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• W3021350158 abstract "The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens. The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens. Post-translational modification (PTM) including phosphorylation (1Alpízar A. Marino F. Ramos-Fernández A. Lombardía M. Jeko A. Pazos F. Paradela A. Santiago C. Heck A.J. Marcilla M. A molecular basis for the presentation of phosphorylated peptides by HLA-B antigens.Mol. Cell. Proteomics. 2017; 16: 181-193Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), ubiquitinylation (2Wei J. Zanker D. Di Carluccio A.R. Smelkinson M.G. Takeda K. Seedhom M.O. Dersh D. Gibbs J.S. Yang N. Jadhav A. Varied role of ubiquitylation in generating MHC class I peptide ligands.J. Immunol. 2017; 198: 3835-3845Crossref PubMed Scopus (24) Google Scholar), acylation (3Beresford G.W. Boss J.M. CIITA coordinates multiple histone acetylation modifications at the HLA-DRA promoter.Nat. Immunol. 2001; 2: 652Crossref PubMed Scopus (156) Google Scholar), deamidation (4McGinty J.W. Marré M.L. Bajzik V. Piganelli J.D. James E.A. T cell epitopes and post-translationally modified epitopes in type 1 diabetes.Current Diabetes Reports. 2015; 15: 90Crossref PubMed Scopus (53) Google Scholar) and other structural modifications (5Sidney J. Vela J.L. Friedrich D. Kolla R. von Herrath M. Wesley J.D. Sette A. Low HLA binding of diabetes-associated CD8+ T-cell epitopes is increased by post translational modifications.BMC Immunol. 2018; 19: 12Crossref PubMed Scopus (24) Google Scholar) has the potential to vastly expand the ligand repertoire presented by cell surface human leukocyte antigen (HLA) class I molecules for recognition by T cells. The alteration of physiochemical properties following PTM may modulate binding affinity to a given HLA allotype (5Sidney J. Vela J.L. Friedrich D. Kolla R. von Herrath M. Wesley J.D. Sette A. Low HLA binding of diabetes-associated CD8+ T-cell epitopes is increased by post translational modifications.BMC Immunol. 2018; 19: 12Crossref PubMed Scopus (24) Google Scholar) as well as eliciting or abrogating T cell responses compared with the native sequence (4McGinty J.W. Marré M.L. Bajzik V. Piganelli J.D. James E.A. T cell epitopes and post-translationally modified epitopes in type 1 diabetes.Current Diabetes Reports. 2015; 15: 90Crossref PubMed Scopus (53) Google Scholar, 6Skipper J. Hendrickson R.C. Gulden P.H. Brichard V. Van Pel A. Chen Y. Shabanowitz J. Wolfel T. Slingluff C.L. Boon T. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.J. Exp. Med. 1996; 183: 527-534Crossref PubMed Scopus (378) Google Scholar, 7Ferris R.L. Hall C. Sipsas N.V. Safrit J.T. Trocha A. Koup R.A. Johnson R.P. Siliciano R.F. Processing of HIV-1 envelope glycoprotein for class I-restricted recognition: dependence on TAP1/2 and mechanisms for cytosolic localization.J. Immunol. 1999; 162: 1324-1332PubMed Google Scholar, 8Yagüe J. Vázquez J. De Castro J.A.L. A post-translational modification of nuclear proteins, N G, N G-dimethyl-Arg, found in a natural HLA class I peptide ligand.Protein Sci. 2000; 9: 2210-2217Crossref PubMed Scopus (26) Google Scholar, 9Meadows L. Wang W. den Haan J.M. Blokland E. Reinhardus C. Drijfhout J.W. Shabanowitz J. Pierce R. Agulnik A.I. Bishop C.E. The HLA-A* 0201-restricted HY antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition.Immunity. 1997; 6: 273-281Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 10Gianfrani C. Troncone R. Mugione P. Cosentini E. De Pascale M. Faruolo C. Senger S. Terrazzano G. Southwood S. Auricchio S. Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope.J. Immunol. 2003; 170: 2719-2726Crossref PubMed Scopus (71) Google Scholar). For example, several citrullinated or phosphorylated peptides have higher binding affinity to HLA-A2 and can upregulate the response of CD8+ T cells compared with their unmodified forms in subjects with type 1 diabetes (4McGinty J.W. Marré M.L. Bajzik V. Piganelli J.D. James E.A. T cell epitopes and post-translationally modified epitopes in type 1 diabetes.Current Diabetes Reports. 2015; 15: 90Crossref PubMed Scopus (53) Google Scholar), and phosphorylation of tumor epitopes has been show to stabilize the peptide-HLA complex (11Petersen J. Wurzbacher S.J. Williamson N.A. Ramarathinam S.H. Reid H.H. Nair A.K. Zhao A.Y. Nastovska R. Rudge G. Rossjohn J. Phosphorylated self-peptides alter human leukocyte antigen class I-restricted antigen presentation and generate tumor-specific epitopes.Pro, Natl. Acad. Sci. U.S.A. 2009; 106: 2776-2781Crossref PubMed Scopus (56) Google Scholar). In addition, selective deamidation of HLA class II-restricted gliadin peptides enhances HLA-DQ2 and HLA-DQ8 binding with concomitant increases in immunogenicity (12Arentz-Hansen H. Körner R. Molberg Ø Quarsten. H Vader W. Kooy Y.M. Lundin K.E. Koning F. Roepstorff P. Sollid L.M. The intestinal T cell response to α-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase.J. Exp. Med. 2000; 191: 603-612Crossref PubMed Scopus (560) Google Scholar). In contrast, the CD8+ T cell response to A-gliadin123–132 is abolished when the position 123 glutamine is deamidated, likely because of reduced HLA binding (10Gianfrani C. Troncone R. Mugione P. Cosentini E. De Pascale M. Faruolo C. Senger S. Terrazzano G. Southwood S. Auricchio S. Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope.J. Immunol. 2003; 170: 2719-2726Crossref PubMed Scopus (71) Google Scholar). Our understanding of the human immunopeptidome—that is, the repertoire of peptides bound to and presented by HLA molecules—has grown rapidly because of increases in the speed and sensitivity of mass spectrometers and improvements in the specific isolation of peptide-HLA (pHLA) complexes from cells and tissues (13Purcell A.W. Gorman J.J. Immunoproteomics mass spectrometry-based methods to study the targets of the immune response.Mol. Cell. Proteomics. 2004; 3: 193-208Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 14Cravatt B.F. Simon G.M. Yates Iii J.R. The biological impact of mass-spectrometry-based proteomics.Nature. 2007; 450: 991Crossref PubMed Scopus (572) Google Scholar, 15Mommen G.P. Frese C.K. Meiring H.D. van Gaans-van den Brink J. de Jong A.P. van Els C.A. Heck A.J. Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD).Pro, Natl. Acad. Sci. U.S.A. 2014; 111: 4507-4512Crossref PubMed Scopus (142) Google Scholar, 16Bassani-Sternberg M. Bräunlein E. Klar R. Engleitner T. Sinitcyn P. Audehm S. Straub M. Weber J. Slotta-Huspenina J. Specht K. Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry.Nat. Communications. 2016; 7: 13404Crossref PubMed Scopus (402) Google Scholar, 17Caron E. Aebersold R. Banaei-Esfahani A. Chong C. Bassani-Sternberg M. A case for a human immuno-peptidome project consortium.Immunity. 2017; 47: 203-208Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 18Purcell A.W. Ramarathinam S.H. Ternette N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics.Nat. Protocols. 2019; 14: 1687Crossref PubMed Scopus (119) Google Scholar, 19Faridi P. Li C. Ramarathinam S.H. Vivian J.P. Illing P.T. Mifsud N.A. Ayala R. Song J. Gearing L.J. Hertzog P.J. Ternette N. Rossjohn J. Croft N.P. Purcell A.W. A subset of HLA-I peptides are not genomically templated: Evidence for cis- and trans-spliced peptide ligands.Sci. Immunol. 2018; 3: eaar3947Crossref PubMed Scopus (109) Google Scholar). These studies rely on computational algorithms to infer the peptide sequence from a reference protein database. Despite PTMs not being explicitly encoded in these databases, such algorithms have the capacity to search for and detect the appropriate mass shift associated with a large array of PTMs (20Witze E.S. Old W.M. Resing K.A. Ahn N.G. Mapping protein post-translational modifications with mass spectrometry.Nat. Methods. 2007; 4: 798Crossref PubMed Scopus (602) Google Scholar, 21Mann M. Jensen O.N. Proteomic analysis of post-translational modifications.Nat. Biotechnol. 2003; 21: 255Crossref PubMed Scopus (1632) Google Scholar, 22Olsen J.V. Mann M. Status of large-scale analysis of post-translational modifications by mass spectrometry.Mol. Cell. Proteomics. 2013; 12: 3444-3452Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). However, except for S/T phosphorylation (23Zarling A.L. Polefrone J.M. Evans A.M. Mikesh L.M. Shabanowitz J. Lewis S.T. Engelhard V.H. Hunt D.F. Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy.Pro, Natl. Acad. Sci. U.S.A. 2006; 103: 14889-14894Crossref PubMed Scopus (136) Google Scholar, 24Abelin J.G. Trantham P.D. Penny S.A. Patterson A.M. Ward S.T. Hildebrand W.H. Cobbold M. Bai D.L. Shabanowitz J. Hunt D.F. Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry.Nat. Protocols. 2015; 10: 1308Crossref PubMed Scopus (65) Google Scholar, 25Cobbold M. De La Peña H. Norris A. Polefrone J.M. Qian J. English A.M. Cummings K.L. Penny S. Turner J.E. Cottine J. MHC class I–associated phosphopeptides are the targets of memory-like immunity in leukemia.Sci. Translational Med. 2013; 5 (203ra125): 203ra125Crossref PubMed Scopus (146) Google Scholar), there have been few systematic studies of PTM peptides in the immunopeptidome. Herein we describe the analysis of several comprehensive immunopeptidomics data sets (our data and those of others), with a focus on assessing the distribution of PTMs across different HLA allotypes. Other than oxidation of methionine deamidation of asparagine and glutamine was the next most prevalent type of modification across all HLA-I allotypes. Detailed analysis of the amino acid residues flanking the site of peptide ligand deamidation revealed a strong prevalence of the known N-linked glycosylation motif NX(S/T), where N is the deamidated asparagine residue and X is any amino acid except proline (26Cao L. Diedrich J.K. Kulp D.W. Pauthner M. He L. Park S.-K.R. Sok D. Su C.Y. Delahunty C.M. Menis S. Global site-specific N-glycosylation analysis of HIV envelope glycoprotein.Nat. Communications. 2017; 8: 14954Crossref PubMed Scopus (131) Google Scholar). Notably no motif was observed flanking the site of glutamine deamidation or for asparagine deamidation in peptides isolated from HLA class II molecules. Subsequent blocking of PNGase activity confirmed the prevalent role of deglycosylation of NX(S/T)-bearing antigenic precursors in the generation of asparagine-deamidated HLA ligands. Although this mechanism is known for a handful of deamidated T cell epitopes (6Skipper J. Hendrickson R.C. Gulden P.H. Brichard V. Van Pel A. Chen Y. Shabanowitz J. Wolfel T. Slingluff C.L. Boon T. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.J. Exp. Med. 1996; 183: 527-534Crossref PubMed Scopus (378) Google Scholar, 7Ferris R.L. Hall C. Sipsas N.V. Safrit J.T. Trocha A. Koup R.A. Johnson R.P. Siliciano R.F. Processing of HIV-1 envelope glycoprotein for class I-restricted recognition: dependence on TAP1/2 and mechanisms for cytosolic localization.J. Immunol. 1999; 162: 1324-1332PubMed Google Scholar, 27Ostankovitch M. Altrich-VanLith M. Robila V. Engelhard V.H. N-glycosylation enhances presentation of a MHC class I-restricted epitope from tyrosinase.J. Immunol. 2009; 182: 4830-4835Crossref PubMed Scopus (23) Google Scholar, 28Altrich-VanLith M.L. Ostankovitch M. Polefrone J.M. Mosse C.A. Shabanowitz J. Hunt D.F. Engelhard V.H. Processing of a class I-restricted epitope from tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic proteases.J. Immunol. 2006; 177: 5440-5450Crossref PubMed Scopus (34) Google Scholar, 29Mosse C.A. Meadows L. Luckey C.J. Kittlesen D.J. Huczko E.L. Slingluff C.L. Shabanowitz J. Hunt D.F. Engelhard V.H. The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol.J. Exp. Med. 1998; 187: 37-48Crossref PubMed Scopus (107) Google Scholar, 30Dalet A. Robbins P.F. Stroobant V. Vigneron N. Li Y.F. El-Gamil M. Hanada K.-i. Yang J.C. Rosenberg S.A. Van den Eynde B.J. An antigenic peptide produced by reverse splicing and double asparagine deamidation.Pro, Natl. Acad. Sci. U.S.A. 2011; 108: E323-E331Crossref PubMed Scopus (103) Google Scholar, 31Selby M. Erickson A. Dong C. Cooper S. Parham P. Houghton M. Walker C.M. Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules.J. Immunol. 1999; 162: 669-676PubMed Google Scholar), these data indicate that the immunopeptidome is substantially enriched for peptides derived from formerly glycosylated proteins and specifically those that have been retro-translocated from the ER and targeted for deglycosylation and degradation in the cytoplasm. These large data sets will help to train algorithms for the prediction of deamidated peptide ligands and the discovery of novel T cell targets. Moreover, our data highlights a relatively unappreciated surveillance mechanism for glycoprotein antigens via the HLA-I antigen presentation pathway. In this study, peptides from three sources were analyzed: (1) HLA-I immunopeptidome; (2) HLA-II immunopeptidome; (3) shot gun proteomics. Each source contains nine data sets. For data sets from HLA-I immunopeptidome, three of them were identified from in-house experiments that involved biological duplicates of 5 × 109 cells which were used for the isolation of each HLA-I allotype (three in total) and analysis of their bound peptides. The remaining data sets from HLA-I immunopeptidome and other two sources come from publicly available data sets. For PNGase inhibition experiments, quadruplicate biological replicates of 1 × 108 cells were tested, with equivalent numbers of control samples (adding equivalent of DMSO instead of PNGase). t Test (32Abelin J.G. Keskin D.B. Sarkizova S. Hartigan C.R. Zhang W. Sidney J. Stevens J. Lane W. Zhang G.L. Eisenhaure T.M. Clauser K.R. Hacohen N. Rooney M.S. Carr S.A. Wu C.J. Mass spectrometry profiling of HLA-associated peptidomes in mono-allelic cells enables more accurate epitope prediction.Immunity. 2017; 46: 315-326Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), one-way ANOVA (33Weinzierl A.O. Rudolf D. Hillen N. Tenzer S. van Endert P. Schild H. Rammensee H.-G. Stevanović S. Features of TAP-independent MHC class I ligands revealed by quantitative mass spectrometry.Eur. J. Immunol. 2008; 38: 1503-1510Crossref PubMed Scopus (58) Google Scholar), and two-way ANOVA (19Faridi P. Li C. Ramarathinam S.H. Vivian J.P. Illing P.T. Mifsud N.A. Ayala R. Song J. Gearing L.J. Hertzog P.J. Ternette N. Rossjohn J. Croft N.P. Purcell A.W. A subset of HLA-I peptides are not genomically templated: Evidence for cis- and trans-spliced peptide ligands.Sci. Immunol. 2018; 3: eaar3947Crossref PubMed Scopus (109) Google Scholar) were used in this study. The Epstein-Barr virus-transformed B-lymphoblastoid cell line C1R, which expresses very low levels of endogenous HLAB*35:03 and low levels HLA-C*04:01 (34Storkus W. Howell D. Salter R. Dawson J. Cresswell P. NK susceptibility varies inversely with target cell class I HLA antigen expression.J. Immunol. 1987; 138: 1657-1659PubMed Google Scholar, 35Zemmour J. Little A. Schendel D. Parham P. The HLA-A, B “negative” mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon.J. Immunol. 1992; 148: 1941-1948PubMed Google Scholar), was stably transfected with either HLA-A*01:01, HLA-A*02:01 or HLA-A*24:02 by electroporation as previously described (36Giam K. Ayala-Perez R. Illing P.T. Schittenhelm R.B. Croft N.P. Purcell A.W. Dudek N.L. A comprehensive analysis of peptides presented by HLA-A1.HLA. 2015; 85: 492-496Google Scholar). Transfected cells were grown in RPMI 1640 (Invitrogen) supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin, 7.5 mm HEPES (Sigma, St Louis, MO), 2 mml-glutamine (MP Biomedical), 75 μm β-mercaptoethanolamine (Sigma), 0.1 mm non-essential amino acids (Invitrogen, Carlsbad, CA) and 10% fetal calf serum (FCS) (RF-10). 0.3 mg/ml hygromycin (Invitrogen) was added to select for stable expression of the transfected HLA-I allotypes, which was confirmed by flow cytometry. Cells were harvested by centrifugation (3724 × g at 4 °C for 15 min), washed with PBS, pelleted by centrifugation (524 × g at 4 °C for 10 min), and snap frozen in liquid nitrogen. Pellets were stored at −80 °C until processing. The pan-caspase inhibitor Z-VAD-FMK (ab120487, Abcam, Cambridge, UK) was used to block PNGase activity, as described by Altrich-VanLith et al. (28Altrich-VanLith M.L. Ostankovitch M. Polefrone J.M. Mosse C.A. Shabanowitz J. Hunt D.F. Engelhard V.H. Processing of a class I-restricted epitope from tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic proteases.J. Immunol. 2006; 177: 5440-5450Crossref PubMed Scopus (34) Google Scholar). Briefly, 1×108 cells were pelleted by centrifugation and resuspended in 10 ml of RF-10 containing either 50 μm Z-VAD-FMK or vehicle control (2.5 μl/ml DMSO) and incubated for 30 min at 37 °C, 5% CO2. The cells were then treated with an isotonic acid stripping buffer (0.066 m Na2HPO4 and 0.131 m citric acid, pH 3.3) to remove existing cell surface HLA class I complexes (37Wang W. Man S. Gulden P.H. Hunt D.F. Engelhard V.H. Class I-restricted alloreactive cytotoxic T lymphocytes recognize a complex array of specific MHC-associated peptides.J. Immunol. 1998; 160: 1091-1097PubMed Google Scholar, 38Luckey C.J. Marto J.A. Partridge M. Hall E. White F.M. Lippolis J.D. Shabanowitz J. Hunt D.F. Engelhard V.H. Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors.J. Immunol. 2001; 167: 1212-1221Crossref PubMed Scopus (73) Google Scholar), resuspended in 10 ml of fresh RF-10 containing the 50 μm Z-VAD-FMK or vehicle alone and incubated at 37 °C, 5% CO2 for 5 h to allow re-expression of HLA class I molecules. Cells were harvested, washed with PBS, snap frozen in liquid nitrogen, and stored at −80 °C until processed. was performed as previously described (18Purcell A.W. Ramarathinam S.H. Ternette N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics.Nat. Protocols. 2019; 14: 1687Crossref PubMed Scopus (119) Google Scholar, 39Illing P.T. Pymm P. Croft N.P. Hilton H.G. Jojic V. Han A.S. Mendoza J.L. Mifsud N.A. Dudek N.L. McCluskey J. HLA-B57 micropolymorphism defines the sequence and conformational breadth of the immunopeptidome.Nat. Communications. 2018; 9: 4693Crossref PubMed Scopus (24) Google Scholar). Briefly, cell pellets were resuspended in lysis buffer (0.5% IGEPAL (Sigma), 50 mm Tris, pH 8 (Sigma), 150 mm NaCl (Merck-Millipore, Darmstadt, Germany) and protease inhibitors (Complete Protease Inhibitor Mixture Tablet [1 tablet per 50 ml solution]; Roche Molecular Biochemicals, Basel, Switzerland)) and incubated for 45 min at 4 °C. Lysates were cleared by centrifugation at 16,000 × g for 40 min at 4 °C. pHLA complexes were immunoaffinity purified from lysates using W6/32 monoclonal antibody (10 mg/109 cells) crosslinked to protein A Sepharose (40Purcell A.W. Gorman J.J. Garcia-Peydró M. Paradela A. Burrows S.R. Talbo G.H. Laham N. Peh C.A. Reynolds E.C. de Castro J.A.L. Quantitative and qualitative influences of tapasin on the class I peptide repertoire.J. Immunol. 2001; 166: 1016-1027Crossref PubMed Scopus (135) Google Scholar). Bound complexes were eluted with 10% acetic acid and fractionated by RP-HPLC as previously described (18Purcell A.W. Ramarathinam S.H. Ternette N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics.Nat. Protocols. 2019; 14: 1687Crossref PubMed Scopus (119) Google Scholar). Briefly, the mixture of eluted peptides, class I heavy chain and β2-microglobulin (β2m) was fractionated by RP-HPLC using a 4.6 × 100 mm monolithic C18 column (Chromolith Speed Rod, Merck-Millipore), an AK̈TAmicro™ HPLC system (GE Healthcare, UK) and mobile phases consisting of buffer A (0.1% trifluoroacetic acid (TFA) [Thermo Scientific, San Jose, CA]) and buffer B (80% acetonitrile (ACN) [Fisher Scientific, Waltham, MA] and 0.1% TFA). Fractions were combined, concentrated by vacuum centrifugation, and reconstituted in 2% v/v acetonitrile in 0.1% v/v aqueous formic acid. Each pool contained 25 fmol/μl iRT peptides (Biognosys, Schlieren, Switzerland (41Escher C. Reiter L. MacLean B. Ossola R. Herzog F. Chilton J. MacCoss M.J. Rinner O. Using i RT, a normalized retention time for more targeted measurement of peptides.Proteomics. 2012; 12: 1111-1121Crossref PubMed Scopus (386) Google Scholar)) as an internal retention time standard. LC-MS/MS of HLA-I bound peptides was carried out using a SCIEX TripleTOF® 6600 equipped with an on-line Eksigent Ekspert nanoLC 415 (SCIEX, Toronto, Canada). 10 μl of each sample was directly loaded onto a trap column (ChromXP C18, 3 μm 120 Å, 350 μm × 0.5 mm [SCIEX]) maintained at an isocratic flow of buffer A (2% v/v acetonitrile in water supplemented with 0.1% v/v formic acid) at 5 μl/min for 10 min and then separated using an analytical column (ChromXP C18, 3 μm 120 Å, 75 μm × 15 cm [SCIEX]) by increasing linear concentrations of buffer B (0.1% v/v formic acid, 80% v/v acetonitrile) at a flow rate of 300 nL/min for 75 min. Up to 20 MS/MS spectra were acquired per cycle using an IDA strategy with accumulation times of 200 ms and 150 ms for MS1 and MS2, respectively. The MS1 scan range was set to 300–1800 m/z and MS2 set to 80–2000 m/z. To prevent multiple sequencing of the same peptide, MS1 masses were excluded for sequencing after two occurrences for 30 s. HLA bound peptides purified from Z-VAD-FMK inhibition experiments (four biological replicates for each condition; Tier 3 analysis) were analyzed using a QTRAP® 6500+ (SCIEX) mass spectrometer equipped with an on-line Eksigent Ekspert nanoLC 415 (SCIEX) using an identical trap-elute schema to the LC-MS/MS experiments above. Data were acquired in positive ion MRM mode (refer to supplemental Table S1 for full transition list parameters) at unit resolution, with a triggered Enhanced Product Ion scan (80–1000 m/z; dynamic fill time; rolling collision energy) for any MRM transition exceeding 1000 counts per second. Data were analyzed in Skyline (v19.1.0.193) and manually assessed for any background or nonspecific interference, as well as ensuring all dot-products were ≥0.8 when comparing against the spectral library of pHLA derived from C1R-A1 cells. Peak areas for transitions were summed and values normalized to levels of iRT peptides present in each sample. MS/MS data were searched against the human proteome [UniProt v2018_11] by PEAKS Studio 8.5 (Bioinformatics Solutions, Toronto, Canada) using the Homo sapiens Uniprot database (71975 entries, dated 2018–11). Both in-house generated MS data files and public MS data files from PRIDE repository (42Vizcaíno J.A. Csordas A. Del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2015; 44: D447-D456Crossref PubMed Scopus (2780) Google Scholar) (PRIDE accession: (HLA-I): PXD004894, PXD000394, PXD005084, PXD004023, PXD008570, PXD008571, PXD008572; (HLA-II): PXD006939, PXD001205; (Proteolysis): PXD000394, PXD009797, PXD004447, PXD003977) were imported into PEAKS Studio 8.5 and subjected to default data refinement. For in-house generated MS data, the parent mass error tolerance was set to 50 ppm and the fragment mass error tolerance to 0.02 Da. For public collected MS data, the parameters were set as based on the default settings for the MS instrument used. For HLA and elastase data sets, enzyme specificity was turned off. For tryptic data sets, enzyme was set to trypsin and 3 missed cleavages were allowed. Oxidation of Methionine, deamidation of Asn or Gln, phosphorylation of Ser, Thr or Tyr and citrullination of Arg were included as variable modifications in the database peptide searches. After this analysis, a PEAKS PTM search was carried out for each data set incorporating all classes of PTMs (in total 313 types). A 1% false discovery rate (FDR, calculated by PEAKS using a target-decoy approach) threshold was applied to allow selection of high-confidence peptides. The differences in flanking residues surrounding the sites of Asn deamidation from different samples (Fig. 4D) and the comparison of the subcellular location of source antigens (Fig. 5B/C) were evaluated using two-way analysis of variance (ANOVA) multiple-comparison Bonferroni test. Differences in the proportion of known N-linked glycosylation sites among the peptides with NX(S/T) motif (Fig. 5A) was evaluated using a multiple-sample t test. PNGase inhibition experiments (Fig. 6C) were assessed by a one-way ANOVA multiple-comparison test. All analyses were calculated using GraphPad Prism 7, and a p value ≤ 0.05 was considered statistically significant.Fig. 5Subcellular location of source proteins and the relationship between deamidation of peptides containing the NX(S/T) motif. A, A high proportion of deamidated HLA-I bound peptides contain a consensus N-linked glycosylation motif which is not reflected in peptides derived from tryptic/elastase digestion of cellular mater" @default.
• W3021350158 created "2020-05-13" @default.
• W3021350158 creator A5002908830 @default.
• W3021350158 creator A5012709797 @default.
• W3021350158 creator A5035495223 @default.
• W3021350158 creator A5055577385 @default.
• W3021350158 creator A5073894470 @default.
• W3021350158 creator A5079919048 @default.
• W3021350158 creator A5087347737 @default.
• W3021350158 creator A5088201972 @default.
• W3021350158 date "2020-07-01" @default.
• W3021350158 modified "2023-10-16" @default.
• W3021350158 title "Immunopeptidomic Analysis Reveals That Deamidated HLA-bound Peptides Arise Predominantly from Deglycosylated Precursors" @default.
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• W3021350158 doi "https://doi.org/10.1074/mcp.ra119.001846" @default.
• W3021350158 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7338083" @default.
• W3021350158 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33451411" @default.
• W3021350158 hasPublicationYear "2020" @default.

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