Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry
2015; Elsevier BV; Volume: 14; Issue: 12 Linguagem: Inglês
10.1074/mcp.m115.052431
ISSN1535-9484
AutoresÉtienne Caron, Daniel J. Kowalewski, Ching Chiek Koh, Theo Sturm, Heiko Schuster, Ruedi Aebersold,
Tópico(s)Multiple Myeloma Research and Treatments
ResumoThe myriad of peptides presented at the cell surface by class I and class II major histocompatibility complex (MHC) molecules are referred to as the immunopeptidome and are of great importance for basic and translational science. For basic science, the immunopeptidome is a critical component for understanding the immune system; for translational science, exact knowledge of the immunopeptidome can directly fuel and guide the development of next-generation vaccines and immunotherapies against autoimmunity, infectious diseases, and cancers. In this mini-review, we summarize established isolation techniques as well as emerging mass spectrometry-based platforms (i.e. SWATH-MS) to identify and quantify MHC-associated peptides. We also highlight selected biological applications and discuss important current technical limitations that need to be solved to accelerate the development of this field. The myriad of peptides presented at the cell surface by class I and class II major histocompatibility complex (MHC) molecules are referred to as the immunopeptidome and are of great importance for basic and translational science. For basic science, the immunopeptidome is a critical component for understanding the immune system; for translational science, exact knowledge of the immunopeptidome can directly fuel and guide the development of next-generation vaccines and immunotherapies against autoimmunity, infectious diseases, and cancers. In this mini-review, we summarize established isolation techniques as well as emerging mass spectrometry-based platforms (i.e. SWATH-MS) to identify and quantify MHC-associated peptides. We also highlight selected biological applications and discuss important current technical limitations that need to be solved to accelerate the development of this field. The immunopeptidome is referred to as the collection of peptides associated with and presented by major histocompatibility complex (MHC) molecules (1.Caron E. Vincent K. Fortier M.-H. Laverdure J.-P. Bramoullé A. Hardy M.-P. Voisin G. Roux P.P. Lemieux S. Thibault P. Perreault C. The MHC I immunopeptidome conveys to the cell surface an integrative view of cellular regulation.Mol. Syst. Biol. 2011; 7: 533Crossref PubMed Scopus (69) Google Scholar, 2.Admon A. Bassani-Sternberg M. The Human Immunopeptidome Project, a suggestion for yet another postgenome next big thing.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 3.Berlin C. Kowalewski D.J. Schuster H. Mirza N. Walz S. Handel M. Schmid-Horch B. Salih H.R. Kanz L. Rammensee H.G. Stevanoviæ S. Stickel J.S. 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These immune peptides are divided in two classes: MHC class I and class II peptides that are distinguishable by (1) their structure, (2) the intracellular pathways by which they are generated, and (3) the type of T lymphocytes that recognize them, reviewed in (12.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 (0) Google Scholar, 13.Cresswell P. Ackerman A.L. Giodini A. Peaper D.R. Wearsch P.A. Mechanisms of MHC class I-restricted antigen processing and cross-presentation.Immunol. Rev. 2005; 207: 145-157Crossref PubMed Scopus (311) Google Scholar). In brief, MHC class I peptides are predominantly 9–12 amino acids in length or slightly longer (14.Rist M.J. Theodossis A. Croft N.P. Neller M.A. Welland A. Chen Z. Sullivan L.C. Burrows J.M. Miles J.J. Brennan R.M. Gras S. Khanna R. Brooks A.G. McCluskey J. Purcell A.W. Rossjohn J. Burrows S.R. 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Immunol. 2009; 46: 1911-1917Crossref PubMed Scopus (26) Google Scholar). Class I peptides are generated mainly following degradation of intracellular proteins by the ubiquitin-proteasome system and are recognized by cytotoxic CD8+ T cells (18.Shastri N. Schwab S. Serwold T. Producing nature's gene-chips: The generation of peptides for display by MHC class I molecules.Annu. Rev. Immunol. 2002; 20: 463-493Crossref PubMed Scopus (239) Google Scholar). MHC class II peptides are 10–25 amino acids in length, derived mainly from protease-mediated degradation of endocytosed proteins of extracellular origin, and are recognized by helper CD4+ T cells. The tissue/cell type distribution also differs for class I and class II peptides: Class I peptides are presented on virtually any nucleated cell, whereas peptides displayed by class II molecules are found on a subset of specialized immune cells such as dendritic cells, macrophages, and B lymphocytes. In recent years, there has been, however, a rapid increase in the number of nonhematopoetic cell types suggested to present peptides on MHC class II molecules (19.Kambayashi T. Laufer T.M. Atypical MHC class II-expressing antigen-presenting cells: Can anything replace a dendritic cell?.Nat. Rev. Immunol. 2014; 14: 719-730Crossref PubMed Scopus (208) Google Scholar). In the human population, the complexity of the MHC immunopeptidome is amplified by the very large genetic pool coding for structurally different class I and class II MHC molecules, termed human leukocyte antigen (HLA) molecules (20.Marsh S. Parham P. Barber L.D. The HLA factsBook.Academic Press. 2000; : 416Google Scholar). In fact, the HLA genes constitute the most polymorphic gene cluster in the human genome. The allelic diversity often alters the structure and specificity of the peptide-binding sites of the HLA molecules (21.Bjorkman P.J. Saper M.A. Samraoui B. Bennett W.S. Strominger J.L. Wiley D.C. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens.Nature. 1987; 329: 512-518Crossref PubMed Scopus (1763) Google Scholar, 22.Garrett T.P. Saper M.A. Bjorkman P.J. Strominger J.L. Wiley D.C. Specificity pockets for the side chains of peptide antigens in HLA-Aw68.Nature. 1989; 342: 692-696Crossref PubMed Scopus (577) Google Scholar). Consequently, each HLA allotype associates with a specific set of peptides bearing conserved amino acids known as '”residues” or HLA binding motif (23.Falk K. Rötzschke O. Stevanović S. Jung G. Rammensee H.G. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules.Nature. 1991; 351: 290-296Crossref PubMed Scopus (2018) Google Scholar, 24.Lazoura E. Lodding J. Farrugia W. Day S. Ramsland P.A. Apostolopoulos V. Non-canonical anchor motif peptides bound to MHC class I induce cellular responses.Mol. Immunol. 2009; 46: 1171-1178Crossref PubMed Scopus (6) Google Scholar). The human genome comprises over 10,000 different HLA allelic forms (http://www.ebi.ac.uk/imgt/hla/stats.html; April 2015), and each person expresses up to six different classical class I allotypes and typically eight different class II allotypes, resulting in a huge HLA peptidomic complexity at the population level (25.Cole D.K. The ultimate mix and match: Making sense of HLA alleles and peptide repertoires.Immunol. Cell Biol. 2015; 93: 515-516Crossref PubMed Scopus (5) Google Scholar). Pioneered by Donald Hunt in the early 1990s, analyses of MHC-associated peptides by data-dependent analysis (DDA) mass spectrometry (MS) have yielded groundbreaking knowledge about the peptide binding motifs of MHC molecules (26.Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Cox A.L. Appella E. Engelhard V.H. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry.Science. 1992; 255: 1261-1263Crossref PubMed Google Scholar). Thanks to the astonishing progress in MS-based technologies over the last decade, hundreds to thousands of MHC-associated peptides can now be identified in a single measurement using optimal biological model systems. More recently, targeted MS techniques have emerged as robust approaches to accurately and reproducibly quantify the dynamics of antigen presentation (27.Croft N.P. Smith S.A. Wong Y.C. Tan C.T. Dudek N.L. Flesch I.E. Lin L.C. Tscharke D.C. Purcell A.W. Kinetics of antigen expression and epitope presentation during virus infection.PLoS Pathog. 2013; 9: e1003129Crossref PubMed Scopus (91) Google Scholar). As a result of such emerging technologies, a better understanding of our immune system as well as clinical applications are expected. In this mini-review, we aim at (1) describing key technical considerations in the selection of appropriate model systems for the exploration of immunopeptidomes, (2) summarizing established methods for the isolation of MHC-associated peptides for mass spectrometric analysis, (3) providing an up-to-date description of standard and emerging MS techniques, and (4) discussing future directions that, if explored, will advance the field. At the genomics level, any living organism can be investigated following robust and efficient extraction of DNA. In contrast, not every biological model system is compatible with the analysis of the immunopeptidome. In principle, class I peptides are expected to be detectable on most cell and tissue types in mammals, as genes coding for MHC class I molecules are expressed in virtually any nucleated cell in jawed vertebrates. However, many technological limitations, as described below, have yet to be overcome (e.g. isolation of peptides, dynamic range of mass spectrometers, software tools) to reach robust and comprehensive analysis of MHC I immunopeptidomes from any mammalian cell type. New methods for the investigation of class I peptides would also be beneficial for the analysis of class II immunopeptidomes as both classes of peptides share generally similar technical limitations. Currently, suitable model cell lines or tissues for immunopeptidome analysis have to express high levels of endogenous MHC molecules. Determining the absolute number of cell surface MHC molecules by flow cytometry and/or mass spectrometry is therefore an important initial step when establishing a new model system (28.Thommen D.S. Schuster H. Keller M. Kapoor S. Weinzierl A.O. Chennakesava C.S. Wang X. Rohrer L. von Eckardstein A. Stevanovic S. Biedermann B.C. Two preferentially expressed proteins protect vascular endothelial cells from an attack by peptide-specific CTL.J. Immunol. 2012; 188: 5283-5292Crossref PubMed Scopus (6) Google Scholar, 29.Apps R. Meng Z. Del Prete G.Q. Lifson J.D. Zhou M. Carrington M. Relative expression levels of the HLA class-I proteins in normal and HIV-infected cells.J. Immunol. 2015; 194: 3594-3600Crossref PubMed Scopus (84) Google Scholar). On average, we noted from pertinent literature reports that the usage of at least ∼5 × 108 cells expressing ∼2 × 105 MHC molecules per cell was a minimum requirement for the exploration of cellular immunopeptidomes (3.Berlin C. Kowalewski D.J. Schuster H. Mirza N. Walz S. Handel M. Schmid-Horch B. Salih H.R. Kanz L. Rammensee H.G. Stevanoviæ S. Stickel J.S. Mapping the HLA ligandome landscape of acute myeloidleukemia: A targeted approach toward peptide-based immunotherapy.Leukemia. 2014; 29: 647-659Crossref PubMed Scopus (0) Google Scholar, 4.Kowalewski D.J. Schuster H. Backert L. Berlin C. Kahn S. Kanz L. Salih H.R. Rammensee H.G. Stevanovic S. Stickel J.S. HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL).Proc. Natl. Acad. Sci. U.S.A. 2015; 112: E166-175Crossref PubMed Scopus (98) Google Scholar). Cell lines expressing low levels of endogenous class I molecules (e.g. C1R cells) but high levels of transfected MHC molecules, either soluble or membrane bound, have also been used in many immunopeptidomics studies (30.Scull K.E. Dudek N.L. Corbett A.J. Ramarathinam S.H. Gorasia D.G. Williamson N.A. Purcell A.W. Secreted HLA recapitulates the immunopeptidome and allows in-depth coverage of HLA A*02:01 ligands.Mol. Immunol. 2012; 51: 136-142Crossref PubMed Scopus (33) Google Scholar, 31.Hickman H.D. Luis A.D. Buchli R. Few S.R. Sathiamurthy M. VanGundy R.S. Giberson C.F. Hildebrand W.H. Toward a definition of self: Proteomic evaluation of the class I peptide repertoire.J. Immunol. 2004; 172: 2944-2952Crossref PubMed Google Scholar, 32.Buchsbaum S. Barnea E. Dassau L. Beer I. Milner E. Admon A. Large-scale analysis of HLA peptides presented by HLA-Cw4.Immunogenetics. 2003; 55: 172-176Crossref PubMed Scopus (19) Google Scholar, 33.Hawkins O.E. Vangundy R.S. Eckerd A.M. Bardet W. Buchli R. Weidanz J.A. Hildebrand W.H. Identification of breast cancer peptide epitopes presented by HLA-A*0201.J. Proteome Res. 2008; 7: 1445-1457Crossref PubMed Scopus (0) Google Scholar, 34.Ben Dror L. Barnea E. Beer I. Mann M. Admon A. The HLA–B* 2705 peptidome.Arthritis Rheum. 2010; 62: 420-429PubMed Google Scholar). This property makes these cells particularly attractive for the analysis of peptides presented by individual class I allotypes as the overexpression provides the flexibility required for the exploration of the allotypes (35.Marcilla M. Alpízar A. Lombardía M. Ramos-Fernandez A. Ramos M. Albar J.P. Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue.Mol. Cell. Proteomics. 2014; 13: 462-474Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 36.Schittenhelm R.B. Lim Kam Sian T.C. C. Wilmann P.G. Dudek N.L. Purcell A.W. Revisiting the arthritogenic peptide theory: Quantitative not qualitative changes in the peptide repertoire of HLA-B27 allotypes.Arthritis Rheum. 2014; 67: 702-713Crossref Scopus (60) Google Scholar) (Fig. 1, upper panel). Analysis of MHC class I and II peptide ligands from cells isolated in mouse primary tissue were reported with limited success given the high number of mice needed to perform an experiment (37.Tan C.T. Croft N.P. Dudek N.L. Williamson N.A. Purcell A.W. Direct quantitation of MHC-bound peptide epitopes by selected reaction monitoring.Proteomics. 2011; 11: 2336-2340Crossref PubMed Scopus (49) Google Scholar, 38.de Verteuil D. 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In terms of translational potential, primary human tissues are highly attractive (Fig. 1, upper panel). In general, ∼1 g of tissue is required to detect hundreds to thousands of MHC class I peptides (41.Dutoit V. Herold-Mende C. Hilf N. Schoor O. Beckhove P. Bucher J. Dorsch K. Flohr S. Fritsche J. Lewandrowski P. Lohr J. Rammensee H.-G. Stevanovic S. Trautwein C. Vass V. Walter S. Walker P.R. Weinschenk T. Singh-Jasuja H. Dietrich P.-Y. Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy.Brain. 2012; 135: 1042-1054Crossref PubMed Scopus (119) Google Scholar). In fact, the amount of starting material needed for the detection of high numbers of peptides is inversely proportional to the expression levels of MHC molecules. For instance, if specific tumor specimens express high levels of MHC molecules, much less material is usually needed to detect high numbers of peptide sequences. However, specific and rigorous assays determining the exact amount and quality of the tissue required for such analyses have yet to be documented to translate the approach more effectively to the clinic. In the early days, papain proteolytic digestion was moderately successful for the isolation of MHC-associated peptides (42.Cresswell P. Turner M.J. Strominger J.L. Papain-solubilized HL-A antigens from cultured human lymphocytes contain two peptide fragments.Proc. Natl. Acad. Sci. U.S.A. 1973; 70: 1603-1607Crossref PubMed Google Scholar, 43.Peterson P.A. Rask L. Lindblom J.B. Highly purified papain-solubilized HL-A antigens contain beta2-microglobulin.Proc. Natl. Acad. Sci. U.S.A. 1974; 71: 35-39Crossref PubMed Google Scholar, 44.Nathenson S.G. Uehara H. Ewenstein B.M. Kindt T.J. Coligan J.E. Primary structural: Analysis of the transplantation antigens of the murine H-2 major histocompatibility complex.Annu. Rev. Biochem. 1981; 50: 1025-1052Crossref PubMed Google Scholar). However, this technique has not been widely used because of the large quantities of starting materials required. In the early 1990s, three main techniques to extract MHC-associated peptides were developed: (1) strong acid elution of MHC class I and II peptides from whole-cell lysate using trifluoroacetic acid (45.Rötzschke O. Falk K. Wallny H.J. Faath S. Rammensee H.G. Characterization of naturally occurring minor histocompatibility peptides including H-4 and H-Y.Science. 1990; 249: 283-287Crossref PubMed Google Scholar, 46.Rötzschke O. Falk K. Deres K. Schild H. Norda M. Metzger J. Jung G. Rammensee H.G. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells.Nature. 1990; 348: 252-254Crossref PubMed Scopus (615) Google Scholar, 47.Falk K. Rötzschke O. Rammensee H.G. Cellular peptide composition governed by major histocompatibility complex class I molecules.Nature. 1990; 348: 248-251Crossref PubMed Scopus (377) Google Scholar), (2) mild acid elution (MAE) of class I peptides (not class II peptides) from the cell surface (48.Storkus W.J. Zeh 3rd, H.J. Salter R.D. Lotze M.T. Identification of T-cell epitopes: Rapid isolation of class I-presented peptides from viable cells by mild acid elution.J. Immunother. Emphasis Tumor Immunol. 1993; 14: 94-103Crossref PubMed Google Scholar), and (3) immunoaffinity purification (IP) of the class I and II MHC peptide complexes from detergent solubilized cell lysates followed by the release of ligands from the isolated complexes (49.Van Bleek G.M. Nathenson S.G. Isolation of an endogenously processed immunodominant viral peptide from the class I H-2Kb molecule.Nature. 1990; 348: 213-216Crossref PubMed Google Scholar) (Fig. 1, middle panel). Nowadays, MAE and IP are the best established and most widely used methods, both still requiring large amounts of starting material (typically 2 × 108 to 1 × 1010 cells). Until recently, MAE was used to isolate MHC class I peptides from various cell lines, bone-marrow-derived dendritic cells, and primary thymocytes (5.Granados D.P. Sriranganadane D. Daouda T. Zieger A. Laumont C.M. Caron-Lizotte O. Boucher G. Hardy M.P. Gendron P. Côté C. Lemieux S. Thibault P. Perreault C. Impact of genomic polymorphisms on the repertoire of human MHC class I-associated peptides.Nat. Commun. 2014; 5: 3600Crossref PubMed Scopus (59) Google Scholar, 38.de Verteuil D. Muratore-Schroeder T.L. Granados D.P. Fortier M.-H. Hardy M.-P. Bramoullé A. Caron E. Vincent K. Mader S. Lemieux S. Thibault P. Perreault C. Deletion of immunoproteasome subunits imprints on the transcriptome and has a broad impact on peptides presented by major histocompatibility complex I molecules.Mol. Cell. Proteomics. 2010; 9: 2034-2047Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 39.Fortier M.H. Caron E. Hardy M.P. Voisin G. Lemieux S. Perreault C. Thibault P. The MHC class I peptide repertoire is molded by the transcriptome.J. Exp. Med. 2008; 205: 595-610Crossref PubMed Scopus (111) Google Scholar, 48.Storkus W.J. Zeh 3rd, H.J. Salter R.D. Lotze M.T. Identification of T-cell epitopes: Rapid isolation of class I-presented peptides from viable cells by mild acid elution.J. Immunother. Emphasis Tumor Immunol. 1993; 14: 94-103Crossref PubMed Google Scholar, 50.Antwi K. Hanavan P.D. Myers C.E. Ruiz Y.W. Thompson E.J. Lake D.F. Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines.Mol. Immunol. 2009; 46: 2931-2937Crossref PubMed Scopus (18) Google Scholar). The MAE approach is cell-surface-specific and can be repeated over time from the same cell population, e.g. to isolate newly generated MHC class I peptides. A significant proportion of peptides is, however, nonspecific for the MHC class I molecule (38.de Verteuil D. Muratore-Schroeder T.L. Granados D.P. Fortier M.-H. Hardy M.-P. Bramoullé A. Caron E. Vincent K. Mader S. Lemieux S. Thibault P. Perreault C. Deletion of immunoproteasome subunits imprints on the transcriptome and has a broad impact on peptides presented by major histocompatibility complex I molecules.Mol. Cell. Proteomics. 2010; 9: 2034-2047Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 39.Fortier M.H. Caron E. Hardy M.P. Voisin G. Lemieux S. Perreault C. Thibault P. The MHC class I peptide repertoire is molded by the transcriptome.J. Exp. Med. 2008; 205: 595-610Crossref PubMed Scopus (111) Google Scholar). In fact, by comparing the number of MAE-extracted peptides from wild-type and β2-microglobulin knockout cells – the latter expressing virtually no detectable level of cell surface MHC class I molecules – about 40–60% of the identified peptides were determined to be contaminants, i.e. non-MHC class I-derived. Two main advantages can be attributed to the IP strategy: (1) the high specificity of the extraction process for peptides associated to MHC molecules and (2) its flexibility (Fig. 1, middle panel). In fact, this method can be used for the isolation of both class I and class II MHC peptides from a range of biological sources such as cell lines, primary tissues and plasma (36.Schittenhelm R.B. Lim Kam Sian T.C. C. Wilmann P.G. Dudek N.L. Purcell A.W. Revisiting the arthritogenic peptide theory: Quantitative not qualitative changes in the peptide repertoire of HLA-B27 allotypes.Arthritis Rheum. 2014; 67: 702-713Crossref Scopus (60) Google Scholar, 51.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 (192) Google Scholar, 52.Bassani-Sternberg M. Barnea E. Beer I. Avivi I. Katz T. Admon A. Soluble plasma HLA peptidome as a potential source for cancer biomarkers.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 18769-18776Crossref PubMed Scopus (86) Google Scholar, 53.Kowalewski D.J. Stevanović S. Biochemical large-scale identification of MHC class I ligands.Methods Mol. Biol. 2013; 960: 145-157Crossref PubMed Scopus (51) Google Scholar). Moreover, up to ∼90% of immunopurified peptides were reported to be specific for the MHC molecules (51.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 (192) Google Scholar, 54.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). Using the standard IP protocol, cells are first treated and lysed with a nondenaturing detergent (53.Kowalewski D.J. Stevanović S. Biochemical large-scale identification of MHC class I ligands.Methods Mol. Biol. 2013; 960: 145-157Crossref PubMed Scopus (51) Google Scholar). MHC peptide complexes are then precipitated by applying the complex lysate to an affinity column coupled with monoclonal antibody (mAb) specific for a certain MHC class or allotype (53.Kowalewski D.J. Stevanović S. Biochemical large-scale identification of MHC class I ligands.Methods Mol. Biol. 2013; 960: 145-157Crossref PubMed Scopus (51) Google Scholar, 55.Kasuga K. Comprehensive analysis of MHC ligands in clinical material by immunoaffinity-mass spectrometry.Methods Mol. Biol. 2013; 1023: 203-218Crossref PubMed Google Scholar). Both cell surface and intracellular MHC molecules are precipitated. HPLC fractionation or filtering is generally used following the IP (and MAE) preparation to separate peptides from larger complex components. Due to high consumption of affinity matrix, this isolation technique typically requires in-house production of mAb from hybridoma cell lines. Of note, papain digestion has also been used in combination with the IP method to cleave and subsequently isolate cell surface MHC-peptide complexes (50.Antwi K. Hanavan P.D. Myers C.E. Ruiz Y.W. Thompson E.J. Lake D.F. Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines.Mol. Immunol. 2009; 46: 2931-2937Crossref PubMed Scopus (18) Google Scholar, 56.Galati G. Arcelloni C. Paroni R. Heltai S. Rovere P. Rugarli C. Manfredi A.A. Quantitative cytometry of MHC class I digestion from living cells.Cytometry. 1997; 27: 77-83Crossref PubMed Scopus (7) Google Scholar). The IP method is highly specific but presumably comes with the cost of a very low yield, i.e. ∼0.5–3% as reported by Hassan et al. (57.Hassan C. Kester M.G. Oudgenoeg G. de Ru A.H. Janssen G.M. Drijfhout J.W. Spaapen R.M. Jiménez C.R. Heemskerk M.H. Falkenburg J.H. van Veelen P.A. Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes.J. Proteomics. 2014; 109: 240-244Crossref PubMed Scopus (31) Google Scholar). This study used a state-of-the-art, quantitatively accurate MS technique–termed selected or multiple reaction monitoring (SRM or MRM, respectively)–to quantify all losses of HLA class I peptides during sample processing. The authors showed, for the first time, that the immunopurification step was at the origin of significant losses during sample handling. Whether the yield of the IP approach is highly variable between different laboratories has yet to be reported. Nevertheless, this study stressed the need for the devel
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