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Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity

2016; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês

10.1074/mcp.m115.055780

ISSN

1535-9484

Autores

Geert P. M. Mommen, Fabio Marino, Hugo D. Meiring, Martien C. M. Poelen, Jacqueline A. M. van Gaans-van den Brink, Shabaz Mohammed, Albert J. R. Heck, Cécile A. C. M. van,

Tópico(s)

Click Chemistry and Applications

Resumo

Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease. Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease. Human Leukocyte Antigen (HLA) 1The abbreviations used are:HLAhuman leukocyte antigenDCdendritic cellETDelectron-transfer dissociationEThcDelectron-transfer/higher-energy collision dissociationGOGene OntologyHCDhigher-energy collision dissociationSCXstrong cation exchange. class II molecules on professional antigen presenting cells such as dendritic cells (DC) expose peptide fragments derived from exogenous and endogenous proteins to be screened by CD4+ T cells (1.Neefjes J. Jongsma M.L. Paul P. Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation.Nat. Rev. Immunol. 2011; 11: 823-836Crossref PubMed Scopus (1114) Google Scholar, 2.Roche P.A. Furuta K. The ins and outs of MHC class II-mediated antigen processing and presentation.Nat. Rev. Immunol. 2015; 15: 203-216Crossref PubMed Scopus (504) Google Scholar). The activation and recruitment of CD4+ T cells recognizing disease-related peptide antigens is critical for the development of efficient antipathogen or antitumor immunity. Furthermore, the presentation of self-peptides and their interaction with CD4+ T cells is essential to maintain immunological tolerance and homeostasis (3.Adamopoulou E. Tenzer S. Hillen N. Klug P. Rota I. a Tietz S. Gebhardt M. Stevanovic S. Schild H. Tolosa E. Melms A. Stoeckle C. Exploring the MHC-peptide matrix of central tolerance in the human thymus.Nat. Commun. 2013; 4: 2039Crossref PubMed Scopus (58) Google Scholar). Knowledge of the nature of HLA class II-presented peptides on DC is of great importance to understand the rules of antigen processing and peptide binding motifs (4.Suri A. Lovitch S.B. Unanue E.R. The wide diversity and complexity of peptides bound to class II MHC molecules.Curr. Opin. Immunol. 2006; 18: 70-77Crossref PubMed Scopus (48) Google Scholar), whereas the identity of disease-related antigens may provide new knowledge on immunogenicity and leads for the development of vaccines and immunotherapy (5.Ovsyannikova I.G. Johnson K.L. Bergen H.R. Poland G.A. Mass spectrometry and peptide-based vaccine development.Clin. Pharmacol. Ther. 2007; 82: 644-652Crossref PubMed Scopus (34) Google Scholar, 6.Thibodeau J. Bourgeois-Daigneault M.C. Lapointe R. Targeting the MHC Class II antigen presentation pathway in cancer immunotherapy.Oncoimmunology. 2012; 1: 908-916Crossref PubMed Scopus (101) Google Scholar). human leukocyte antigen dendritic cell electron-transfer dissociation electron-transfer/higher-energy collision dissociation Gene Ontology higher-energy collision dissociation strong cation exchange. Mass spectrometry (MS) has proven effective for the analysis HLA class II-presented peptides (4.Suri A. Lovitch S.B. Unanue E.R. The wide diversity and complexity of peptides bound to class II MHC molecules.Curr. Opin. Immunol. 2006; 18: 70-77Crossref PubMed Scopus (48) Google Scholar, 7.Purcell A.W. Isolation and characterization of naturally processed MHC-bound peptides from the surface of antigen-presenting cells.Methods Mol. Biol. 2004; 251: 291-306PubMed Google Scholar, 8.Lippolis J.D. White F.M. Marto J.A. Luckey C.J. Bullock T.N. Shabanowitz J. Hunt D.F. Engelhard V.H. Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets.J. Immunol. 2002; 169: 5089-5097Crossref PubMed Scopus (71) Google Scholar). MS-based ligandome studies have demonstrated that HLA class II molecules predominantly present peptides derived from exogenous proteins that entered the cells by endocytosis and endogenous proteins that are associated with the endo-lysosomal compartments (4.Suri A. Lovitch S.B. Unanue E.R. The wide diversity and complexity of peptides bound to class II MHC molecules.Curr. Opin. Immunol. 2006; 18: 70-77Crossref PubMed Scopus (48) Google Scholar). Yet proteins residing in the cytosol, nucleus or mitochondria can also be presented by HLA class II molecules, primarily through autophagy (9.Dengjel J. Schoor O. Fischer R. Reich M. Kraus M. Müller M. Kreymborg K. Altenberend F. Brandenburg J. Kalbacher H. Brock R. Driessen C. Rammensee H.-G. Stevanovic S. Autophagy promotes MHC class II presentation of peptides from intracellular source proteins.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 7922-7927Crossref PubMed Scopus (514) Google Scholar, 10.Münz C. Antigen processing for MHC class II presentation via autophagy.Front. Immunol. 2012; 3: 1-6Crossref PubMed Scopus (82) Google Scholar, 11.Deretic V. Saitoh T. Akira S. Autophagy in infection, inflammation and immunity.Nat. Rev Immunol. 2013; 13: 722-737Crossref PubMed Scopus (1326) Google Scholar). Multiple studies have mapped the HLA class II ligandome of antigen presenting cells in the context of infectious pathogens (12.Stenger R.M. Meiring H.D. Kuipers B. Poelen M. van Gaans-van den Brink J.A. Boog C.J. de Jong A.P. van Els C.A. Bordetella pertussis proteins dominating the major histocompatibility complex class II-presented epitope repertoire in human monocyte-derived dendritic cells.Clin. Vaccine Immunol. 2014; 21: 641-650Crossref PubMed Scopus (8) Google Scholar), autoimmune diseases (13.Bergseng E. Dørum S. Arntzen M.Ø. Nielsen M. Nygård S. Buus S. de Souza G.a. Sollid L.M. 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.Immunogenetics. 2014; 67: 73-84Crossref PubMed Scopus (64) Google Scholar, 14.Depontieu F.R. Qian J. Zarling A.L. McMiller T.L. Salay T.M. Norris A. English A.M. Shabanowitz J. Engelhard V.H. Hunt D.F. Topalian S.L. Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12073-12078Crossref PubMed Scopus (84) Google Scholar, 15.Benham H. Nel H.J. Law S.C. Mehdi A.M. Street S. Ramnoruth N. Pahau H. Lee B.T. Ng J. G Brunck M.E. Hyde C. Trouw L.A. Dudek N.L. Purcell A.W. O'Sullivan B.J. Connolly J.E. Paul S.K. Lê Cao K.A. Thomas R. Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients.Sci. Transl. Med. 2015; 7: 290ra87Crossref PubMed Scopus (238) Google Scholar, 16.Fissolo N. Haag S. de Graaf K.L. Drews O. Stevanovic S. Rammensee H.G. Weissert R. Naturally presented peptides on major histocompatibility complex I and II molecules eluted from central nervous system of multiple sclerosis patients.Mol. Cell. Proteomics. 2009; 8: 2090-2101Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 17.Hill J. a Southwood S. Sette A. Jevnikar A.M. Bell D. a Cairns E. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule.J. Immunol. 2003; 171: 538-541Crossref PubMed Scopus (572) Google Scholar) or cancer (14.Depontieu F.R. Qian J. Zarling A.L. McMiller T.L. Salay T.M. Norris A. English A.M. Shabanowitz J. Engelhard V.H. Hunt D.F. Topalian S.L. Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12073-12078Crossref PubMed Scopus (84) Google Scholar, 18.Chornoguz O. Gapeev a. O'Neill M.C. Ostrand-Rosenberg S. Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides That Activate Tumor-specific T Cells From Breast Cancer Patients.Mol. Cell. Proteomics. 2012; : 1457-1467Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 19.Dengjel J. Decker P. Schoor O. Altenberend F. Weinschenk T. Rammensee H.G. Stevanovic S. Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry.Eur. J. Immunol. 2004; 34: 3644-3651Crossref PubMed Scopus (18) Google Scholar), or those that are essential for self-tolerance in the human thymus (3.Adamopoulou E. Tenzer S. Hillen N. Klug P. Rota I. a Tietz S. Gebhardt M. Stevanovic S. Schild H. Tolosa E. Melms A. Stoeckle C. Exploring the MHC-peptide matrix of central tolerance in the human thymus.Nat. Commun. 2013; 4: 2039Crossref PubMed Scopus (58) Google Scholar, 20.Collado J.A. Alvarez I. Ciudad M.T. Espinosa G. Canals F. Pujol-Borrell R. Carrascal M. Abian J. Jaraquemada D. Composition of the HLA-DR-associated human thymus peptidome.Eur. J. Immunol. 2013; 43: 2273-2282Crossref PubMed Scopus (32) Google Scholar). Notwithstanding these efforts, and certainly not in line with the extensive knowledge on the HLA class I ligandome (21.Granados D.P. Laumont C.M. Thibault P. Perreault C. The nature of self for T cells—a systems-level perspective.Curr. Opin. Immunol. 2015; 34: 1-8Crossref PubMed Scopus (55) Google Scholar), the nature of the HLA class II-presented peptide repertoire and particular its relationship to the cellular source proteome remains poorly understood. To advance our knowledge on the HLA-DR ligandome on activated DC without having to deal with limitations in cell yield from peripheral human blood (12.Stenger R.M. Meiring H.D. Kuipers B. Poelen M. van Gaans-van den Brink J.A. Boog C.J. de Jong A.P. van Els C.A. Bordetella pertussis proteins dominating the major histocompatibility complex class II-presented epitope repertoire in human monocyte-derived dendritic cells.Clin. Vaccine Immunol. 2014; 21: 641-650Crossref PubMed Scopus (8) Google Scholar, 21.Granados D.P. Laumont C.M. Thibault P. Perreault C. The nature of self for T cells—a systems-level perspective.Curr. Opin. Immunol. 2015; 34: 1-8Crossref PubMed Scopus (55) Google Scholar, 22.van Haren S.D. Herczenik E. ten Brinke A. Mertens K. Voorberg J. Meijer A.B. HLA-DR-presented peptide repertoires derived from human monocyte-derived dendritic cells pulsed with blood coagulation factor VIII.Mol. Cell. Proteomics. 2011; 10 (M110.002246)Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) or tissue isolates (3.Adamopoulou E. Tenzer S. Hillen N. Klug P. Rota I. a Tietz S. Gebhardt M. Stevanovic S. Schild H. Tolosa E. Melms A. Stoeckle C. Exploring the MHC-peptide matrix of central tolerance in the human thymus.Nat. Commun. 2013; 4: 2039Crossref PubMed Scopus (58) Google Scholar), we explored the use of MUTZ-3 cells. This cell line has been used as a model of human monocyte-derived DCs. MUTZ-3 cells can be matured to act as antigen presenting cells and express then high levels of HLA class II molecules, and can be propagated in vitro to large cell densities (23.Masterson A.J. Sombroek C.C. de Gruijl T.D. Graus Y.M.F. van der Vliet H.J.J. Lougheed S.M. van den Eertwegh A.J.M. Pinedo H.M. Scheper R.J. MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+precursors.Blood. 2002; 100: 701-703Crossref PubMed Scopus (123) Google Scholar, 24.Hoefnagel M.H. Vermeulen J.P. Scheper R.J. Vandebriel R.J. Response of MUTZ-3 dendritic cells to the different components of the Haemophilus influenzae type B conjugate vaccine: towards an in vitro assay for vaccine immunogenicity.Vaccine. 2011; 29: 5114-5121Crossref PubMed Scopus (13) Google Scholar, 25.Hoonakker M.E. Verhagen L.M. Hendriksen C.F. van Els C.A. Vandebriel R.J. Sloots A. Han W.G. In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: a proof of principle.Biologicals. 2015; 43: 100-109Crossref PubMed Scopus (10) Google Scholar). We also evaluated the performance of complementary and hybrid MS fragmentation techniques electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD) (26.Frese C.K. Altelaar A.F. van den Toorn H. Nolting D. Griep-Raming J. Heck A.J. Mohammed S. Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higher-energy collision dissociation tandem mass spectrometry.Anal. Chem. 2012; 84: 9668-9673Crossref PubMed Scopus (216) Google Scholar), and higher-energy collision dissociation (HCD) to sequence and identify the HLA class II ligandome. Together this workflow allowed for the identification of an unprecedented large set of about 14 thousand unique peptide sequences presented by DC derived HLA-DR molecules, providing an in-depth view of the complexity of the HLA class II ligandome, revealing underlying features of antigen processing and surface-presentation to CD4+ T cells. Institutional principles of RIVM relating to the use of material and data obtained from human subjects, including prototype cell lines obtained via cell bank catalogues, are in agreement with the guidelines expressed for Good Clinical Practice expressed in the Declaration of Helsinki. The deposited MUTZ-3 cell line, a human HLA-DR10, -DR11, -DR52 (HLA-DRB1*10, HLA-DRB1*11, HLA-DRB3*01) positive acute myelo-monocytic leukemia serving as a dendritic cell model (kindly provided by Dr. R. Scheeper, VU University Medical Center, Amsterdam), was grown under maintenance conditions in roller bottles in α-Minimum Essential Medium (Gibco, Thermo Fisher Scientific, Bremen, Germany), supplemented 20% heat-inactivated FBS (Hyclone, Logan, USA), 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), 2 mm l-glutamine (Gibco), and 25 U/ml GM-CSF (Pepotech, London, UK)1. MUTZ-3 cells were induced into an immature DC state by a 5-day exposure to 1000 U/ml GM-CSF (100 ng/ml), 1000 U/ml IL-4 (20 ng/ml) and 2.5 ng/ml TNF-α. Immature MUTZ-3 DC were matured by increasing the concentration of TNFα to 75 ng/ml for 20 h. During this maturation phase BCG antigens were present as an antigenic pulse (kindly provided by Camille Locht, Institut Pasteur de Lille, France). The mature state was verified based on the expression of DC-associated maturation markers CD40, CD80, CD83, and CD86 by flow cytometry (data not shown). The large bulk of 1.2 × 109 cells stimulated MUTZ-3 was washed in ice cold PBS and snap frozen before lysing and solubilizing of cell membrane proteins with Nonidet P40 containing IP lysisbuffer (Thermo Fisher Scientific). After removal of the nonsolubilized fraction using ultracentrifugation, HLA class II molecules were immunoprecipitated from the MUTZ-3 cell lysate using the HLA-DR-specific monoclonal antibody L243. An aliquot of the MUTZ-3 cell lysate after HLA-DR pull down was used for proteomics. HLA class II molecules and associated peptides were eluted with 10% acetic acid and peptides were collected by passage over a 10-kDa mw cutoff membrane and concentrated using vacuum centrifugation. The MUTZ-3 cell lysate was diluted in 2 m urea, 50 mm ammonium bicarbonate containing one tablet of EDTA-free protease inhibitor mixture (Sigma-Aldrich, Zwijndrecht, The Netherlands) and one tablet of PhosSTOP phosphatase inhibitor mixture (Roche, Almere, The Netherlands). Cysteine residues were reduced and alkylated using 200 mm dithiotreitol (Sigma-Aldrich) and 200 mm iodoacetamide (Sigma-Aldrich). The proteins were digested with Lys-C (Roche) at an enzyme/protein ratio of 1:75 for 4 h at 37 °C. Two times diluted samples were digested with trypsin (Roche) overnight at 37 °C at an enzyme/protein ratio of 1:100. Peptide mixtures were desalted using a 1-cc Sep Pack C18 columns (Waters, Etten-Leur, The Netherlands) according manufacture's protocol. HLA-DR eluted peptides were fractionated by strong cation exchange (SCX) chromatography (27.Meiring H.D. Soethout E.C. Poelen M.C.M. Mooibroek D. Hoogerbrugge R. Timmermans H. Boog C.J. Heck A.J.R. de Jong A.P.J.M. van Els C.A.C.M. Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.Mol. Cell. Proteomics. 2006; 5: 902-913Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The system consists of a Hypercarb™ trapping column (200 μm I.D., 5 mm, 7 μm particle size, Thermo Fisher Scientific) and SCX analytical column (200 μm I.D., 12 cm, polysulfoethyl aspartamide, 5 μm, PolyLC, Columbia, USA). The peptides were separated by a linear salt gradient ramping to 500 mm KCl in 0.1 m HOAc and 35% acetonitrile at a column flow rate of 2 μl/min. A total number of 26 fractions (2 min per fraction) were collected, dried down using a vacuum centrifuge, and reconstituted. Based on the LC-MS/MS signal intensities of pre-analyzed sample aliquots the 10 most informative fractions were selected for analysis. Tryptic peptides from the MUTZ-3 digest were fractioned by SCX using a ZorbaxBioSCX-Series II column (0.8 mm I.D., 50 mm, 3.5 μm particle size, Agilent Technologies, Waldbronn, Germany). A multistep gradient up to 500 mm Nacl in 0.05% formic acid 20% acetonitrile was used to separate the tryptic peptides (28.Meiring H.D. Soethout E.C. de Jong A.P. van Els C.A. Targeted identification of infection-related HLA class I-presented epitopes by stable isotope tagging of epitopes (SITE).Curr. Protoc. Immunol. 2007; (Chapter 16, Unit 16.3)Crossref PubMed Scopus (7) Google Scholar). Fractions were pooled based on their UV signal intensity to a total of 10 fractions. For the HLA ligands, each individual SCX fraction was analyzed in triplicate by nanoscale LC-MS using a Thermo Scientific EASY-nLC 1000 (Thermo Fisher Scientific) and ETD enabled LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with either EThcD, HCD or ETD fragmentation. The system comprises an in-house packed 20 mm × 100 μm ID trapping column (Reprosil C18, 3 μm, Dr Maisch, Ammerbuch, Germany) and a 50 cm × 50 μm ID analytical column (Poroshell 120 EC C18, 2.7 μm, Agilent Technologies) heated to 40 °C. The gradient for the separation linearly ranged from 7% to 30% of solvent B in 90 min at a flow rate of 100 nl/min. The column effluent was directly electro-sprayed into the MS using a gold-coated fused silica tapered tip of ∼5 μm I.D. Full MS spectra (m/z 300 to 1,500) were acquired in the Orbitrap at 60,000 resolution (FWHM). The 10 most abundant precursor ions were selected for either data-dependent EThcD, HCD or ETD fragmentation (isolation width of 1.5 Th) at an abundance threshold of 500 counts. Fragment ions were detected in the Orbitrap analyzer at 15,000 resolution (FWHM). The automatic gain control (AGC) target in MS/MS was set to 3 × 105 for EThcD, 7 × 104 for HCD, and 1 × 105 ETD. The maximum ion accumulation time for MS scans was set to 250 ms and for MS/MS scans to 1500 ms. For EThcD, modified instrument firmware was used to allow all-ion HCD fragmentation after an initial ETD. The HCD normalized collision energy was set to 32%. The ETD reaction time was set to 50 ms and supplemental activation and charge dependent activation time was enabled. Precursor ions with unknown and +1 charge states were excluded from MS/MS analysis. Dynamic exclusion was enabled (exclusion size list 500) with a repeat count of 1 and an exclusion duration of 60 s. The SCX fractions of the tryptic digested MUTZ-3 cells were analyzed by LC-MS/MS using an Agilent 1290 Infinity System (Agilent Technologies) modified for nanoflow LC (passive split) connected to a TripleTOF analyzer (Sciex, Nieuwerkerk aan den Ijssel, The Netherlands). Peptides were eluted using a similar trapping and analytical column system and LC gradient conditions as described above. A voltage of 2.7 kV was applied to the needle. The survey scan was from 375 to 1250 m/z and the high resolution mode was utilized, reaching a resolution of up to 40,000. Tandem mass spectra were acquired in high sensitivity mode with a resolution of 20,000. The 20 most intense precursors were selected for subsequent fragmentation using an information dependent acquisition, with a minimum acquisition time of 50 ms. The raw files collected from the TripleTOF were first recalibrated based on five background ions with m/z values of 391.2847, 445.12003, 51913882, 593.15761, 667.17640. The calibrated raw files were converted to mgf by the AB Sciex MS Data Converter (version 1.3 beta) program before analysis with Proteome Discoverer 1.4. RAW files acquired with the Orbitrap Elite were directly analyzed with Proteome Discoverer 1.4 software package (Thermo Fisher Scientific) using default settings unless otherwise stated. For the EThcD and ETD spectra the nonfragment filter was added with the following settings: The precursor peak was removed within a 1 Da window, charged reduced precursors and neutral loss peaks were removed within a 0.5 Da window. MS/MS scans were searched against the human Uniprot database (2012, 20,205 entries) using the SEQUEST HT mode (Proteome Discoverer 1.4, Thermo Fisher Scientific). Precursor ion and MS/MS tolerances were set to 3 ppm and 0.02 Da, respectively. In SEQUEST, spectrum matching was set to one for c and z ions for ETD data, b and y ions for HCD and b, y, c, and z for EThcD. The data were searched with no enzyme specificity, methylation and dimethylation (Arg, Lys) acetylation (N terminus, Lys), cysteinylation (Cys), deamidation (Asn, Gln, Arg) and oxidation (Met) set as variable modifications. The allowed peptide length was set between 6 and 30 amino acids, the typical length distribution of HLA class II peptides. An additional search was performed with no enzyme specificity and phosphorylation (Ser, Thr, Tyr), deamidation (Asn), and oxidation (Met) set as dynamic modification. PTM assignments were validated manually. The data sets were searched (separately) against the full reversed database, and the Percolator software was used to rescore and filter the peptide-to-spectrum matches (PSM) to a < 1% false discovery rate (FDR) (29.Käll L. Canterbury J.D. Weston J. Noble W.S. MacCoss M.J. Semi-supervised learning for peptide identification from shotgun proteomics datasets.Nat. Methods. 2007; 4: 923-925Crossref PubMed Scopus (1368) Google Scholar). SEQUEST searching combined with Percolator is particularly useful for the analysis of EThcD data and boosts the performance of HLA ligandome identification while maintaining stringency, as validated elsewhere (30.Mommen 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).Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 4507-4512Crossref PubMed Scopus (113) Google Scholar). The peptide identification list was additionally filtered for Xcorr score ≥ 1.5. The final refiltered peptide identification list was used as the HLA-DR ligandome for further analysis. TripleTOF data files were analyzed using identical settings unless otherwise stated. Precursor ion tolerance was set to 20 ppm and the MS/MS tolerance to 0.15 Da. In SEQUEST, spectrum matching was set to 1 for y and b ions. The data were searched with specificity for trypsin and enabling 2 miss cleavages. Oxidation (Met), N-terminal acetylation, phosphorylation (Ser, Thr, Tyr), methylation (Arg, Lys), dimethylation (Arg, Lys) were set as dynamic modification and carbamidomethylation (Cys) was set as a static modification. The amount of HLA-DR peptides presented at the cell surface expressed as copy number per cell were estimated based on the MS intensities provided by proteome discoverer and known amounts of the synthetic peptides angiotensin-III and oxytocin, which were spiked in each fraction prior to LC-MS analysis. CELLO2GO (31.Yu C.S. Cheng C.W. Su W.C. Chang K.C. Huang S.W. Hwang J.K. Lu C.H. CELLO2GO: a web server for protein subCELlular LOcalization prediction with functional gene ontology annotation.PLoS ONE. 2014; 9: e99368Crossref PubMed Scopus (279) Google Scholar) was used for protein subcellular localization prediction. The peptide binding affinities and the 9 a.a. binding core for HLA-DR10, HLA-DR11, and HLA-DR52 were predicted using the NetMHpan-3.0 algorithm (32.Karosiene E. Rasmussen M. Blicher T. Lund O. Buus S. Nielsen M. NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including all three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ.Immunogenetics. 2013; 65: 711-724Crossref PubMed Scopus (193) Google Scholar). Peptides with a moderate to high binding affinity (IC) < 1000 nm were considered as potential binder for a particular allele. The GibbsCluster-1.0 algorithm (33.Andreatta M. Lund O. Nielsen M. Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach.Bioinformatics. 2013; 29: 8-14Crossref PubMed Scopus (77) Google Scholar) was for simultaneous alignment and clustering of complete date set of HLA-DR-associated peptides. Gibbs clustering was performed using default settings (33.Andreatta M. Lund O. Nielsen M. Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach.Bioinformatics. 2013; 29: 8-14Crossref PubMed Scopus (77) Google Scholar), with preference of hydrophobic amino acids at position P1, the number of clusters set to 1–4, λ set to 0.8. Sequence logo's we created using IceLogo (34.Colaert N. Helsens K. Martens L. Vandekerckhove J. Gevaert K. Improved visualization of protein consensus sequences by iceLogo.Nat. Methods. 2009; 6: 786-787Crossref PubMed Scopus (522) Google Scholar), with p values set to 0.005. Following accepted procedures the protein/peptide abundances were estimated based on their spectral counts normalized to the protein length (35.Bassani-Sternberg M. Pletscher-Frankild S. Jensen L.J. Mann M. Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation.Mol. Cell. Proteomics. 2015; 14: 658-673Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). The mass spectrometry data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (36.Vizcaíno J.A. Côté R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Pérez-Riverol Y. Reisinger F. Ríos D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-9Crossref PubMed Scopus (1595) Google Scholar) with the data set identifier PXD002951. HLA-DR-peptides were isolated from human matured MUTZ-3 DCs and prefractionated by strong cation exchange chromatography prior to analysis by reversed phase LC-MS/MS, employing the complementary peptide fragmentat

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