Proteomic analyses identify major vault protein as a prognostic biomarker for fatal prostate cancer.

The demographic shift toward an older population will increase the number of prostate cancer cases. A challenge in the treatment of prostate cancer is to avoid undertreatment of patients at high risk of progression following curative treatment. These men can benefit from early salvage treatment. An explorative cohort consisting of tissue from 16 patients who underwent radical prostatectomy, and were either alive or had died from prostate cancer within 10 years postsurgery, was analyzed by mass spectrometry analysis. Following proteomic and bioinformatic analyses, major vault protein (MVP) was identified as a putative prognostic biomarker. A publicly available tissue proteomics dataset and a retrospective cohort of 368 prostate cancer patients were used for validation. The prognostic value of the MVP was verified by scoring immunohistochemical staining of a tissue microarray. High level of MVP was associated with more than 4-fold higher risk for death from prostate cancer (hazard ratio = 4.41, 95% confidence interval: 1.45-13.38; P = 0.009) in a Cox proportional hazard models, adjusted for Cancer of the Prostate Risk Assessments Post-surgical (CAPRA-S) score and perineural invasion. Decision curve analyses suggested an improved standardized net benefit, ranging from 0.06 to 0.18, of adding MVP onto CAPRA-S score. This observation was confirmed by receiver operator characteristics curve analyses for the CAPRA-S score versus CAPRA-S and MVP score (area under the curve: 0.58 versus 0.73). From these analyses, one can infer that MVP levels in combination with CAPRA-S score might add onto established risk parameters to identify patients with lethal prostate cancer.

The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense.

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status.IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

EpitoCore: Mining Conserved Epitope Vaccine Candidates in the Core Proteome of Multiple Bacteria Strains.

In reverse vaccinology approaches, complete proteomes of bacteria are submitted to multiple computational prediction steps in order to filter proteins that are possible vaccine candidates. Most available tools perform such analysis only in a single strain, or a very limited number of strains. But the vast amount of genomic data had shown that most bacteria contain pangenomes, i.e., their genomic information contains core, conserved genes, and random accessory genes specific to each strain. Therefore, in reverse vaccinology methods it is of the utmost importance to define core proteins and core epitopes. EpitoCore is a decision-tree pipeline developed to fulfill that need. It provides surfaceome prediction of proteins from related strains, defines core proteins within those, calculate their immunogenicity, predicts epitopes for a given set of MHC alleles defined by the user, and then reports if epitopes are located extracellularly and if they are conserved among the core homologs. Pipeline performance is illustrated by mining peptide vaccine candidates in Mycobacterium avium hominissuis strains. From a total proteome of ~4,800 proteins per strain, EpitoCore predicted 103 highly immunogenic core homologs located at cell surface, many of those related to virulence and drug resistance. Conserved epitopes identified among these homologs allows the users to define sets of peptides with potential to immunize the largest coverage of tested HLA alleles using peptide-based vaccines. Therefore, EpitoCore is able to provide automated identification of conserved epitopes in bacterial pangenomic datasets.

Proteomic and Metabolomic Analysis of Azospirillum brasilensentrC Mutant under High and Low Nitrogen Conditions.

Azospirillum brasilense is a diazotrophic microorganism capable of associating with roots of important grasses and cereals, promoting plant growth and increasing crop yields. Nitrogen levels and the Ntr regulatory system control the nitrogen metabolism in A. brasilense. This system comprises the nitrogen regulatory proteins GlnD, which is capable of adding uridylyl groups to the PII proteins, GlnB (PII-1) and GlnZ (PII-2), under limiting nitrogen levels. Under such conditions, the histidine kinase NtrB (nitrogen regulatory protein B) cannot interact with GlnB and phosphorylate NtrC (nitrogen regulatory protein C). The phosphorylated form of NtrC acts as a transcriptional activator of genes involved in the metabolism of alternative nitrogen sources. Considering the key role of NtrC in nitrogen metabolism in A. brasilense, in this work we evaluated the proteomic and metabolomic profiles of the wild-type FP2 strain and its mutant ntrC grown under high and low nitrogen. Analysis of the integrated data identifies novel NtrC targets, including proteins involved in the response against oxidative stress (i.e., glutathione S-transferase and hydroperoxide resistance protein), underlining the importance of NtrC to bacterial survival under oxidative stress conditions.

A high-throughput pipeline for validation of antibodies.

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry.

Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography-mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa.

Proteome analysis of an Escherichia coli ptsN-null strain under different nitrogen regimes.

The carbohydrate-uptake phosphorelay PTS system plays a key role in metabolic regulation in Bacteria controlling the utilization of secondary carbon sources. Some bacteria, such as Escherichia coli, encode a paralogous system named PTSNtr (nitrogen related PTS). PTSNtr is composed of EINtr (ptsP), NPr (ptsO), and EIIANtr (ptsN). These proteins act as a phosphorelay system from phosphoenolpyruvate to EINtr, NPr and them to EIIANtr. PTSNtr is not involved in carbohydrate uptake and it may be dedicated to performing regulatory functions. The phosphorylation state of EINtr is regulated by allosteric binding of glutamine and 2-oxoglutarate, metabolites whose intracellular levels reflect the nitrogen status. Although PTSNtr is designated as having nitrogen-sensory properties, no major effect of this system on nitrogen regulation has been described in E. coli. Here we show that an E. coli ptsN deletion mutant has impaired growth in minimal medium. Proteome analysis of the ∆ptsN strain under different nitrogen regimes revealed no involvement in regulation of the canonical nitrogen regulatory (Ntr) system. The proteomic data support the conclusion that ptsN is required to balance the activities of the sigma factors RpoS and RpoD in such way that, in the absence of ptsN, RpoS-dependent genes are preferentially expressed. SIGNIFICANCE: The nitrogen related PTSNtr phosphorelay system has been hypothesized to participate in the control of nitrogen metabolism. Here we used a proteomics approach to show that an Escherichia coli ptsN null strain, which misses the final module of PTSNtr phosphorelay, has no significant effects on nitrogen metabolism under different nitrogen regimes. We noted that ptsN is required for fitness under minimal medium and for the proper balance between RpoS and sigma 70 activities in such way that, in the absence of ptsN, RpoS-dependent genes are preferentially expressed.

The SysteMHC Atlas project.

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts.

Renal Amyloidosis Associated With 5 Novel Variants in the Fibrinogen A Alpha Chain Protein.

INTRODUCTION: Fibrinogen A alpha chain amyloidosis is an autosomal dominant disease associated with mutations in the fibrinogen A alpha chain (FGA) gene, and it is the most common cause of hereditary renal amyloidosis in the UK. Patients typically present with kidney impairment and progress to end-stage renal disease over a median time of 4.6 years. METHODS: Six patients presented with proteinuria, hypertension, and/or lower limb edema and underwent detailed clinical and laboratory investigations. RESULTS: A novel FGA gene mutation was identified in each case: 2 frameshift mutations F521Sfs*27 and G519Efs*30 and 4 single base substitutions G555F, E526K, E524K, R554H. In 5 subjects, extensive amyloid deposits were found solely within the glomeruli, which stained specifically with antibodies to fibrinogen A alpha chain, and in one of these cases, we found coexistent fibrinogen A alpha chain amyloidosis and anti-glomerular basement membrane antibody disease. One patient was diagnosed with light-chain amyloidosis after a bone marrow examination revealed a small clonal plasma cell population, and laser microdissection of the amyloid deposits followed by liquid chromatography and tandem mass spectrometry identified kappa light chain as the fibril protein. DISCUSSION: We report 6 novel mutations in the FGA gene: 5 were associated with renal fibrinogen A alpha chain amyloidosis and 1 was found to be incidental to light-chain amyloid deposits discovered in a patient with a plasma cell dyscrasia. Clinical awareness and suspicion of hereditary amyloidosis corroborated by genetic analysis and adequate typing using combined immunohistochemistry and laser microdissection and mass spectrometry is valuable to avoid misdiagnosis, especially when a family history of amyloidosis is absent.

Strong Clonal Relatedness between Serum and Gut IgA despite Different Plasma Cell Origins.

Mucosal antigens induce generation of lamina propria plasma cells (PCs) that secrete dimeric immunoglobulin A (IgA) destined for transport across the epithelium. In addition, blood contains monomeric IgA. To study the relationship between mucosal and systemic antibody responses, we took advantage of celiac disease patient samples for isolation of gut PCs as well as serum IgA and IgG reactive with a gluten-derived peptide or the autoantigen transglutaminase 2. Proteomic analysis of serum IgA revealed antigen-specific V-gene preferences, which matched those found in gut PCs. Further, gut PC CDR-H3 sequences were abundant in serum IgA but also detectable in serum IgG. Our data indicate that the same B cell clones that give rise to gut PCs also contribute to the serum antibody pool. However, serum IgA antibodies had a molecular composition distinct from that of IgA antibodies secreted in the gut, suggesting that individual B cell clones give rise to different PC populations.

Dynamics of the Escherichia coli proteome in response to nitrogen starvation and entry into the stationary phase.

Nitrogen is needed for the biosynthesis of biomolecules including proteins and nucleic acids. In the absence of fixed nitrogen prokaryotes such as E. coli immediately ceases growth. Ammonium is the preferred nitrogen source for E. coli supporting the fastest growth rates. Under conditions of ammonium limitation, E. coli can use alternative nitrogen sources to supply ammonium ions and this reprogramming is led by the induction of the NtrC regulon. Here we used label free proteomics to determine the dynamics of E. coli proteins expression in response to ammonium starvation in both the short (30min) and the longer (60min) starvation. Protein abundances and post-translational modifications confirmed that activation of the NtrC regulon acts as the first line of defense against nitrogen starvation. The ribosome inactivating protein Rmf was induced shortly after ammonium exhaustion and this was preceded by induction of other ribosome inactivating proteins such as Hpf and RaiA supporting the hypothesis that ribosome shut-down is a key process during nitrogen limitation stress. The proteomic data revealed that growth arrest due to nitrogen starvation correlates with the accumulation of proteins involved in DNA condensation, RNA and protein catabolism and ribosome hibernation. Collectively, these proteome adaptations will result in metabolic inactive cells which are likely to exhibit multidrug tolerance.

Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.

Mechanisms underlying AQP4 accumulation in astrocyte endfeet.

The brain-blood interface holds the key to our understanding of how cerebral blood flow is regulated and how water and solutes are exchanged between blood and brain. The highly specialized astrocytic membranes that enwrap brain microvessels are salient constituents of the brain-blood interface. These endfoot membranes contain a distinct set of molecules that is anchored to the subendothelial basal lamina forming an endfoot-basal lamina junctional complex. Here we explore the mechanisms underpinning the formation of this complex. By use of a tailor made model system we show that endothelial cells promote AQP4 accumulation by exerting an inductive effect through extracellular matrix components such as agrin, as well as through a direct mechanical interaction with the endfoot processes. Through the compounds they secrete, the endothelial cells also increase AQP4 expression. The present data suggest that the highly specialized gliovascular interface is established through inductive processes that include both chemical and mechanical factors. GLIA 2015;63:2073-2091.

Frequency of root resorption following trauma to permanent teeth.

This retrospective study evaluated the frequency of development of root resorption in dental trauma cases involving supporting tissue. For 249 traumatized teeth of 125 patients aged between 7 and 51 years, we collected data on the gender and age of the patient, the teeth involved, the type of trauma, and the period between dental injury and initial examination. Radiographic parameters examined in relation to root resorption included the presence of inflammatory external root resorption, internal root resorption, replacement resorption, and canal calcification. Data were analyzed by chi-squared test, Fisher's exact test, and mult iple logistic regression (P < 0.05). The results indicated that there was a significant relationship between the period from the date of injury until initial examination and the occurrence of inflammatory external resorption (P = 0.0199), as well as the type of injury (P = 0.0406). Furthermore, external resorption was most frequently associated with intrusive luxation (92.8%), followed by avulsion (89.0%), lateral luxation (80.2%), and extrusive luxation (77.4%). Among the types of dental injury, replacement resorption was observed more frequently in cases of avulsion (87.2%). The only factor that was significantly associated with this type of resorption was the type of injury (P < 0.0001). Root resorption is observed more frequently and its risk of development is higher in cases of severe trauma, especially avulsion and intrusive luxation.

Intrathecal BCR transcriptome in multiple sclerosis versus other neuroinflammation: Equally diverse and compartmentalized, but more mutated, biased and overlapping with the proteome.

The mechanisms driving the intrathecal synthesis of IgG in multiple sclerosis (MS) are unknown. We combined high-throughput sequencing of transcribed immunoglobulin heavy-chain variable (IGHV) genes and mass spectrometry to chart the diversity and compartmentalization of IgG-producing B cells in the cerebrospinal fluid (CSF) of MS patients and controls with other neuroinflammatory diseases. In both groups, a few clones dominated the intrathecal IGHV transcriptome. In most MS patients and some controls, dominant transcripts matched the CSF IgG. The IGHV transcripts in CSF of MS patients frequently carried IGHV4 genes and had more replacement mutations compared to controls. In both groups, dominant IGHV transcripts were identified within clusters of clonally related B cells that had identical or related IGHV transcripts in the blood. These findings suggest more pronounced affinity maturation, but an equal degree of diversity and compartmentalization of the intrathecal B-cell response in MS compared to other neuroinflammatory diseases.

Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog.

Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

Different binding motifs of the celiac disease-associated HLA molecules DQ2.5, DQ2.2, and DQ7.5 revealed by relative quantitative proteomics of endogenous peptide repertoires.

Celiac disease is caused by intolerance to cereal gluten proteins, and HLA-DQ molecules are involved in the disease pathogenesis by presentation of gluten peptides to CD4(+) T cells. The α- or ß-chain sharing HLA molecules DQ2.5, DQ2.2, and DQ7.5 display different risks for the disease. It was recently demonstrated that T cells of DQ2.5 and DQ2.2 patients recognize distinct sets of gluten epitopes, suggesting that these two DQ2 variants select different peptides for display. To explore whether this is the case, we performed a comprehensive comparison of the endogenous self-peptides bound to HLA-DQ molecules of B-lymphoblastoid cell lines. Peptides were eluted from affinity-purified HLA molecules of nine cell lines and subjected to quadrupole orbitrap mass spectrometry and MaxQuant software analysis. Altogether, 12,712 endogenous peptides were identified at very different relative abundances. Hierarchical clustering of normalized quantitative data demonstrated significant differences in repertoires of peptides between the three DQ variant molecules. The neural network-based method, NNAlign, was used to identify peptide-binding motifs. The binding motifs of DQ2.5 and DQ7.5 concurred with previously established binding motifs. The binding motif of DQ2.2 was strikingly different from that of DQ2.5 with position P3 being a major anchor having a preference for threonine and serine. This is notable as three recently identified epitopes of gluten recognized by T cells of DQ2.2 celiac patients harbor serine at position P3. This study demonstrates that relative quantitative comparison of endogenous peptides sampled from our protein metabolism by HLA molecules provides clues to understand HLA association with disease.

Identification of rare alternative splicing events in MS/MS data reveals a significant fraction of alternative translation initiation sites.

Integration of transcriptome data is a crucial step for the identification of rare protein variants in mass-spectrometry (MS) data with important consequences for all branches of biotechnology research. Here, we used Splooce, a database of splicing variants recently developed by us, to search MS data derived from a variety of human tumor cell lines. More than 800 new protein variants were identified whose corresponding MS spectra were specific to protein entries from Splooce. Although the types of splicing variants (exon skipping, alternative splice sites and intron retention) were found at the same frequency as in the transcriptome, we observed a large variety of modifications at the protein level induced by alternative splicing events. Surprisingly, we found that 40% of all protein modifications induced by alternative splicing led to the use of alternative translation initiation sites. Other modifications include frameshifts in the open reading frame and inclusion or deletion of peptide sequences. To make the dataset generated here available to the community in a more effective form, the Splooce portal (http://www.bioinformatics-brazil.org/splooce) was modified to report the alternative splicing events supported by MS data.

HLA-DQ molecules as affinity matrix for identification of gluten T cell epitopes.

Even though MHC class II is a dominant susceptibility factor for many diseases, culprit T cell epitopes presented by disease-associated MHC molecules remain largely elusive. T cells of celiac disease lesions recognize cereal gluten epitopes presented by the disease-associated HLA molecules DQ2.5, DQ2.2, or DQ8. Employing celiac disease and complex gluten Ag digests as a model, we tested the feasibility of using DQ2.5 and DQ2.2 as an affinity matrix for identification of disease-relevant T cell epitopes. Known gluten T cell epitope peptides were enriched by DQ2.5, whereas a different set of peptides was enriched by DQ2.2. Of 86 DQ2.2-enriched peptides, four core sequences dominated. One of these core sequences is a previously known epitope and two others are novel epitopes. The study provides insight into the selection of gluten epitopes by DQ2.2. Furthermore, the approach presented is relevant for epitope identification in other MHC class II-associated disorders.