MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/x01/MSV000079539/primaryOK20016000ProteomicsDr Matthias MannQ ExactiveHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=3cbc93bc952444dd8c5d5e47226c3b62MassIVE46MS:1002864 - No post-translational-modifications are included in the identified peptides of this datasetHLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins - those with at least fivefold higher density score than expected for their abundance - we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use.Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation.Arsenical encephalopathy.Bassani-Sternberg Michal M, Pletscher-Frankild Sune S, Jensen Lars Juhl LJ, Mann Matthias MCHERIAN C P CPAntigen Processings, antigen presentation, Leukocyte, human being, immune cell, protein complex, Modern, Proteins, Gene, protein, Spectrum Analyses, PMN cell, protein-containing complex, Spectrum Analysis, White Blood Corpuscle, Human, Spectroscopy, Antigen Presentations, protein polypeptide chains, MS, native protein, Homo sapiens, resilient, natural protein, polypeptide chain, tough, Protein, Mass, Antigen, Gene Products, Corpuscles, Analysis, protein aggregate, Blood Corpuscle, Man, Mass Spectrum Analyses, Mass Spectroscopy, Mass Spectrum Analysis, Blood Cells, Mass Spectrum, strong, Blood Cell, leucocyte, Man (Taxonomy), Analyses, Spectrometry, proteins, PMNC, White Blood Corpuscles, human, White Blood Cells, Protein Gene Products, Gene Proteins, white blood cell, Corpuscle, Modern Man, White Blood, Antigen Processing, White, Blood Corpuscles, antigen processing., White Blood Cell, humans, polymorphonuclear cellextent, Antigenic Determinants, IPP2A2, Thymus-Dependent Lymphocytes, Therapeutic Effects, determination, Effects, Ass-1, selection process, Neoplasms, Degradation, Benign Neoplasm, FBN, Gene, protein, T-Lymphocyte, Tumor, Antigenic Specificity, protein-containing complex, Normalcies, Malignant, PHAPII, 5730420M11Rik, Polypeptides, protein polypeptide chains, Determinant, sampling, peptido, resilient, polypeptide chain, T Lymphocyte, ECTOL1, tough, AA408052, Line, Gene Products, fold, Analysis, Work Flow, protein aggregate, immature T cell, Effect, WMS, Normalities, average, strong, SET, pheromone catabolism, peptides, Malignancy, TAF-I, T-Cells, ligand, Spectrum, ipp2a2, 2pp2a, T, Antigenic, proteins, Protein Degradations, COXA_DROME, Protein Degradation, CG10574, OCTD, DmelCG4299, Neoplasias, ASS, mitochondrial electron transport chain, Halflife, IGAAD, set, T Cells, Use, Halflifes, 2PP2A, DmelCG10574, malignant neoplasm, taf-ibeta, mature T cell, COX, Half Life, Therapeutic Use, dSET, dSet, peptidos, Malignancies, GPHYSD2, T Lymphocytes, Thymus Dependent Lymphocytes, Lymphocyte, SGS, Cancer, Tumors, phapii, Individual Health, Epitope, Degradations, Malignant Neoplasm, Data Set, completeness, protein complex, Proteins, igaad, Cell Lines, total expressed protein, StF-IT-1, Normalcy, Protein Digestion, Digestions, Cell, Peptide, predicted, group, ACMICD, Spectroscopy, mitochondrial respiratory chain, Workflows, MT, Benign, Half-Lifes, native protein, natural protein, Proteolyses, I-2PP2A, T-Cell, Digestion, Protein, Dm I-2, chemical analysis, I2PP2A, Neoplasm, MFS1, median, Individual, Determinants, 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peptido, T Lymphocyte, Line, Analysis, Work Flow, protein aggregate, immature T cell, Effect, WMS, average, strong, SET, Man (Taxonomy), peptides, TAF-I, T-Cells, T, proteins, COXA_DROME, Protein Degradation, DmelCG4299, IGAAD, set, T Cells, Halflifes, DmelCG10574, malignant neoplasm, Half Life, Therapeutic Use, Malignancies, GPHYSD2, Thymus Dependent Lymphocytes, SGS, Tumors, phapii, Epitope, Degradations, Data Set, completeness, Modern, StF-IT-1, colon carcinoma cell line, Normalcy, colon cancer cell line, predicted, ACMICD, Spectroscopy, Workflows, Benign, Half-Lifes, T-Cell, Digestion, median, Individual, Therapeutic Effect, Lines, Antigenic Determinant, T-cell, HLA-DR-associated protein II, DI-2, Va, I-2Dm, Protein Digestions, Thymus-Dependent, Spectrometry, colonic carcinoma cell line, Benign Neoplasms, MASS, CG4299, T-lymphocyte, human, Thymus-Dependent Lymphocyte, Malignant Neoplasms, I-2PP1, mitochondrial respiratory supercomplex, Health, multicellular organismal protein catabolic process, T cell, TAF-IBETA, Cells, Therapeutic., TAF-Ibeta, T Cell, Polypeptide, DmelCG14724, i2pp2a, Proteomes, Work Flows, humans, Thymus-Dependent Lymphocytes, human being, Effects, Neoplasms, Degradation, Benign Neoplasm, FBN, Gene, Antigenic Specificity, protein-containing complex, Normalcies, Malignant, PHAPII, Human, Determinant, resilient, polypeptide chain, Homo sapiens, ECTOL1, tough, Gene Products, Man, Normalities, pheromone catabolism, Malignancy, ligand, Spectrum, ipp2a2, 2pp2a, Antigenic, Protein Degradations, CG10574, OCTD, Neoplasias, mitochondrial electron transport chain, Halflife, Use, 2PP2A, taf-ibeta, mature T cell, COX, dSET, dSet, peptidos, T Lymphocytes, Lymphocyte, Cancer, Individual Health, Malignant Neoplasm, protein complex, Proteins, igaad, Cell Lines, total expressed protein, Protein Digestion, Digestions, Cell, Peptide, group, mitochondrial respiratory chain, MT, native protein, natural protein, Proteolyses, I-2PP2A, Protein, Dm I-2, chemical analysis, I2PP2A, Neoplasm, MFS1, Determinants, T lymphocyte, WMS2, pheromone catabolic process, Uses, primary cancer, ensemble, Normality, tend, Specificity, Lymphocytes, Cancers, mitochondrial respiratory chain supercomplex, malignant tumor, Protein Gene Products, Gene Proteins, dSET/TAF-Ibeta, sample collection, 2610030F17Rik, CG14724, Therapeutic, Modern Man, SSKS, Peptid, assay, AA407739, NeoplasiaMass Spectrum Analysis, Mass Spectrum, strong, Antigen Processings, antigen presentation, Analyses, protein complex, Proteins, Spectrometry, Gene, proteins, protein, Spectrum Analyses, protein-containing complex, Spectrum Analysis, Protein Gene Products, Spectroscopy, Gene Proteins, Antigen Presentations, protein polypeptide chains, MS, native protein, resilient, natural protein, polypeptide chain, tough, Protein, Mass, Gene Products, Antigen Processing, Analysis, protein aggregate, antigen processing., Mass Spectrum Analyses, Mass Spectroscopy16PXD000394trueMass spectrometry of HLA-I peptidomes reveals strong effects of protein abundance and turnover on antigen presentationHLA class I molecules reflect the health state of cells to cytotoxic T-cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (P<0.0001). When analyzing over-presented proteins - those with at least five-fold higher density score than expected for their abundance â?? we noticed that they are degraded almost 3 hours faster than similar but non-presented proteins (top 20% abundance class; median half-life 20.8h vs. 23.6h, p<0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain and nucleosomal proteins as particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our dataset. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides, suggesting the usefulness of combining MS-analysis with immunogenesis prediction for ranking and selection of epitopes for therapeutic use.Mon Feb 29 09:30:00 GMT 2016MSV00007953925576301