) ETD works best on multiply charged ions of 3+ and above, whereas CID works best on 1+ to 3+ ions; (2) amino acid side chains and important modifications such as phosphorylated amino acid residues are left intact by ETD, producing a rich ladder of c and z ions for sequencing. CID produces mostly b- and y-type ions by collision with gases. Work reported here compares DDNLMS3 data (and another neutral loss–based technique referred to as multistage activation, also utilized in the work of Mann and colleagues) generated on a series of human kinases with results obtained by data-dependent ETD. The different dissociation techniques were found to be complementary, and a number of novel phosphorylation sites were identified on such well-known kinases as PKB and SAPK2a.

The Wadsworth Center is the most comprehensive state health laboratory in the country, and is dedicated to science in the pursuit of health. It fulfills its mission of protecting and promoting the health of New Yorkers through analysis, research and education combined with comprehensive clinical and environmental laboratory evaluation and accreditation programs. Our scientists study public health issues, from drug resistance to emerging infections and environmental toxicants as well as basic biological processes. The Core is a resource for investigators within the Wadsworth Center as well as those from outside institutions who require advanced light microscopic (LM) and/or image analysis as part of their research programs. Our goal is to operate a user friendly full-service Core for our user community, including a comprehensive educational program. This includes the latest in imaging equipment and methods, and in image analysis software and methods. In the event that current instrumentation cannot answer the question posed by the user, we seek to develop innovative solutions. One example of this is optical projection tomography, this modality bridges the resolution gap between NMR/CAT scans and traditional LM. No matter the specimen or the question(s) being asked we provide the technologies and assistance to get the answers. A few recent examples are: Imaging water borne contaminants to identify the organism responsible for sickening the patrons of a water park. Imaging motile bacteria at “near” video rates and then tracking them to investigate the cellular effects of an antibiotic. Visualizing in 3D, multiple biomolocules/fluorophores to investigate neuronal function in brain slices. Automatically counting cells outgrowing from explants in order to map quantitative trait loci between inbred strains of mice in response to vascular endothelial growth factor. Measuring the fluidity of lipid bi-layer using FRAP to determine how lipid mobility controls the diffusion of small biopolymer adsorbates.

2,5-Dihydroxybenzoic acid (DHB) is the matrix of choice for carbohydrate and glycopeptide analysis, but due to the inhomogeneous surface morphology of samples prepared with DHB, it is typically incompatible with automated measurements. We describe a simple and rapid method for the analysis of glycoproteins, which combines (a) reducing the complexity of the digest mixtures with glyco-specific enrichment and (b) subsequent LC-MALDI-TOF MS/MS analysis with DHB as MALDI matrix. All samples were prepared on hydrophobic sample plates with hydrophilic anchors 400 or 600 μm in diameter confining the sample dimensions. In a first step, the matrix was applied to the 384 sample spots (“anchors”) of a microtiter plate–shaped MALDI target. The LC eluate from CAP-RP-HPLC subsequently dissolved the DHB matrix confined to the hydrophobic boundaries of the anchors. Co-crystallization of glycopeptides in DHB suitable for the automated analysis was achieved. This method was applied to recombinant human inte-grin alpha and beta; glycosylation sites were identified and described. The MALDI-MSMS spectra of glycopeptides (N-linked type) include information about the structure of peptide moiety as well as the glycan part of the molecules. MALDI-TOF/TOF spectra permitted (a) the detection of N-linked glycopeptides by a neutral loss analysis across the entire LC-MALDI-MS/MS dataset, (b) the determination of the molecular weight of the pure peptide chain by typical fragmentation patterns, (c) the identification of the peptide part of the fragmented glycopeptide by means of simple database searching, and (d) initial information about the glycan composition and the attachment site. LC-MALDI-TOF/TOF on DHB matrix preparation is a powerful approach for the detailed characterization of glycoproteins.

Affinity purification is a powerful tool for protein enrichment in proteomics studies. We here present a fully automated system for purification of His-tag proteins and IgG using Ni2+/Co2+ and Protein A magnetic beads, respectively. Reagents for His-tag protein or IgG purification are pre-dispensed in a sealed cartridge for automated runs on a Magtration 12GC robot. The automated purification is based on Magtration technology to perform magnetic bead separation similar to a filtration process in a pipette tip. An optimized protocol has been developed for the automated protein purification. High protein purity and yields were obtained using this automated system. His-tag protein human galectin-1 was purified to approx. 1.6 mg with 12 samples processed in parallel within 30 min on the 12GC robot. This system was also used to screen the expression of His-tag water-soluble proteins and inclusion bodies in bacterial cells, even at a very low expression level. Using Protein A magnetic beads and corresponding pre-filled reagent cartridges, various amounts of human serum (15–80 μL) and the magnetic beads (100–200 mg) were tested on the robotic system. With 30 μL serum and 150 mg magnetic beads, we purified IgG with a high yield of 230 μg. A total of approx. 2.8 mg IgG can be obtained within 60 min with 12 samples run in parallel on the robot. The magnetic beads after the affinity purification can be regenerated by automation for repeated use. Magtration robotic system can be extended for purification of GST-tag fusion proteins and Immunoprecipitation by automation. We have provided an automated protein purification system with a Magtration robot and pre-filled reagent cartridges for rapid and multiparallel processing of different proteins.

Protein phosphorylation plays a significant role in regulating cellular processes such as signal transduction, cell division, cell motility, apoptosis, metabolism, differentiation, gene regulation, and carcinogenesis. Typically, there are 10–20% of proteins which are phosphorylated. Due to the low level of phosphoproteins in the presence of overwhelming amounts of non-phosphorylated proteins, as well as those proteins’ wide dynamic variation over time, identification of phosphopeptides is still a formidable task. In addition, phosphopeptides often have poor ionization efficiency in MS analysis. Thus, a highly sensitive detection method plus phosphopeptide enrichment is extremely important for a successful phosphopeptide identification. Currently, immobilized metal affinity chromatography (IMAC) is the method of choice for enriching phosphopeptides from complex biological samples. Typically, nickel, iron, and gallium–based IMAC shows significant binding of non-phosphorylated peptides that have multiple acidic residues. Forest White et al. used a kind of chemistry to put methyl esters onto those acidic groups (D and E) to solve the problem of nonspecific binding to the IMAC beads. However, this approach brings in a lot of side reactions to that chemistry, and raises issues of how complete the modifications are. Recently, several papers and posters have been published demonstrating the unique ability of titanium dioxide and zirconium dioxide to selectively retain phosphopeptides contained in complex biological mixtures. In this application, a TiO2-based IMAC method was successfully developed to enrich phosphopeptides and adapted to a complex biological sample, Saccharomyces. Trapping phosphopeptides are demonstrated via the analysis protein CaO19_4593 (gi|68466366), a family of GTPase-activating proteins which contains multiple kinase-binding domains.

Protein N-myristoylation is an early event in acylprotein biosynthesis. There are many known N-myristoylproteins such as cAMP-dependent kinase, calcineurin B, p60src, cytochrome b5 reductase, p56LSTRA/TCK, MuLV gag, M-PMV gag, BLV gag, MMTV gag, FeLV gag, BaEV gag, HTLV gag, HIV gag, HIV 3′-orf, Hepatitis B Virus pre-S1, Polyoma Virus VP2, SV40 Virus VP2, Poliovirus VP4, Bovine Enterovirus VP4, bovine brain G0 α-subunit. A number of these N-myristoylated proteins are protein kinases or phosphatases that have important roles in modulating cellular metabolism. A variety of observations suggest that N-myristoylation may participate in the juxtapositioning of these acylproteins and other components of cellular regulatory circuits. This might occur either by directing these proteins to particular cellular membranes, or by permitting them to interact with other polypeptides that reside in either the cytoplasm or membranes. N-myristoylpeptides are being used to investigate the roles of the N-myristoylation. We present here a simple procedure of making the N- myristoylated peptides in this Laboratory. Briefly, the fully protected peptides (~0.05 mmoles) on resins (~0.1 g) using 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry are de-Fmoced by 20% piperidine in dimethylformamide (DMF). After washed resins with DMF and dichloromathane (DCM), resins are mixed with 5 mL of 50% chloroform in DMF containing ~5 g myristic anhydride and kept at 65°C for 1.5 h. After that, resins are washed several times with the hot 50:50 chloroform: DMF solution. Finally N-myristoylpeptides are cleaved of resins and deprotected by a standard cleaving procedure. Compare to the procedure used by Dr. Glaser which uses pyridine at 23°C for overnight with mixing, our method is faster and do not use pyridine.

The application of multireaction monitoring (MRM) for proteomics analysis is a quite recent development. The sensitivity of four orders of magnitude, reproducibility, and the option of quantification as well as high throughput make MRM a valuable tool for measuring specific proteins. We present a bioinformatics workflow for the determination of MRM candidates that reduces the number of nonproteotypic peptide candidates considerably, enabling the analysis of complex and highly homologous protein families. A peptide candidate has to be unique for its targeted protein in respect to the proteome of the organism. Polymorphisms of the target protein are an important issue, when coverage of all alleles of the target protein is desired rather than rudimentary genotyping by MS. Both aspects can be solved by using annotated protein databases. When using absolute quantification (e.g., the AQUA technique of Kirkpatrick et al.), the ability of synthesis for the stable isotope-labeled peptides has to be taken into account additionally. The selection of suitable fragment ions can be done by evaluating previously acquired spectra or MS/MS fragment prediction. The result has been compared to measurements of human cytochrome P450 (CYP450). CYP450 comprises families (over 200 protein members known today) with low evolutionary conservation and thus high homology. Developing antibodies specific for one protein can be a daunting task; this holds especially true for the human CYP2 family. By targeting only proteotypic peptides using mass spectrometry, antibody-related problems are avoided. As a proof of concept, the analysis of highly polymorphic human CPY2D6 is depicted by ESI-MRM/MS/MS analysis.

Tuberculin PPD Bovis, Intradermic (purified protein derivative of Mycobacterium bovis strain AN-5) was widely used for the Tuberculin test in cattle. However, protein components of PPD remain unknown since it is extremely difficult to separate PPD proteins by traditional protein fractionation techniques. A pilot mass spectrometric protein identification of PPD using the new protein fractionation system of Beckmen Coulter’s ProteomeLab PF 2D is reported here. ProteomeLab PF 2D is a two-dimensional liquid chromatographic system to fractionate complex proteomes. This system consists of a chromatofocusing column to separate the protein based on charge/pI in the first dimension and a reversed-phase column to separate the protein based on hydrophobicity in the second dimension. The advantages of this technique include high loading capacity and better detection of hydrophobic and low molecular weight proteins. Up to 800 protein fractions are collected. So far, 50 proteins such as cfp10, MPB 53, MPB 70, and others have been identified using MALDI-TOF or FT-ICR MS with Sequest or Mascot search engines. This study has demonstrate that ProteomeLab PF 2D with mass spectrometry is a potentially powerful tool to identify proteins from a large proteome.

Epicocconone, a naturally occurring fluorophore that alters its fluorescent character upon binding to proteins is the basis for a number of new labeling technologies ranging from liquid protein quantitation to gel stains and cell stains. LavaPurple is a highly sensitive total protein gel stain that may be used as a stain for nitrocellulose and PVDF blots. The high sensitivity of the stain, coupled with the unique reversible covalent binding of epicocconone, make it an ideal blot stain as it is easily removed by pH change thus not interfering with downstream immuno-staining, mass spectrometry or Edman sequencing. Aim: This study measures the sensitivity of LavaPurple as a blot stain on both nitrocellulose and PVDF membranes, as well as the compatibility of the stain with immuno-staining. Method: a dilution series of Low molecular weight SDS-PAGE calibration markers (GE Healthcare) were separated by electrophoresis and electro-blotted onto either nitrocellulose or PVDF. The blots were then stained with LavaPurple and image analysis performed to determine sensitivity and linearity of the stain. Cell lysate from ovarian cancer cells and rat fibroblasts were separated by 2D GE and blotted onto both nitrocellulose and PVDF membranes before staining with LavaPurple. The blots were then used for immuno-staining and the efficiency of visualization compared with and without total protein pre-staining. Results: LavaPurple was able to visualise proteins on blots at less than 250pg/band loaded on a 1D gel and did not inhibit subsequent antibody binding to target proteins on either 1D or 2D blots. Furthermore the stain could be easily removed from the blot for subsequent staining with other fluorophores. Conclusions: LavaPurple is a highly sensitive stain that is suitable for total protein visualization on blots either for spot removal or prior to immuno-staining.

The proteolytic activities of the ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) protein families play important roles in normal and numerous pathological conditions. The ADAM family members have potential implications in the metastasis of human tumor cells via cell adhesion and protease activities. This family is characterized by the presence of both disintegrin and metalloproteinase domains, responsible for the adhesive and proteolytic properties, respectively. MMPs are a family of proteases responsible for the degradation of the extracellular matrix to allow cell growth and to facilitate remodeling. Under pathological conditions these proteases are involved in many diverse processes from tumor cell migration to cartilage destruction in rheumatoid arthritis. The gene expression levels of eight ADAMs, previously described to possess proteolytic activities; and seven MMPs, previously described to have significant roles in the remodeling of the extracellular matrix during the metastatic process, were analyzed by Real Time PCR using an ABI SDS Prism 7000. RNA was isolated from multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic normal tissue and tumor samples. This method allowed for detected changes in mRNA expression of the individual metalloproteainase genes to be compared between normal and metastatic states, and also between tissue and cultured cells. Based on the preliminary results, there are substantial differences in the level of ADAM and MMP mRNA expression between tissue and cell lines. In general, the level of expression is several fold higher in cultured cells compared to the isogenic tissue they are derived from. We are currently performing in situ fluorogenic enzyme assays on cultured cells to determine if the elevated expression in accompanied by enhanced proteolytic activity.

Reliable identification and characterization of protein kinase substrates is of paramount importance to deciphering cellular signaling pathways. Previously, an artificial phosphomonoester-binding molecule, 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato Zn2 complex (a.k.a. Phos-tag) was shown to bind to phenylphosphate under physiological conditions with a Kd of 25 nM. A fundamental challenge in chemical biology is efficiently performing comprehensive profiling of the activity of artificial chemical structures, such as the Phos-tag molecule, against the hundreds or even thousands of biological targets they could potentially interact with. A peptide microarray-based phosphorylation site screening assay was thus performed to determine the binding selectivity of fluorophore-conjugated Phos-tag molecule, based upon direct detection of binding through readout of fluorescent signal generated by the reporter group. Epitope mapping was performed on a comprehensive list of phosphorylation sites known to be expressed in the human proteome, including serine, threonine and tyrosine phosphorylated residues. The collected data was evaluated using a combination of different in-house software packages resulting in scores for each amino acid residue relative to the phosphorylation site. The study demonstrates that fluorophore-conjugated Phos-tag exhibits very broad substrate specificity and does not appear to depend upon a specific sequence surrounding the targeted phosphorylation site. This relatively low degree of sequence restriction at the phosphorylation recognition site suggests that Phos-tag is broadly suitable for universal enrichment and detection of phosphorylation sites on phosphoproteins and phosphopeptides. The fluorescent Phos-tag technology is easy to perform, cost effective and should allow rapid large-scale screening of protein and peptide phosphorylation using a variety of solid-phase assay formats.

Agrobacterium tumefaciens is a typical soil bacterium that causes crown gall disease in a variety of plant species. A. tumefaciens is capable of recognizing wound sites on a plant by detecting chemicals produced during the wound response of the plant. Laceration of the plant tissue causes the production of phenols and sugar molecules, which in turn trigger not only the chemotaxis of the bacteria towards the injury, but the processing of the tumor-inducing plasmid (Ti plasmid) as well as the expression of certain virulence genes. It is known that the bacterim inserts a segment of its Ti plasmid, the transfer DNA (T-DNA), into the plant at the wound site. The integration of the T-DNA allows for the production of certain plant hormones. It is the uncontrolled plant cell division stimulated by the overproduction of growth hormones that leads to the formation of tumors. Through experimental analysis we seek to identify mutant lines of Arabidopsis that are resistant to transformation, or hypersensitive to transformation. Identification of resistant or hypersensitive plant types is done through root tumor assays as well as GUS assays. In using these methods, it was determined that mutants show evidence of increased resistance while other mutants exhibit susceptibility to transformation similar to wild types. There is little known about the mechanism behind A. tumefacien attachment and transfer of T- DNA into plant cells, and it is our hope that identification of Arabidopsis genes associated with transformation in this screen will allow greater understanding of how the bacterial genetic material is transferred into the plant genome.

Multidimensional protein identification technology (MudPIT) is a platform integrating multidimensional liquid chromatography and tandem mass spectrometry for protein profiling. As MudPIT becomes increasingly popular, the full characterization of large datasets remains challenging. A total of 36 MudPIT technical replicates from two biological samples were acquired on XPs and LTQs ion-trap tandem mass spectrometers. This study evaluated the applicability of label-free quantitative proteomic information by fitting mean and standard deviation of normalized spectral abundance factors (NSAFs) to a power law global error model (PLGEM). Protein fractions from affinity purifications of 293 cells expressing various Flag-tagged human Mediator subunits were enzymatically digested and analyzed by MudPIT. A total of 475 unique proteins were identified from 36 runs, resulting in a 36 × 475 matrix of relative abundance (NSAF values). We observed a “goodness of fit” when PLGEM was applied to the entire 36 × 475 dataset, and subsets of the data containing only NSAF values from XP or from LTQ, indicating that NSAF datasets are compatible with this modeling scheme. Specifically, log-log plots of standard deviation vs. mean NSAFs revealed a linear dependence, with slopes ranging from 0.66 to 0.75 and an adjusted r2 equaled to 0.99. In addition, the residuals were found to be normally distributed across the entire dynamic range of average NSAF values. Similar ranges and distribution were also observed in Affymetrix GeneChip datasets. This study demonstrates the utilities of NSAFs and PLGEM in statistically analyzing MudPIT datasets. With additional modeling using datasets from more complex samples or with “spiked in” experiments, this approach has a potential to become an integral data-mining tool for proteomics datasets acquired from one mass spectrometer as well as across multiple instrumentation platforms.

In order to elucidate the signaling pathway of the human mPR-α protein, a membrane progesterone receptor, a strain of S. cerevisiae overexpressing this protein was generated. Initial global screens using 2D gels, a phospho-specific stain, and tandem MS/MS identified 35 proteins exhibiting alterations in phosphorylation state in response to progesterone activation. Among these were Pil1p and Lsp1p, components of a newly discovered immobile protein mass, the eisosome, purported to denote endocytotic sites. However, due to their similar molecular weights, pI values, and a shared sequence identity of 72%, the separation achieved using 2D gel electrophoresis was insufficient to identify which of these two proteins was exhibiting a change in its phosphorylation state. The experiment presented here utilizes a strain of yeast containing an endogenously TAP (tandem affinity purification)-tagged version of Lsp1p. Since Pil1p and Lsp1p interact, the purification pulled out both of these eisosome proteins. Subsequent separation using 1D gels distinguished the two proteins due to the additional molecular mass imparted to Lsp1p by the tag. Both proteins were then excised, digested in-gel with trypsin, and analyzed via LC-MS/MS using a Q-Star XL mass spectrometer. A total of six unique phosphopeptides were identified: five in Pil1p and one in Lsp1p. The tag was then swapped onto Pil1p in order to identify any additional sites on Lsp1p that may have been suppressed due to the presence of the tag. The findings presented here represent a significant step towards our ultimate goal of monitoring changes in phosphoproteins in response to progesterone activation.

Mutations in the human Huntingtin gene cause the neurological disorder Huntington’s disease (HD). Specifically, the expansion of a trinucleotide repeat (CAG) in excess of 39 leads to a disease state in individuals. Therefore, determining the number of repeats in subjects is important, since the number of repeats has an effect on the severity of the pathology and the number of repeats shows a high degree of instability between generations. The present study utilizes fragment analysis and DNA sequencing to confirm the exact number of repeats present in exon 1 of the human Huntingtin gene in the transgenic mice used in a drug study. Since genotyping with an automated capillary electrophoresis instrument can be very precise, but not necessarily accurate as to the exact number of bases in the fragment, some of the PCR products were also sequenced. By the use of a 3730 DNA Analyzer, the actual fragment sizes as determined by DNA sequencing were found to be in close agreement with the predicted sizes based upon the fragment analysis—for example, 506 and 501 bases, respectively. The use of a mixture of sequencing kits, BigDye Terminator v3.1 and dGTP BigDye Terminator v3.0, without additives was found to be the best method to sequence over 100 trinucleotide repeats.

Phosphorylation is one of the most common of the reversible post-translational modifications, but also one of the most challenging. During the last few years, phosphopeptide analysis has benefited from the rapid improvement in biological mass spectrometry, but also from innovations such as immobilized metal affinity chromatography, the development of antibodies specific for phosphorylated amino acids, and most recently, chromatography using TiO2. During our analysis of the saliva phosphoproteome, we were concerned that there is a bias against monophosphorylated peptides when using TiO2 columns. We therefore decided to explore alternatives that rely on fractionation rather than affinity purification. Using the approach of Roepstorff and colleagues of packing columns in pipette tips to make disposable microcolumns, and combining strong cationic exchange (SCX) together with hydrophilic interaction liquid chromatography (HILIC), we have been able to fractionate peptides from digests of total saliva, and isolate phosphopeptides in a few fractions with a minimum of non-phosphorylated peptides. In preliminary experiments using about 6 μg of saliva, we have been able to map out phosphorylation sites from proteins such as the salivary acid proline-rich phosphoprotein 1/2, the salivary α-amylase, and statherin. A comparison of isolation of phosphopeptides using TiO2 vs. a combination of SCX and HILIC will be presented.

Protein phophorylation is a widespread post-translational modification that plays a crucial role in many cellular processes. However the analysis of phosphopeptides and reproducibility of methods is not easy because of many factors, among which relatively low abundance makes it a challenging analytical process. We present an automated method for the phophopeptide analysis using an immobilized metal affinity chromatography (IMAC) column to enrich phosphopeptides and subsequent reverse-phase HPLC analysis. The recently developed IMAC is an analytical HPLC column that can be used repeatedly and reproducibly. By using a dual pump and automatic sample injections, the entire phophopeptide analysis is automated and is completed in about an hour. We used tryptic digestions of beta-casein as the model to evaluate nonspecific binding of some nonphosphorylated and all phosphorylated peptides to the IMAC column under the loading conditions (20 mmol/L formic acid). Under the release conditions at pH 9 with ammonium formate, all the expected phosphopeptides are released and recovered quantitatively. The data are presented to show the specificity, recovery, and reproducibility of the method.

The complex methodology used in many large-scale quantitative proteomics experiments often dictates an experimental design with small sample size and limited repetition. Variation in a complex dataset may hopefully arise from changes caused by the experimental perturbation, but could also arise due to technical noise (poor sample prep, run-to-run variation) and biological noise (normal differences between samples, especially present in clinical samples). Here, we apply principle component analysis (PCA) and unsupervised hierarchical clustering (HC) to the data generated from multi-variable difference gel electrophoresis (DIGE) experiments. This global perspective on multivariable datasets assesses whether the variation in the system describes the biological signal, rather than being derived from technical/biological noise whereby “significant” changes may arise stochastically. Although we use DIGE datasets as examples (due to the low technical noise), these issues are germane to all proteomics experimental platforms. Examples will be shown from experiments containing samples from microorganisms, tissue culture, and clinical samples, where these tools were instrumental in demonstrating sample outliers, fouled samples, as well as variation in sample preparation that overrides the variation from biological treatment (despite standard biological tests for sample validity). Experiments contained high-resolution datasets from multiple variables, with hundreds to thousands of protein forms monitored within each sample. Even with simple, two-condition experiments (e.g., WT vs. KO), these tests provide essential quality assurance and quality control. One set of experiments focuses on familial pulmonary arterial hypertension associated with a mutation in the bone morphogenetic protein receptor 2. Affected individuals carrying the mutation, familial obligates (carrying the mutation but asymptomatic), and a control set (married into the family) are all inter-compared. PCA and HC were used to assess the efficacy of drug treatment and the effect of inter-personal variation among the normal samples.

A gel-based method for a mass spectrometric (MS) site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling was performed using matrix-assisted laser desorption/ionization MS and MS/MS. The method proved to be fast and sensitive, and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant protein, both comprising N-glycans of a highly heterogeneous nature. To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low-abundant (50–80 ng/mL) N-glycosylated plasma protein was purified in an individual-specific manner from five healthy individuals using IgG depletion and immuno-affinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and tri-antennary complex oligosaccharides. Additionally, it was shown that platelet-derived TIMP-1 displayed a similar glycoprofile. This is the first study to investigate the glycosylation of naturally occurring human TIMP-1 and the high similarity of the glycoprofiles showed that individual-specific glycosylation variations of TIMP-1 are minimal. In addition, the results showed that TIMP-1 derived from platelets and plasma is similarly glycosylated. This comprehensive and rapid glycoprofiling of a low-abundant glyco-protein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus lead to early detection and monitoring of diseases.

Here, we report the isolation and identification of a number of low-molecular-weight proteins (<10 kDa) from pancreatic β-cell granules. To date, most reported studies have employed two-dimensional electrophoresis to separate insulin granule proteins. However, standard 2DE is unable to resolve proteins of molecular mass <10 kDa, and we designed our current proteomic analysis to target the pool of small regulatory peptides contained within the β-cell granule. We used a single-step method to enrich granules for analysis by LC-MALDI and LC-MS, with subsequent protein sequencing. Our results indicate the β-cell granule to be a rich source of peptides, obtained proteolytically from larger precursor molecules. We fully describe two novel peptides, both likely derived from intragranular proteolytic processing of pro-IGF-II. It is commonly accepted that intracellular proteolysis occurs in neuroendocrine and endocrine cells, liberating physiologically active secretory peptides from larger precursors. Processing begins co-translationally at the rough endoplasmic reticulum, where the signal peptide is removed from the prepro-hormone by a signal peptidase. The pro-hormone is then routed through the Golgi apparatus before being co-packaged with proteases into secretory vesicles destined either for the constitutive or regulatory pathway. The majority of the endoproteolytic processing is thought to occur in these secretory vesicles, with controlled proteolysis at the C-terminal side of dibasic amino acids (usually RR or KR), as well as at selected mono-basic residues (both of which are also recognized by trypsin), and more specific proteolytic motifs. Our methods allowed us to obtain information about the intact molecules (including proteolytic PTMs) that would otherwise be lost using proteomic methods that utilize tryptic digestion (e.g., MuDPIT or PMF).

The evolution of proteomics-based technologies has led to the development of powerful analytical methods for the analysis of post-translational modifications such as phosphorylation. Chemical derivatization strategies that involve beta-elimination and Michael addition chemistries as well as chromatographic enrichment approaches using immobilized metal affinity chromatography have been successfully applied to global phosphoproteome analysis. Recently, enrichment of phosphorylated peptides using TiO2 prior to mass spectrometric analysis has been shown to be a selective and robust method for phosphoprotein characterization. We present an automated workflow using TiO2 microcolumns for the enrichment of phosphorylated peptides derived from in-gel digested proteins in combination with the Investigator Proteomic System (Genomics Solutions, Ann Arbor, MI). A pool of beta casein and ovalbumin (10 μg each) was used as a standard to test the automated phosphoprotein analysis workflow. This same workflow was then successfully applied to characterize in vitro and in vivo phosphorylation events of selected 14-3-3 proteins derived from Arabidopsis thaliana. Samples were separated by either 1D or 2D SDS-PAGE, stained with Pro-Q Diamond (phosphoprotein-specific) fluorescent stain, and imaged using either a Typhoon 9400 scanner or the Investigator ProPic (Genomic Solutions). Putative phosphoprotein-containing spots were then excised and digested in-gel with trypsin using the Investigator ProPic and ProGest, respectively. Tryptic digests from each protein spot were processed with the ProMS workstation using TiO2 microcolumns (Glygen Corp., Columbia, MD) for phosphopeptide enrichment prior to MALDI-TOF/TOF analysis (ABI 4700 Proteomics Analyzer).

A pilot protein profiling of one of the smallest organism ureaplasma urealyticum, a pathogen causing urogenital and placental infection/inflammation, using the new protein fractionation system of Beckmen Coulter’s ProteomeLab PF 2D is reported here. ProteomeLab PF 2D is a two-dimensional liquid chromatographic system to fractionate complex proteomes. This system consists of a chromatofocusing column (250 × 2.1 mm) to separate the protein based on charge/pI and a nonporous reversed-phase column (4.6 × 33 mm) to separate the protein based on hydrophobicity. The advantages of this technique include high loading capacity and better detection of hydrophobic and extreme PI and molecular weight proteins. Up to 1000 protein fractions are collected. So far, 30 proteins such as GroEL, heat shock protein, protein L7/L12, and others have been identified using MALDI-TOF or FT-ICR MS with Sequest or Mascot search engines. This study has demonstrate that ProteomeLab PF 2D with mass spectrometry and some precautions is a potentially powerful tool to identify proteins from a large proteome.

Mass spectrometry has established itself as the primary technique for identifying proteins due to its unparalleled speed, sensitivity, and specificity. Strategies for digestion of the proteins use a specific protease that cleaves at predictable residues along the peptide backbone, providing smaller stretches of peptide sequence more amenable to mass spectrometry analysis. When coupled with protein-level pre-fractionation strategies, such as one-dimensional PAGE, thus reducing the complexity of the protein mixture, this approach has proven highly successful in comprehensive protein identification and characterization. The downside of this approach is the number of gel samples, or fractions, to be analyzed by the LC-MS/MS system. With typical analytical HPLC run times of 45 min to 1 h, the amount of time required to analyze one top-level sample can be prohibitive. Here we describe the use of elevated flow rates combined with nanoscale columns packed with sub-2-μm particles for rapid separations using a nanoUPLC system. Increasing the flow rate to 900 nL/min and running a very rapid gradient over 8 min on a 75 μm × 15 cm column, allows high-quality peptide separations to be achieved with a sample-to-sample inject time of 10 min. This, combined with an orthogonal acceleration time-of-flight mass spectrometer, using a newly developed high-speed data-dependent MS/ MS approach fragmenting up to eight precursor ions per second, allows for the rapid characterization of simple protein mixtures, such as those obtained from 1D gel bands. We will present data from standard tryptic digests of known proteins and simple mixtures of protein digests used in the development of this method, and data from in-gel digests of 1D gel bands.

Although high-density whole-genome SNP scans are available for association studies, the tagging SNP approach used to design many of these panels from International HapMap Project data may miss a substantial number of coding functional variations of drug metabolism enzymes (DME). In fact, more than 40 DME genes are not covered by the HapMap Project, probably due to the difficulties in assay design for these highly homologous gene families. Additionally, many of these technologies do not provide detection in a high number of known DME genes, leading to further gaps in whole-genome scans. Of the polymorphic putative functional DME variants not typed in Hap-Map, a large proportion is untagged by any combination of HapMap SNPs. Therefore, to correlate phenotypes to putative functional DME variations in pharmacogenomic studies, direct genotyping of these functional SNPs will be necessary. Applied Biosystems has developed a panel of N = 2394 TaqMan Drug Metabolism Genotyping Assays to interrogate putative functional variations in N = 220 DME genes. At the National Cancer Institute’s Core Genotyping Facility, an automated, high-throughput pipeline has been created to genotype these assays on the International HapMap Project population. DNA sample preparation and handling, assay set-up, genotype analysis, and data publishing at SNP500 Cancer Database (http://snp500cancer.nci.nih.gov), have all been automated. Using a series of custom-designed methods on five Beckman Coulter Biomek FXs, a Laboratory Information Management System, and analysis software, >650,000 genotypes have been obtained and analyzed by a single person in about 8 weeks. Using this pipeline, a completion rate of >99% and no Mendelian inheritance errors were observed. Furthermore, the CGF has implemented quality-controlled, automated pipelines for sample receiving, quantification, numerous DNA handling procedures, genotyping, and analysis for all samples and studies processed.

The fungal pathogen Cryptococcus neoformans often causes life-threatening meningitis in immunocompromised individuals. Extracellular proteins are critical to the pathogenesis of cryptococcosis, and both secreted and cell wall-bound proteins contribute to this pathogenicity. Unfortunately, only a fraction of the C. neoformans extracellular proteome has been characterized to date. To further elucidate the extracellular proteome of C. neoformans, we conducted an analysis involving both collection of secreted proteins and enzymatic (proteolytic) released proteins from the outer cell wall. Peptides were proteolyzed from live cells, then purified by offline high pressure liquid chromatography, and subsequently identified by LC-MS/MS. In order to assure only cell wall and secreted proteins were identified, Gray’s Reagent was used to stain cells and confirm membrane integrity. Cells were also treated with Brefeldin A, an inhibitor of protein trafficking through the classical ER/Golgi-dependent export pathway, and compared with untreated cells to assess secretion through this pathway. Interestingly, a number of the proteins identified as cell surface in this study were present in the Brefeldin-treated cells; this extracellular presentation may imply non-classical secretion. Also, a number of the proteins identified as extracellular in this study are generally considered cytosolic; however, cytosolic proteins can be transported to the surface as a means of performing additional functions; these have been referred to as “moonlighting proteins.” Unlike previous studies of either secreted proteins or proteins released from isolated cell walls, the novel use of proteases on whole cells in vivo without disrupting internal compartments allows for a more complete snapshot of the extracellular proteome of C. neoformans. Furthermore, this in vivo strategy may allow rectification of proteins misannotated as intracellular. Finally, the Brefeldin A may give clues as to the route each protein takes to reach the surface of C. neoformans.

The application of 2DE to membrane proteins is hampered by the limited protein solubility. In addition, the gel matrix is a limiting factor in combination with downstream analytical techniques such as mass spectrometry. Free-Flow Electrophoresis (FFE) in the isoelectric focusing (IEF) mode provides a carrier-free alternative to 2DE. IEF-FFE has proven useful for soluble proteins since the methodology is not limited in pH or size range. Various detergents have been applied to handle more hydrophobic proteins under denaturing conditions. However, a prerequisite for IEF-FFE is the maintained solubility of the proteins at their pI. This is not the case for many membrane proteins which have high tendency to precipitate at their respective pI. Here we present Interval Zone FFE (IZ-FFE) as a novel mode of FFE which facilitates separation of charged molecular species including proteins and protein complexes. In contrast to IEF, the separation is carried out at a constant pH relying on the net charges of the proteins. The applied pH may be significantly different from the pI of the proteins to be separated thus maintaining proteins in solution that would otherwise precipitate. Resolution and solubility may be increased further under denaturing conditions using urea and/or thiourea and by including non-ionic or zwitterionic detergents. IZ-FFE was applied to a total protein extract from HeLa cells and compared to IEF-FFE. A significant increase in protein solubility was observed in the IZ-FFE mode. Separations were performed using detergent-free media at high loading rates without observing precipitation. This was in contrast to IEF-FFE where precipitation was observed even when detergents were included in the media. The detergent-free separation media facilitated the coupling to RPLC-MS/MS since no detergent removal was required and the proteins were maintained in solution.

Tyrosine phosphorylation plays a pivotal role in signal transduction, which affects all aspects of cell life. Recently, mass spectrometry (MS) has been a powerful tool for identification of tyrosine phosphorylation. Yet the MS approach is still far from being routine due mainly to low phosphorylation stoichiometry at any given site. The goal of this work was to identify the phosphotyrosine (pY) residues on a human sorting nexin protein using nanoLC coupled with a triple-quadrupole linear ion-trap mass spectrometer. The initial identification was performed at enhanced survey MS (EMS)-driven information-dependent acquisition (IDA) mode. The subsequent validation and further identification of low-stoichiometry pY sites were carried out using both precursor ion scan-triggered IDA and targeted multiple reaction monitoring (MRM)-triggered IDA. Compared to EMS-IDA analysis, the triple quadrupole–based precursor ion scan and MRM-driven IDA analyses provide significantly increased selectivity and sensitivity for detection of pY residues. The immunoprecipitation samples from lysates of two transfected cell lines with Src kinase and ACK2 kinase were separated by SDS gel. The bands of interest (1–2 μg) were subjected to tryptic digestion/extraction. The extracts were split into three fractions for nanoLC-MS/MS operated at EMS-IDA, precursor ion scan IDA, and targeted MRM-IDA. The IDA files were used for database searches, and the spectra for identified pY peptides were manually inspected and validated. The results showed that five pY residues were identified by EMS-IDA and validated by the combined PTM discovery workflow for the Src kinase–catalyzed sample. The estimated stoichiometry of identified pY residues is from 3% to 25%. Furthermore, two pY sites were identified by the PTM discovery approach for the ACK2 kinase–catalyzed sample. The results demonstrate that the triple quadrupole–based precursor ion scan and MRM-driven IDA provide excellent utilities for improved detection of phosphotyrosine residues without additional steps for enrichment of phosphopeptides.

Complex proteomic digests can require a combination of either multi-dimensional biphasic packed beds or longer reverse-phase beds (greater than 15 cm) to achieve adequate separation. Multi-dimensional and extended-length columns are time-consuming and expensive to produce; column failure of one section of the packed bed requires replacement of the entire column. An attractive alternative to a single biphasic or long-bed column is based on efficient column-to-column coupling, as employed with conventional-bore high-performance liquid chromatography. Such connections in nanobore LC are enabled by coupling shorter-bed columns via optically clear high-pressure zero-dead-volume (ZDV) unions. This approach facilitates extended-column configurations and column swap-out during system maintenance. Using two clear ZDV unions to achieve flush connections to column bed termini, two conventional 10-cm-bed nanobore columns were coupled and connected to the bed terminus of a 10-cm nanobore column with integrally fritted tip. Analytical performance of this extended-bed column was then compared with a single 30-cm-bed nanobore column with integrally fritted tip. Chromatographic data from single- and extended-bed configurations were nearly identical, with negligible peak-tailing and resolution loss. The first section of the column is readily removed, replaced, and effectively integrates guard column performance into the analytical column bed.

An important part of proteomics is to study changes in protein levels between samples from different cells or tissues where ideally all proteins present in the sample are monitored. There are two main methods that allow for both global scanning for significantly varying proteins, and targeted profiling of proteins of interest. One is based on 2D gel electrophoresis and image analysis of labeled proteins. The other method is based on LC-MS/MS analysis of either unlabeled peptides or peptides derived from isotopically labeled proteins or peptides. In this study, the non-labeling approach was used, involving DeCyder MS Differential Analysis Software (DeCyder MS), for automated detection and relative quantitation of LC-MS data acquired from trypsin-digested proteins. The study method aimed to identify and quantitate differentially regulated proteins following administration of recombinant human growth hormone (rhGH). Two groups of hypophysectomized male Sprague Dawley rats were daily subcutaneously injected during 8 d with rhGH and saline, respectively. The total soluble protein content of nucleus accumbens was extracted, digested with trypsin, and analyzed by 2D LC-MS/MS. In the first dimension, the peptides were separated by micropreparative cation exchange chromatography using an Ettan LC System. Collected fractions were analyzed in the second dimension using nanoscale RPC on Ettan NanoLC connected to an ion-trap mass spectrometer. Acquired LC-MS data were evaluated using DeCyder MS, resulting in the detection of more than 5000 peptides, of which several were found to be significantly regulated. Several of these peptides could be correlated to proteins involved in synaptic plasticity as a response to growth factors.

Keyhole limpet hemocyanin (KLH) conjugated peptides are routinely used as antigens to generate antibody. In the traditional method, peptides are dissolved in phosphate-buffer saline (PBS) and then mixed with 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) conjugated KLH to make KLH conjugated peptides. If the peptide is not soluble in PBS, it can be dissolved in 6 M guanidine-HCl/0.01 M phosphate buffer, pH 7.0. However, there are peptides which are insoluble in both buffers. We present here a procedure of making the KLH conjugated peptides using dimethylformamide (DMF) instead of PBS or 6 M guanidine-HCl to increase solubilities of peptides. Additionally, the last desalt step in the traditional method is eliminated. Briefly, 5 mg of peptide is dissolved in 100 μL of DMF. This solution is added to 1 ml of purified KLH-MBS slowly. The pH of the solution is adjusted to ~7.0 immediately by adding either 2 N NaOH or 0.5 N HCl. The solution is then stirred or rotated for 3 h or overnight at 4°C. Many peptides which do not dissolve in 6 M guanidine-HCl will be soluble in DMF. Some peptide solutions may give cloudy or gelly appearance, nevertheless, peptides do react with KLH to some extend and solutions will become clear. The reaction can be kept longer for such peptides. Also, some peptide-KLHs do form precipitates which can be left in the final solution. Finally, 2 ml of 0.1 M ammonium bicarbonate is added to the peptide/KLH solution, and the solution is lyophilized. Compare to the traditional method, our method is faster and peptides with solubility problems in PBS or 6 M guanidine-HCl can be used.

Prion diseases are a class of neurodegenerative diseases in which conversion of a normal cellular glycoprotein, the prion protein (PrPc), to a misfolded form (PrPSc) generates a protein that is believed to be the sole component of the transmissible agent of the diseases. It is the accumulation of PrPSc in the CNS that causes neurodegeneration and gliosis, which are characteristic of the diseases. Conformational changes in PrPc that lead to PrPSc cause the protein to become proteinase resistant and to form fibrils. PrPc is a glycoprotein expressed by many cells, including neurones and muscle cells. However, it is predominantly expressed at synapses, suggesting that, functionally, PrPc is important for neuronal activity. The fusion glycoprotein was expressed in CHO cells and purified with protein G beads. The aim of this study has been to characterize the N-linked oligosaccharides on the prion protein. The released and permethylated N-linked oligosaccharides were analyzed by both MALDI-MS and ESI-MS. The structure of N-linked glycans was confirmed by MS/MS analysis of permethylated glycans. The N-linked profiling experiment produced a complicated set of data with more than twenty different oligosaccharides that are present on this glycoprotein. The main structures were the fucosylated biantennary structure, with one galactose, and the fucosylated biantennary structure, with two galactose residues. Larger triantennary and tetra-antennary structures were also detected. The structure of different forms of prion proteins will be investigated.

Depletion of high abundance proteins from human plasma and serum has become a valuable tool in the search for protein biomarkers. However, some speculate the existence of potential biomarkers associated with the depleted proteins. For example, human plasma albumin is a well-known carrier of proteins and peptides. Previous studies have identified a limited set of non-specifically bound proteins associated with proteins removed from serum and plasma, but the dynamic range of the depleted fraction may affect its accuracy and content. Therefore, an exhaustive analysis of the depleted proteins is important to address this challenge. Different elution methods and gradients will be evaluated to selectively elute non-specifically bound proteins and peptides from the depletion resin without co-eluting the twenty, targeted proteins. The identifications from this analysis should produce a more complete set of non-specifically bound proteins and peptides.

Enterovirus 71 (EV71) is one of the 69 enteroviruses belonging to the picornavirus family. We have previously generated a recombinant baculovirus encoding P1 and 3CD genes for co-expressing P1 and 3CD proteins for VLP assembly in insect cells. In this study, we further characterized the VLP after ultracentrifugation purification, and evaluated the immunological properties of VLPs in Balb/c mice. The immunization experiments show that the VLP induced stronger anti-VP1 antibody titer and neutralization titers compared to the inactivated EV71 virus and lysed VLP. The antibodies induced by EV71 VLP were also capable of neutralizing live EV71 of different strains. Moreover, the VLP was able to induce cellular immunities as judged by helper T cell proliferation assay and cytokine assay. These data collectively confirmed that EV71 VLP produced from baculovirus/insect cells is able to elicit humoral and cellular immunities in mice, implicating its potentials as a vaccine against EV71 infection.

Interest in tyrosine phosphorylation patterns has been sparked since increases in phosphotyrosine have been associated with malignancy and tumor formation. We have employed several methods to investigate the phosphotyrosine profile of Src-transformed cells, including an extensive study using the methods published by Rush et al.1 In order to streamline the Rush et al. protocol, we have generated peptides by running the lysate approximately 1.5 cm into a 10% bis-tris polyacrylamide gel (with reduction and alkylation prior to the gel) followed by in-gel digestion. Phosphotyrosine-containing peptides from tryptic digestions were enriched using either the pY100 antibody or the 4G10 antibody. Phosphotyrosine-containing peptides were eluted off the beads as described by the Rush et al. protocol or with the addition of an acetonitrile elution of the beads. Samples were analyzed on a Thermo LTQ by reverse-phase chromatography using data-dependent analysis. The results were searched using the Sequest algorithm and the mouse subset of the Uniref100 database with concatenated reverse database searching for false-positive estimation. Results were filtered in a two-step process based on multiple Sequest scores, with increased stringency for novel sites. Results from multiple biological replicates were compared with those that we obtained using the Rush et al. protocol, and they produced comparable results. Peptides accepted from multiple replicates resulted in a false-positive rate of less than 3%. The addition of an acetonitrile wash increased the elution of peptides from the beads and increased identifications. This procedure saved 1–2 d for sample preparation with equivalent or improved results. We are currently exploring digestion of whole-cell lysates in trifluoroethanol to further streamline this protocol, and the use of the LTQ-Orbitrap to more confidently identify the sites.

The objective of this study was to elucidate the role that Fanconi anemia complementation group A protein (FANCA) plays in cells upon DNA damage through identification of DNA damage dependent protein interactions with FANCA using the Tandem Affinity Purification (TAP) methodology. Fanconi anemia (FA) is a rare autosomal recessive disease that is characterized by aplastic anemia, congenital abnormalities, birth defects and increased susceptibility to cancer. FA patients are assigned to one of twelve complementation groups, with about 65% of patients falling into complementation group A. FA patients share the common phenotypic feature of FA of bone marrow failure, and cells from FA patient’s exhibit sensitivity to DNA crosslinking agents such as Mitomycin C (MMC) and Diepoxybutane. TAP was used in order to purify the protein complexes twice, and to maintain protein interactions based on post translational modifications. The TAP FANCA construct contains two tags, a streptavidin binding peptide (SBP) and a calmodulin binding peptide (CBP) which allow for the two-step purification. Human embryonic kidney cells (HEK 293) were transiently transfected with either a negative control or the TAP FANCA construct. Half of the FANCA transfected HEK 293 plates were treated with MMC twenty-four hours post transfection to induce DNA damage. The transfected cells were collected forty-eight hours post transfection, and the FANCA interacting proteins were TAP purified and identified using Mass Spectrometry. MMC treatment induced a novel interaction between FANCA and Huntingtin (HTT) that was not found in the untreated FANCA or negative control samples. The DNA damaged induced FANCA/HTT interaction was confirmed through western blot. The role of the FANCA/HTT interaction in DNA damage is not known. More studies are underway to determine if this interaction is important for DNA damage recognition or repair.

Folate deficiency (FD) alters hepatic methionine metabolism and is associated with increased hepatocellular apoptosis. Additionally, mice deprived of folate showed increased oxidative damage in brain tissue leading to cognitive impairment. Most previous studies have focused independently on either liver, the main tissue of folate storage and metabolism, or brain, where folate regulates neurogenesis and programs cell death. The aim of this study was to apply a powerful and rapid proteomics approach to understand potential subcellular correlations of folate deficiency in both brain and liver of the same rat. This approach combined a new density based sample fractionation technology (Edge Technology) with other conventional proteomics techniques, such as western blot analysis, 2DE and mass spectrometry. The brain and the liver from individual rats, fed normal or FD diets for 6 weeks, were homogenized and then fractionated using the Edge 200 Separation System. Subsequently, all fractions from brain and liver, from control and treated rats, were analyzed by western blot using two markers of oxidative stress: glutathione peroxidase 1 (GPx1) and Glucose-Regulated Protein 75 (GRP75). Certain fractions were selected based on western blot analysis and were further analyzed by 2DE. Protein spots of interest were identified by MALDI-TOF/TOF. The results demonstrated that Edge technology provides a powerful density based separation and enrichment method for rapid screening of potential FD markers and their possible correlations to both liver and brain diseases.

Chemically induced β-elimination of phosphate from serine and threonine coupled with Michael addition has emerged as a chemical strategy to address both the ion suppression and the gas-phase lability of the phosphate group. In previous work, we have adopted the chemistry to solid-phase derivatization on C18 ZipTip pipette tips using barium hydroxide as elimination base and 2-amino-ethanethiol as nucleophile.1 The utility of the protocol for improved phosphopeptide detection by signal enhancement was demonstrated with low-level amounts of tryptic protein digests, and the resultant increased MS/MS spectral information content greatly facilitated mapping of the site of phosphorylation. In this report, we have focused on chemistry optimization of O-phospho and O-GlcNAc modified peptides reported as resistant to β-elimination, i.e., those containing the modified residues followed by proline. Conclusive mapping of these phosphorylation sites has become increasingly important in view of the fact that phosphorylated Ser/Thr-Pro motifs are substrates for prolyl cis/trans isomerase Pin1.2 Similarly, unambiguous site determination of O-GlcNAc modifications has more recently attracted considerable interest because of the global and often site-specific reciprocal relationship between O-GlcNAc and O-phosphate in many cellular responses.3 We have used a panel of model peptides to define the optimal reaction conditions for both concurrent and consecutive elimination/Michael addition reactions and employed carbon/C18 mixed-phase ZipTips to afford efficient binding of small hydrophilic peptides.

Analysis of complex proteomes often results in long protein lists, but falls short in measuring the validity of identification and quantification results on a greater number of proteins. Biological and technical replicates are mandatory, as is the combination of the MS data from various workflows (gels, 1D-LC, 2D-LC), instruments (TOF/TOF, trap, qTOF or FTMS), and search engines. We describe a database-driven study that combines two workflows, two mass spectrometers, and four search engines with protein identification following a decoy database strategy. The sample was a tryptically digested lysate (10,000 cells) of a human colorectal cancer cell line. Data from two LC-MALDI-TOF/TOF runs and a 2D-LC-ESI-trap run using capillary and nano-LC columns were submitted to the proteomics software platform ProteinScape. The combined MALDI data and the ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen), and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined maximum false-positive rate of 5%. ProteinScape combined the data to one LC-MALDI and one LC-ESI dataset. The initial separate searches from the two combined datasets generated eight independent peptide lists. These were compiled into an integrated protein list using the ProteinExtractor algorithm. An initial evaluation of the generated data led to the identification of approximately 1200 proteins. Result integration on a peptide level allowed discrimination of protein isoforms that would not have been possible with a mere combination of protein lists.

Asthma is a complex genetic disease that is under the influence of many genes, and it has been proposed that different genes act in different families and different individuals. The aim of the present study was to investigate at the genomic level the genes that may participate in the causation and origins of asthma using microarray technology. A better understanding of these processes has the potential to identify markers of disease and new therapeutic targets. Seven atopic asthmatics and five non-asthmatics underwent bronchoscopy to obtain bronchial biopsies. Total RNA was purified from bronchial biopsies, and biotynilated complementary RNA was prepared and hybridized to Affymetrix Hum 133 two plus chips (Affymetrix, Santa Clara CA). Using the multi-chip average procedure, we compared the mRNA expression profiles of 47,000 genes and/or EST sequences of atopic asthmatic with normal controls. Four hundred eighty genes had twofold mean regulation expression differences or greater. Three hundred fifty-two genes were upregulated, including surface molecules involved in T- cell and B-cell activation, cytokines, extracellular matrix proteins, intracellular signaling products, and transcription factors. In conclusion, atopic asthmatics show a number of activated proinflamatory pathways that participate in T- and B-cell activation and airway remodeling. These differentially expressed genes identify potential molecular targets for preventive and therapeutic options in asthma.

Melanoma causes the highest rate of skin cancer related fatalities. If detected and treated early, melanoma is mostly curable. Unfortunately, once metastasis has occurred melanoma is often fatal. The biochemical pathways involved in the development from primary to metastatic melanoma is an area under intense investigation. A high-throughput proteomics approach has been applied to better understand the processes that underlie tumor formation and progression. Analyses of global melanoma proteome expression patterns are compared between the primary melanoma cell line WM-115 and the metastatic melanoma cell line WM-266-4, where both cell lines derived from the same patient. Total cell lysates were separated by two dimensional-gel electrophoresis (2DE). After protein differential expression analysis, protein spots of interest were excised, digested with trypsin and analyzed by LC-MS/MS. The mass data were searched against an in-house NCBInr database using the Mascot search engine for protein identification. Comparing the spots between gels, 470 spots were matched for both gels, while 109 spots were up-regulated and 15 spots down-regulated in metastatic melanoma. We identified proteins involved in tumor progression, metabolism, signal transduction, DNA binding, as well as structural and heat shock proteins. This study establishes a step forward in the development of the metstatic melanoma protein database, the understanding of the chemical pathways that lead from primary to metastatic melanoma, and identifying new targets for inhibitor development.

Conventional methodology of protein mass spectrometry using collision induced dissociation (CID) has been widely adopted for confident identification and characterization of proteins in recent years. However, CID-based protein analysis works best with relatively short peptides and many important post translational modifications are not preserved during CID-based analyses. A new fragmentation method, electron transfer dissociation (ETD), has been developed recently and introduced to linear ion trap mass spectrometer. In ETD, peptide backbone is fragmented along pathways that are analogous to those observed in ECD. ETD has been reported to not only favor multiply charged, relatively large peptides, but also preserve post translational modifications. Thus, ETD is drawing more and more attention as mass spectrometry based methods for protein identification and analysis of post-translational modifications. However, current experimental strategies, which perfectly match CID-based analysis, are not optimized for ETD. This lack of efficient experimental strategies that enhance ETD-based analysis could significantly limit the benefits which ETD can provide for the analysis of proteins. The purpose of this study is to optimize experimental strategies for the comprehensive analysis of the ABRF sPRG Protein Standard and to compare sensitivity of CID and ETD activation methods using various concentrations of this standard. Protease digested ABRF sPRG 49 protein standard mixture was analyzed on a Finnigan LTQXL with ETD employing the combination of CID and ETD activation types. Comparison of data generated by ETD and CID indicates complementarity of amino acid sequence coverages in the resultant datasets. Many peptides only show strong fragmentation patterns by either one or the other method. However, improved protein sequence coverage was enabled by alternating between CID and ETD activation types. Increased protein sequence coverage not only allowed a more thorough characterization of the 49 proteins in the standard, but also reduced the number of false positive protein hits.

Quantitative LC-MS/MS analyses of human plasma are predominantly challenged by the wide dynamic range of proteins and extensive biological sample variation, which make systematic detection and quantitation of low-abundant proteins for biomarker discovery quite difficult. In a proof-of-concept study, plasma samples were processed by alternative multi-dimensional workflows that first utilized immunodepletion of twenty abundant plasma proteins (ProteoPrep 20, Sigma). Multiple recombinant proteins were spiked into depleted plasma samples at varying levels to determine detection limits of subsequent SDS-PAGE + LC-MS/MS vs. LC-MS/MS only. After SDS-PAGE, entire lanes were cut into uniform slices, and each slice was digested with trypsin. Digests were subsequently analyzed by nanocapillary reverse-phase chromatography directly coupled to an ESI-LTQ-FT mass spectrometer (Thermo) operating in data-dependent mode. In addition, aliquots of each sample were either digested in solution or as single in-gel digests after brief electrophoresis in an SDS gel, to evaluate advantages and complications of multi-fraction vs. single-fraction LC-MS/MS analysis of a proteome. Resultant LC-MS/MS data were analyzed in a blinded fashion using the Elucidator protein expression data-analysis system. Using the PeakTeller algorithm and statistical analysis tools, we were able to effectively organize the data to distinguish controls from spiked samples and estimate the relative abundances of spiked-in proteins in different samples. Detection limits, protein coverage, and reproducibility of the alternative workflows were compared. This study demonstrates that reproducible relative quantitation can be achieved in complex mixtures such as plasma, using a combination of abundant protein depletion followed by different workflow approaches and data analysis using the Elucidator system.

CD4+ T helper cells are the key player for control and regulation of adaptive immune responses. In a healthy immune system they protect the body against a variety of pathogens but CD4+ T cells are also involved in pathogenesis and perpetuation of diverse autoimmune diseases like chronic rheumatic inflammation. Consequently, analysis of T cell activation has the potential to assist the development T cell-directed therapies and diagnostic approaches. Membrane proteins are of specific interest because they represent 60% of known drug targets and have also the potential to serve as diagnostic markers. The membrane proteomes of a well characterized set of freshly isolated and CD3/CD28-stimulated human CD4+ T cells were analyzed by high resolution 2D-IEF-SDS-PAGE. Membrane proteins were enriched by differential centrifugation and following Triton-X114 extraction. Reproducibility of this isolation method for membrane proteins and 2D PAGE was proven by comparison of proteomes of resting and activated CD4+ T cell samples derived from individual human donors. Using differential centrifugation and detergent extraction protein spots were displayed in proteomes that were not detectable or underrepresented in 2D gels of whole cellular lysates without any fractionation. Furthermore, for quality control of the procedure a selection of well known surface antigens was quantitatively measured by FACS analysis prior to electrophoresis. These antigens were stained by large 2D-PAGE western blot of the separated T cell proteins identified by mass spectrometry. They were assigned to the corresponding 2D gel maps for quantitative spot analysis. Recovery as well as expression ratios of surface antigens between resting and activated T cells were compared.

P. aeruginosa is an opportunistic human pathogen and a major cause of death in patients with cystic fibrosis. Recently, an elaborate regulatory network has been discovered that regulates transition between two different virulent modes of the bacterium, one that causes an acute and another that causes chronic infection. In response to unknown environmental signals this network modulates activity of a single protein, RsmA. RsmA is a translational regulator and an RNA binding protein. The objective of this work is to identify direct mRNA targets of RsmA and to elucidate the mechanisms underlying RsmA-mediated regulation. Using a data-independent, label-free LC-MS method, protein expression patterns were monitored in wt, rsmA mutant, and rsmYZ double mutant (analogous to a strain overexpressing RsmA) cultures of P. aeruginosa. Separate analyses were carried out for the cell-associated and secreted proteins. LC-MS data were acquired using a nano-ACQUITY UPLC system and a QTof Premier mass spectrometer operating in alternate scanning mode. In parallel, pull down experiments were carried out in which RNA species that co-purified with His-tagged RsmA were isolated, reverse transcribed, cloned and sequenced. Comparison of the proteomics results and the pull down data revealed that majority of the proteins that were differentially expressed in the proteomics study were also identified among the pulled-down RNA species, including mRNAs encoding major virulence factors. These results suggest that RsmA regulates all of the differentially regulated genes directly by binding their cognate mRNAs. In addition, comparison of the proteomics and the pull down data showed that RsmA binds mRNAs of genes that are regulated either positively or negatively by RsmA, suggesting at least two different mechanisms for RsmA-mediated translational regulation.

Biomarker discovery and application in toxicological studies requires rapid and accurate methods to assess environmental risk. The emerging field of proteomics now includes powerful analytical tools involving mass spectrometry that can be utilized in toxicological studies. We are developing a focused proteomic technique using ESI- MS/MS to quantify the abundance of estrogen receptor (ER-alpha, ER-beta a, and ER-beta b) proteins in tissues of largemouth bass (LMB; Micropterus salmoides), a popular game fish that is used as a toxicological model. LMB ER sequences were cloned into expression vectors and recombinant proteins expressed for all three ER isoforms. Recombinant LMB ER proteins were purified to use as standards. In silico trypsin digestions using ProteinProspector and MS-Digest of each LMB ER isoform were compared to identify peptides unique to each LMB ER. The peptide 406-LIFAQDLILDR-418 distinguishes LMB ER-alpha from the other two LMB ER isoforms, and analysis with purified LMB ER-alpha digests shows that this peptide ionizes consistently. For quantification, this peptide was synthesized with heavy isotopes (2nd leucine) to produce a peptide with a mass of 1322.6 Da, a difference of approximately 6 Da when compared to the endogenous natural peptide mass of 1316.58 Da. A standard curve using the heavy isotope ER-alpha peptide showed linearity over five orders of magnitude and is detectable in the attomole range. Complex protein mixtures from the liver, gonad, and brain will be isolated using either whole-tissue protein or nuclear protein fractions for optimal compatibility with MS and ER peptide identification. In addition, Westerns blots are being developed for all three LMB ERs in parallel to determine the most sensitive method of ER detection. These techniques are powerful toxicological tools to rapidly identify changes in biomarker proteins such as ER isoforms.

Proteomics laboratories and proteomics service facilities produce a large volume of data that originate from diverse sources, involve the participation of several members of the lab, and are communicated to clients and collaborators of the laboratory. Laboratory information management systems (LIMS) play a key role in collating and tracking the flow of data through the laboratory. The cost of off-the-shelf LIMS systems can be significant and compounded by further customization, development, and installation costs. Fields that employ rapidly changing technologies in support of research require information management that is stringent and secure, but also must be adaptable. We have developed an open-source scientific data management system for proteomics that is capable of acting as a stand-alone LIMS system, or working in conjunction with other LIMS systems. We support sample processing workflow, protocols, and datasets generated by LC-ESI, LC-MALDI, MALDI, 1D-GELS, 2D-GELS, and 2D-gel image analysis. The workflow supports input from existing data analysis and allows batch analysis of proteomics data to be queued to search engines and other tools. It supports extensive data curation, has a simple Web browser interface that allows tiered, secure access to data and functions, and also allows export of data from individual spectra up to full project files. The source code is available from Proteomecommons.org. Installation of the software can be performed through https://www.prime-sdms.org/main.htm.

RNA quality is directly correlated to the success of various applications, such as microarray or real time qPCR-based gene expression analyses, cDNA library construction, Northern analyses, and RNAse protection assays, which utilize RNA samples from various organisms, tissues, cell lines, and precious biological samples. We present a novel method for examining RNA integrity and purity using capillary electrophoresis that is more cost-effective and scalable than current standard methods. This highly sensitive method uses less material, uncovers impurities following RNA purification, and detects degradation resulting from nuclease contamination. Total RNA and cRNA, derived from various tissues and cell lines, were stained with the dye YOYO-1 and run through a custom polymer formulation on a capillary electrophoresis platform. Using downstream analysis software, we resolved RNA species and their relative quality based on parameters such as size, profile, peak area, and peak height. Our results highlight the potential for high-throughput capillary electrophoresis as a much more discriminatory and cost-saving method in evaluating RNA quality.

The objective is to develop a simple, real-time method for monitoring protease activity. Proteolytic digestion prior to identification by MS is pivotal to contemporary proteomics. Proteolytic digestions are taken between 1 and 18 h to complete, depending on the protein, enzyme activity and reaction conditions. Ensuring that digestion is stopped at the optimal time is important as failure, partial digestion or over-digestion can waste MS-time and make results difficult to interpret. Monitoring proteolysis is also important in areas ranging from digestibility studies of foods to removal of tags or fusions from recombinant proteins. Standard techniques for monitoring proteolysis (e.g., HPLC, circular dichroism, SDS-PAGE) are slow, may require expensive instrumentation, can be inaccurate and/or are unsuitable for real-time monitoring. Epicocconone yields an intensely red-fluorescent product on reversible binding to lysine, arginine and histidine residues in the hydrophobic environments around proteins. This feature provides a novel approach for staining and quantification of proteins and peptides in solutions, gels, blots, and cells that is compatible with downstream processes such as HPLC, MS, Edman degradation and Western blotting. LavaDigest is a new product that is based on epicocconone which measures the hydrophobicity loss as a decrease in fluorescence as a protein or proteome is digested. This provides a simple, approach to monitoring proteolytic activity. LavaDigest appears suitable for most proteins and proteases tested; it does not interfere with proteolytic activity, is compatible with down-stream proteomic analysis and can be used to derive kinetic parameters of proteolytic activity. LavaDigest has been used to compare the rates of degradation of different proteins, by a variety of proteases under different conditions (pH, surfactants, temperature, etc). The assay is unique in that it can be used to tracelessly follow the activity of proteases on native substrates.

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA) and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)n oligonucleotides using surface plasmon resonance spectroscopy. A single NC protein is able to bind to more than one immobilized oligonucleotide. However, if oligonucleotides are immobilized at low densities (~10RUs), then NC no longer displays this behavior. As NC is proposed to be the nucleic acid binding domain of Gag, we would expect Gag to show the same behavior. To investigate the stoichiometry and cooperativity of Gag binding to oligonucleotides, we must ensure that Gag is unable to bind to two oligonucleotides at once. In addition, we need to know accurately how much oligonucleotide is immobilized so we can calculate the stoichiometry of Gag binding. We developed a method where we can determine precisely the amount of oligonucleotide immobilized at very low-density surfaces (0.1–2 RUs). In this approach, we used electrospray ionization Fourier transform mass spectrometry to determine that two and four molecules of NC bind to d(TG)5 and d(TG)10. We were then able to use NC injections to calibrate the immobilized oligonucleotide and determine an accurate measurement of the surface density. Using this approach, we have measured the binding of Gag to d(TG)n. Gag binds to a 5-mer and a 20-mer with a stoichiometry greater than 1 and 4, respectively. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules bind to the Gag already bound to the oligonucleotide, forming oligo-Gag-Gag complexes.

Human amyloidosis describes a condition where one of some 23 known proteins form non-soluble pathological fibrils in various organs and tissues throughout the body. A common feature of these amyloid deposits is distinctive green birefringence when stained with Congo Red. Staining is performed on formalin fixed paraffin embedded (FFPE) tissue sections of tissue biopsies or on subcutaneous abdominal fat aspirates. This method is combined with clinical, immunohistochemical, and genetic information to diagnose the condition and in some cases, identify the amyloid involved. Definitive identification of the amyloid protein is essential for proper therapies in treating patients. Identification in the cases where the other methods give ambiguous results, involve solubilizing and extracting the amyloid proteins from the fixed sections or from fat aspirates. In an attempt to reduce the processing time of these types of clinical samples and still provide definitive identification, we explored in-situ enzymatic digestion methods that exploit the speed and sensitivity of nanoRPLC-ESI-tandem mass spectrometry. Using previously diagnosed clinical samples, we tested a variety of conditions to perform trypsin digestion directly on laser capture microdisected (LCM) generated samples and on fat aspirate samples with the goal of having a robust method suitable for mass spectrometry based identification. Trypsin digestions were performed using, ammonioum bicarbonate, Tris, Tris EDTA/ ± zwittergent 3–16, with and without solubilization steps of hexafluoroisopropanol, acetonitrile, or DMSO. A commercially available FFPE solubilization kit, Liquid Tissue, was also tested. The efficacies of these treatments are judged by performing Sequest or Mascot searches of LTQ generated MS/MS spectra, noting the presence of peptides from the expected amyloid proteins. This method has been successfully utilized to correctly identify amyloids comprised of transthyretin (TTR), serum amyloid A (SAA), lambda and kappa light chains. Controls taken from regions adjacent to the amyloid plaques were consistently devoid of the amyloid proteins.

Ovarian Cancer is the fifth leading cause of death from all cancers in women. Platinum-based chemotherapy is the primary treatment for ovarian cancer. Most patients with the disease are initially very responsive to chemotherapeutic treatment. However, the majority of ovarian cancer patients eventually relapses and becomes refractory to additional treatment. This drug-resistance is a major impediment to the successful treatment of ovarian cancer. To date the mechanisms of drug-resistance are poorly understood. To elucidate the underlying mechanisms in which drug-resistance is developed in ovarian cancer cells, global protein expression pattern changes in drug-sensitive and -resistant ovarian cancer cells need to be established. In this study, we approached ovarian cancer resistance using proteomics technology to quantitatively profile the global protein expression levels of two pairs of ovarian cancer cell lines, with cisplatin drug treatment. A2780 and 2008 human ovarian cancer cell lines were chosen as cisplatin-sensitive and A2780/CP and 2008/C13*5.25 were their resistant counterpart. Statistical analysis was carried out by ANOVA models and these were fit using Proc_Mixed in SAS. Through our efforts to identify those protein changes associated with cisplatin drug-resistance, we identified and quantified over ~2000 proteins where 855 proteins were identified with high confidence (Priority 1) and 760 of them showed significant expression changes (False Discovery Rate is less than 0.05). Based on the data that we obtained, we were able to select a panel of potential proteins that could play a role in cisplatin drug resistance. Inhibition assays targeting select proteins from our panel were carried out, to determine whether down-regulation of these proteins would be successful in reversing cisplatin resistance. Preliminary data showed a very promising effect in reversing the cisplatin resistance in the resistant cells when the combination of cisplatin and TETA, an inhibitor of antioxidant Superoxide dismutase (SOD1) protein, were used.

In mammals, cytosine methylation at CpG sites is an important epigenetic mechanism for altering gene expression in normal and cancer cells. Sequencing of PCR products from bisulfite-treated genomic DNA provides the most direct measurement of methylation at individual cytosines. To compare the relative ability of pyrosequencing and fluorescent dideoxynucleotide sequencing to accurately quantify cytosine methylation, I designed primers for amplification of a 197-bp region within the IGF2 gene. Two sets of primers were synthesized: (1) a set containing a 5′-biotin on the reverse primer (used for streptavidin purification of the template strand prior to pyrosequencing) and (2) a set containing the M13 forward and M13 reverse primer sequences as 5′ extensions of the forward and reverse PCR primers, respectively. Only the first set was used to generate PCR products for pyrosequencing; both sets were used to make amplicons for fluorescent DNA sequencing. Following PCR amplification of eight bisulfite-treated human genomic DNAs, templates were purified and subjected to both DNA sequencing technologies. Pyrosequencing was able to measure percent methylation at 7 CpG sites, while the fluorescent chemistry was able to interrogate up to 12 sites, due in part to its longer read length. The percent methylation measured by each technology differed for a given CpG site and genomic DNA: the value obtained by pyrosequencing was usually 10–15% less than that obtained by the fluorescent chemistry. In order to determine which chemistry measures percent methylation more accurately, experiments with known ratios of fully methylated and unmethylated templates are underway.

We report a case of a 54-year-old white female with a seven year history of angiolymphoid hyperplasia with eosinophilia. (Figures 1, 2) A proteomic evaluation of the tumor was performed with the intention of identifying targets for therapy. The tissue had high expressions of the following: platelet derived growth factor-alpha(PDGFR-α), platelet derived growth factor-beta (PDGFR-β), c-kit (CD 117), angiotensin converting enzyme, p-mTOR (SER 2448) and p-NF-κB p-p65. The signal transducers PDGFR-α and PDGFR-β are expressed in the cytoplasm of numerous mast cells. c-kit, another signal transducer, is expressed in the plasmalemmal compartment (cell membrane) by an appreciable number of mast cells. The transactivator, ACE, is expressed in the plasmalemmal compartment of the lesional endothelial cells. The downstream effector, p-mTOR is expressed in the nuclear, cytoplasmic and plasmalemmal compartments of the lesional endothelial cells. And, an antiapoptotic/tumorigenic factor, NF-κB p-p65 is expressed in the nuclear and cytoplasmic compartments of endothelial growth cells. Imitanib mesylate (Gleevec) targets c-kit, PDGFR-α and PDGFR-β. ACE inhibitors, such as enalapril, target angiotensin converting enzyme. Sirolimis (Rapamycin) antagonizes p-mTOR whichinhibits mast cells and lymphocytes via reduced IL-5 and vascular endothelial growth factor (VEGF). Bortezomib (Velcade) blocks the activation of NF-κB which interrupts VEGF production and endothelial cell stimulation. FIGURE 1FIGURE 2

Cytokines and chemokines are responsible for regulating inflammation and the immune response. Cytokine and chemokine release is typically quantitated by ELISA or Western blot analysis. In order to expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based ExcelArray antibody sandwich microarrays. Each slide consists of 16 subarrays (wells) printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the “sandwich” ELISA, in which an analyte protein is “sandwiched” between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked DyLight 649 dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. The Jurkat cell line was used as a model for human T cell leukemia and the A549 cell line was used as model for human non-small cell lung cancer in our experiments. In order to evoke a cytokine/chemokine response, cells were stimulated with Tumor Necrosis Factor alpha (TNFα), Phorbol-12-myristate-13-acetate (PMA, TPA) and phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angiogenesis and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-α, and MIP-1α, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell culture supernatants for the profiling of cellular inflammatory mediator release.

We set out to develop an easy-to-use system for in vitro synthesis of recombinant eukaryotic proteins containing posttranslational modifications. A wide range of eukaryotic proteins require such modifications (e.g., phosphorylation, glycosylation, signal peptide cleavage) for correct folding or to display full functional activity. To optimize expression, a number of expression constructs were generated using polymerase chain reaction (PCR). These constructs encoded proteins carrying combinations of C- or N-terminal His- or Strep-affinity tags. Promoters added during the PCR enabled the efficiency of expression and proportion of soluble protein delivered by each construct to be tested directly in small-scale synthesis reactions. Once the makeup of the optimal construct was determined, it was cloned into an expression plasmid for larger-scale syntheses. A range of proteins—including kinases, membrane proteins, and transcription factors— were expressed to test the robustness and applicability of the method. Optimized Spodoptera frugiperda cell lysates, which contain all the cellular machinery required for posttranslational modifications, were used for expression. The presence of post-translational modifications in synthesized proteins was demonstrated using epitope-specific antibodies (phosphoproteins), enzymatic treatment (glycoproteins), and activity assays (luciferase). The ability to use PCR products in small-scale expression screening reactions speeded up the entire process of construct optimization considerably. It was possible to synthesize a wide range of post-translationally modified proteins in high yields (up to 40 μg/mL reaction), significantly higher than those obtained in a comparison with a rabbit-reticulocyte lysate-based system. This system offers a range of benefits for eukaryotic protein expression, including simplified expression screening, a fast procedure, robust and high-yield expression, and no requirement for specialized equipment.

Imaging Mass Spectrometry (IMS) using matrix-assisted laser desorption ionization (MALDI) is being investigated using a linear ion trap with the overall goal of analyzing the distribution of various targeted therapies within patients’ tumors. The drug chosen for evaluation here is erlotinib (Tarceva) which targets the epidermal growth factor receptor (EGFR), a type I receptor tyrosine kinase (TK) involved in cellular differentiation and proliferation, by binding to the ATP pocket and inhibiting the autophosphorylation of the receptor. Tarceva has demonstrated clinical activity in non-small cell lung cancer, head and neck cancer, and ovarian cancer in Phase II studies. The sensitivity and MSn capabilities of the Finnigan LTQ are exploited for the unambiguous determination of the distribution of this drug within human pancreatic tumors. Custom software was used to acquire data while rastering the tissue and data visualization software used to display the 2- and 3-dimensional images. SCID mice bearing patients’ pancreas tumors grown as xenografts were dosed once with 2.5 mg Tarceva in 6% Captisol, or Captisol alone, delivered by oral gavage. Sixteen hours following the administration of treatment or the vehicle, the pancreas tumors were resected, snap frozen immediately, and then 10-μ-thick tissue sections were prepared with a cryotome and placed on various MALDI surfaces. Nebulized matrix was applied with a commercial airbrush. Peaks corresponding to the m/z of both Tarceva and its O-demethylated metabolite, OSI-420, were found distributed in Tarceva-treated tumor, but not in the control sample. Results from single reaction monitoring (SRM) experiments on the drug precursor at m/z 394 and the metabolite at m/z 380 were mapped to visualize the drug distribution within the tissue. The results confirm that the Finnigan LTQ with vMALDI source mass spectrometer can be used to localize Tarceva and its metabolite in a xeno-graft tumor model.

Protein modification by ubiquitin is an important event that represents a highly versatile means for regulation of protein function. Ubiquitination acts at multiple levels to regulate gene expression, including targeting for degradation, proteolytic activation, and intracellular localization. Mapping of protein ubiquitination sites is important because it can facilitate the elucidation of mechanisms of this modification, yielding insights into cell regulation. Recently, mass spectrometry has been employed for determination of ubiquitination sites; however, this protein analysis approach is incomplete and not saturated for any given protein. Existing targeted mass spectrometric approaches can monitor only a few predicted ubiquitinated peptides at any given time, thus requiring multiple analysis and samples. Additionally, proteins are typically enzymatically digested with trypsin. However, ubiquitinated peptides are modified on their lysine residues with the addition of GG motifs, thus generating large tryptic peptides that are often multiply charged. Hence, MS/MS spectra can be complicated and challenging to interpret. Here we employ a unique approach to target and sequence sites of ubiquitination on proteins and their relevant peptides. Ubiquitinated proteins were purified and digested with various enzymes to obtain varying lengths of peptides. Sample analysis was performed using reverse-phase nano-HPLC interfaced to a hybrid quadrupole linear ion trap mass spectrometer. MRM-initiated detection and sequencing workflow was used to monitor 70–100 predicted ubiquitinated peptide masses and their fragment ions in one analysis for simultaneous detection, identification, and relative quantification of the peptides. A number of ubiquitinated peptides were identified by this analysis, and the results will be discussed. This targeted approach has been shown to provide a high-throughput analysis in addition to better specificity and sensitivity for ubiquitination site mapping.

Phosphorylation is an important regulator of cell function in eukaryotes. This post-translational modification can alter protein localization, regulate protein function, and stabilize and mediate their interactions. Due to their associated negative charge, phosphopeptides are often poorly ionized compared to their non-phosphorylated counterparts, and their analysis is often complicated due to their low cellular abundance. Therefore, it is critical to selectively enrich the phosphopeptides prior to MS analysis. In this study, we have evaluated a new way of enriching phosphopeptides by using a metal oxide–based solid-phase extraction (SPE) microscale device where the eluent is analyzed by LC MALDI MS. This sorbent has a high affinity for phosphopeptides, and the problem of acidic peptide adsorption associated with immobilized metal-ion affinity chromatography, the previous technique, is greatly minimized. Furthermore, recent developments in MALDI and LC-MALDI spotting devices allow the coupling of the off-line chromatographic separation step to the subsequent MS analysis. Data will be presented showing the enrichment of phosphopeptides from a digest of a single phosphoprotein, beta casein, analyzed by LC-MALDI MS, where enhanced performance was observed in terms of the selectivity of the phosphopeptides. Further analysis of more complex protein mixtures (six and twelve standard proteins) which contain phosphopeptides will be presented, demonstrating the selectivity achieved using an aromatic carboxylic acid additive.

One of the main goals in proteomics is to extract and collect all the functional information available in existing databases in relation to a defined set of identified proteins. Due to the huge amount of data available, it is not possible to gather up this information by hand; we need to have automatic methods for addressing this task. Protein information and knowledge extractor (PIKE) solves this problem by accessing several public information systems and databases automatically through the Internet and retrieving all functional information available on the different repositories, and then clustering this information according to the pre-selected criteria. The PIKE bioinformatics tool, accessible through http://proteo.cnb.uam.es:8080/pike, uses the Java and XML languages. Starting with a selected group of identified proteins, listed as NCBI nr, uniprot, and/or ipi (http://www.ebi.ac.uk/IPI/IPIhelp.html) accession codes, PIKE retrieves all relevant information stored in databases by choosing the correct pathway and/or the best information source. Once the search is done, a typical PIKE output shows a report table with an entry for each protein containing all extracted information. The report contains a large amount of meaningful protein features, such as (1) function information, (2) sub-cellular location, (3) tissue specificity, (4) links with other repositories, such as Mendelian Inheritance in Man (OMIM) or Kyoto Encyclopaedia of Genes and Genomes (KEGG), and (5) gene ontology tree classification. The table is exportable in CSV and text file formats, and, more important, it is possible to export it in PRIDE XML (http://www.ebi.ac.uk/pride/) format for results integration into the information stored in other applications such as ProteinScape.

DNA footprinting is a powerful tool to study regions of DNA where proteins bind. Traditionally, target DNA fragments are labeled with 32P, incubated with the protein of interest, then subjected to chemical nuclease attack. The DNA probe is then purified and nicked fragments analyzed on sequencing gels to reveal regions of DNA protected by the protein from attack. The traditional method is a time-consuming and difficult protocol requiring specialized skill. Proteins with low dissociation constants require an additional preparatory separation before analysis, which reduces the yield of bound probe. The isotope is labile, involves safety considerations, and requires a separate labeling and purification step. The finished gels must be dried down and exposed. This adds time to the overall process, varies according to the potency of the isotope, and decreases resolution. Normalization and alignment of free and bound probes is difficult. The one-dimensional nature of the single label necessitates separate analysis of each DNA strand, and the analytical gel limits the length of the analytical window. We have developed a novel method of DNA footprinting that utilizes the multi-dimensional nature of fluorophores to label both strands of the DNA target, which permits differential analysis of both strands simultaneously. Labeled primers are commercially available for use as PCR primers, permitting simultaneous labeling and amplification of the DNA probe. The use of the chemical nuclease 1,10-phenathroline-copper facilitates in-gel attack of free and bound probe, which increases the yield of bound probe and reduces perturbation of the three-dimensional conformation of the complex. Samples can be analyzed rapidly by standard automated sequencing instrumentation with baseline, base pair resolution. Software data manipulation reduces normalization issues. The result is high-quality data acquisition in a matter of hours instead of days.

Capabilities for quantitative mass spectrometry–based proteomic profiling of formalin-fixed archival tissue have been developed. Using Liquid Tissue reagents and protocols, we have effectively profiled and validated known and novel cancer biomarkers across a wide variety of fixed cancer tissue samples. Recently developed applications include quantification of protein expression in fixed archival tissue with an emphasis on oral cavity (head and neck squamous cell carcinoma—HNSCC) and breast cancer. Spectral count bioinformatics provides a non-labeling method for quantitative proteomics of formalin-fixed histologically-defined oral cavity cancer. Results indicate many proteins were differentially expressed in cells obtained by laser microdissection of normal, highly differentiated, moderately differentiated, and poorly differentiated HNSCC fixed tissue. Candidate protein biomarkers found to be differentially expressed in the process of HNSCC progression were confirmed and validated by immunohistochemistry on large panels of HNSCC tissue. In addition, we have developed and evaluated a method for direct detection and absolute quantification by selected reaction monitoring (SRM) of HER2 directly in formalin-fixed paraffin-embedded breast cancer tissue using a stable isotope standard peptide derived from HER2. Soluble protein extracts from a collection of breast cancer tissues known to express a range of HER2 were prepared using Liquid Tissue reagents, and quantitative levels were determined. Results demonstrate the ability to quantitate HER2 expression in Liquid Tissue extracts from fixed tissue sections that correlate with standard IHC and indicate the ability to quantify HER2 in immunohistochemical-negative cells. These cumulative results demonstrate development of technologies for quantitative proteomic analysis of proteins that can be applied to the vast worldwide formalin-fixed tissue archives.

Protein Microarrays enable the analysis of multiple binding events in parallel. Based on patented UNIclone technology, we have established a reproducible protein biochip platform to determine specific binding profiles to multiple targets in a quantitative way. The UNIchip protein biochips provide a novel and unique tool for antibody characterization and ranking. Potential off-target activities can be detected by establishing a quantitative binding profile of an antibody to 400 different, unrelated targets. Together with on-chip determination of sensitivity, linearity and dynamic range the quantitative fingerprint enables a unique performance ranking and specificity analysis of antibodies. We have analyzed the performance of three block-buster biotherapeutics directed against TNF-alpha. The biotherapeutics show a clear differentiation both with regard to their binding profile to unrelated proteins as well as their binding affinity to the cognate target on the biochip. The quantitative fingerprint also offers a novel strategy for antibody pair selection in ELISA development.