Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics

Nature Protocols volume 14, pages 1687–1707 (2019)Cite this article

Abstract

Peptide antigens bound to molecules encoded by the major histocompatibility complex (MHC) and presented on the cell surface form the _targets of T lymphocytes. This critical arm of the adaptive immune system facilitates the eradication of pathogen-infected and cancerous cells, as well as the production of antibodies. Methods to identify these peptide antigens are critical to the development of new vaccines, for which the goal is the generation of effective adaptive immune responses and long-lasting immune memory. Here, we describe a robust protocol for the identification of MHC-bound peptides from cell lines and tissues, using nano-ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (nUPLC–MS/MS) and recent improvements in methods for isolation and characterization of these peptides. The protocol starts with the immunoaffinity capture of naturally processed MHC-peptide complexes. The peptides dissociate from the class I human leukocyte antigens (HLAs) upon acid denaturation. This peptide cargo is then extracted and separated into fractions by HPLC, and the peptides in these fractions are identified using nUPLC–MS/MS. With this protocol, several thousand peptides can be identified from a wide variety of cell types, including cancerous and infected cells and those from tissues, with a turnaround time of 2–3 d.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: MHC class I and II antigen-presentation pathways.
Fig. 2: Workflow for MHC-associated peptide purification, identification and validation by nUPLC–MS/MS.
Fig. 3: Generation of MHC immunoaffinity column.
Fig. 4: MHC immunoaffinity purification.
Fig. 5: Overview of anticipated results.

Similar content being viewed by others

References

  1. Dudek, N. L., Croft, N. P., Schittenhelm, R. B., Ramarathinam, S. H. & Purcell, A. W. A systems approach to understand antigen presentation and the immune response. Methods Mol. Biol. 1394, 189–209 (2016).

    CAS  PubMed  Google Scholar 

  2. Ternette, N. et al. Early kinetics of the HLA class I-associated peptidome of MVA.HIVconsv-infected cells. J. Virol. 89, 5760–5771 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Mommen, G. P. et al. Sampling from the proteome to the human leukocyte antigen-DR (HLA-DR) ligandome proceeds via high specificity. Mol. Cell. Proteomics 15, 1412–1423 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Mommen, G. P. et al. Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD). Proc. Natl Acad. Sci. USA 111, 4507–4512 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Schittenhelm, R. B., Sian, T. 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 Rheumatol. 67, 702–713 (2015).

    CAS  PubMed  Google Scholar 

  6. Giam, K. et al. A comprehensive analysis of peptides presented by HLA-A1. Tissue Antigens 85, 492–496 (2015).

    CAS  PubMed  Google Scholar 

  7. Caron, E. et al. An open-source computational and data resource to analyze digital maps of immunopeptidomes. Elife 4, e07661 (2015).

  8. 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 14, 658–673 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Ternette, N. et al. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells. Eur. J. Immunol. 46, 60–69 (2016).

    CAS  PubMed  Google Scholar 

  10. Bassani-Sternberg, M. et al. Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nat. Commun. 7, 13404 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Shao, W. et al. The SysteMHC Atlas project. Nucleic Acids Res. 46, D1237–D1247 (2017).

    PubMed Central  Google Scholar 

  12. Abelin, J. G. et al. Mass spectrometry profiling of HLA-associated peptidomes in mono-allelic cells enables more accurate epitope prediction. Immunity 46, 315–326 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Khodadoust, M. S. et al. Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens. Nature 543, 723–727 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Liepe, J. et al. A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 354, 354–358 (2016).

    CAS  PubMed  Google Scholar 

  15. Altman, J. D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    CAS  PubMed  Google Scholar 

  16. McHeyzer-Williams, M. G., Altman, J. D. & Davis, M. M. Enumeration and characterization of memory cells in the TH compartment. Immunol. Rev. 150, 5–21 (1996).

    CAS  PubMed  Google Scholar 

  17. Rodenko, B. et al. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat. Protoc. 1, 1120–1132 (2006).

    CAS  PubMed  Google Scholar 

  18. Sheikh, Q. M., Gatherer, D., Reche, P. A. & Flower, D. R. Towards the knowledge-based design of universal influenza epitope ensemble vaccines. Bioinformatics 32, 3233-3239 (2016).

  19. Clemens, E. B. et al. Towards identification of immune and genetic correlates of severe influenza disease in indigenous Australians. Immunol. Cell Biol. 94, 367–377 (2016).

    CAS  PubMed  Google Scholar 

  20. Assarsson, E. et al. Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans. J. Virol. 82, 12241–12251 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Parra-Lopez, C. et al. Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein. J. Biol. Chem. 281, 14907–14917 (2006).

    CAS  PubMed  Google Scholar 

  22. Nardin, E. H. et al. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types. J. Immunol. 166, 481–489 (2001).

    CAS  PubMed  Google Scholar 

  23. Purcell, A. W., McCluskey, J. & Rossjohn, J. More than one reason to rethink the use of peptides in vaccine design. Nat. Rev. Drug Discov. 6, 404–414 (2007).

    CAS  PubMed  Google Scholar 

  24. Brennick, C. A., George, M. M., Corwin, W. L., Srivastava, P. K. & Ebrahimi-Nik, H. Neoepitopes as cancer immunotherapy _targets: key challenges and opportunities. Immunotherapy 9, 361–371 (2017).

    CAS  PubMed  Google Scholar 

  25. Verdegaal, E. M. E. & van der Burg, S. H. The potential and challenges of exploiting the vast but dynamic neoepitope landscape for immunotherapy. Front. Immunol. 8, 1113 (2017).

    PubMed  PubMed Central  Google Scholar 

  26. Nepom, G. T. MHC class II tetramers. J. Immunol. 188, 2477–2482 (2012).

    CAS  PubMed  Google Scholar 

  27. Crawford, F. et al. Specificity and detection of insulin-reactive CD4+ T cells in type 1 diabetes in the nonobese diabetic (NOD) mouse. Proc. Natl Acad. Sci. USA 108, 16729–16734 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Coles, R. M. et al. Virus infection expands a biased subset of T cells that bind tetrameric class I peptide complexes. Eur. J. Immunol. 33, 1557–1567 (2003).

    CAS  PubMed  Google Scholar 

  29. Gray, C. M. et al. Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART). J. Immunol. 162, 1780–1788 (1999).

    CAS  PubMed  Google Scholar 

  30. Bieganowska, K. et al. Direct analysis of viral-specific CD8+ T cells with soluble HLA-A2/Tax11-19 tetramer complexes in patients with human T cell lymphotropic virus-associated myelopathy. J. Immunol. 162, 1765–1771 (1999).

    CAS  PubMed  Google Scholar 

  31. Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998).

    CAS  PubMed  Google Scholar 

  32. Reguzova, A. Y., Karpenko, L. I., Mechetina, L. V. & Belyakov, I. M. Peptide-MHC multimer-based monitoring of CD8 T-cells in HIV-1 infection and AIDS vaccine development. Expert Rev. Vaccines 14, 69–84 (2015).

    CAS  PubMed  Google Scholar 

  33. Bacher, P. & Scheffold, A. Flow-cytometric analysis of rare antigen-specific T cells. Cytometry A 83, 692–701 (2013).

    PubMed  Google Scholar 

  34. Gojanovich, G. S. et al. The use of peptide-major-histocompatibility-complex multimers in type 1 diabetes mellitus. J. Diabetes Sci. Technol. 6, 515–524 (2012).

    PubMed  PubMed Central  Google Scholar 

  35. Castelli, C. et al. Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes. J. Exp. Med. 181, 363–368 (1995).

    CAS  PubMed  Google Scholar 

  36. Storkus, W. J., Zeh, 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. 14, 94–103 (1993).

    CAS  Google Scholar 

  37. Storkus, W. J., Zeh, H. J., Maeurer, M. J., Salter, R. D. & Lotze, M. T. Identification of human melanoma peptides recognized by class I restricted tumor infiltrating T lymphocytes. J. Immunol. 151, 3719–3727 (1993).

    CAS  PubMed  Google Scholar 

  38. Barnea, E. et al. Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor-specific antigens. Eur. J. Immunol. 32, 213–222 (2002).

    CAS  PubMed  Google Scholar 

  39. Buchsbaum, S. et al. Large-scale analysis of HLA peptides presented by HLA-Cw4. Immunogenetics 55, 172–176 (2003).

    CAS  PubMed  Google Scholar 

  40. Wahl, A. et al. HLA class I molecules consistently present internal influenza epitopes. Proc. Natl Acad. Sci. USA 106, 540–545 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Bassani-Sternberg, M. et al. Soluble plasma HLA peptidome as a potential source for cancer biomarkers. Proc. Natl Acad. Sci. USA 107, 18769–18776 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Scull, K. E. et al. Secreted HLA recapitulates the immunopeptidome and allows in-depth coverage of HLA A*02:01 ligands. Mol. Immunol. 51, 136–142 (2012).

    CAS  PubMed  Google Scholar 

  43. Yaciuk, J. C. et al. Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells. J. Virol. 88, 12992–13004 (2014).

    PubMed  PubMed Central  Google Scholar 

  44. Norcross, M. A. et al. Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity. Aids 26, F21–F29 (2012).

    CAS  PubMed  Google Scholar 

  45. Trolle, T. et al. The length distribution of class I–restricted T cell epitopes is determined by both peptide supply and MHC allele–specific binding preference. J. Immunol. 196, 1480–1487 (2016).

    CAS  PubMed  Google Scholar 

  46. Kaabinejadian, S. et al. Immunodominant West Nile virus T cell epitopes are fewer in number and fashionably late. J. Immunol. 196, 4263–4273 (2016).

    CAS  PubMed  Google Scholar 

  47. Abelin, J. G. et al. Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry. Nat. Protoc. 10, 1308–1318 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Gilchuk, P. et al. Discovering naturally processed antigenic determinants that confer protective T cell immunity. J. Clin. Invest. 123, 1976–1987 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Corr, M. et al. Endogenous peptides of a soluble major histocompatibility complex class I molecule, H-2Lds: sequence motif, quantitative binding, and molecular modeling of the complex. J. Exp. Med. 176, 1681–1692 (1992).

    CAS  PubMed  Google Scholar 

  50. Joyce, S. & Nathenson, S. G. Methods to study peptides associated with MHC class I molecules. Curr. Opin. Immunol. 6, 24–31 (1994).

    CAS  PubMed  Google Scholar 

  51. Hunt, D. F. et al. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, 1261–1263 (1992).

    CAS  PubMed  Google Scholar 

  52. Engelhard, V. H. et al. Mass spectrometric analysis of peptides associated with the human class I MHC molecules HLA-A2.1 and HLA-B7 and identification of structural features that determine binding. Chem. Immunol. 57, 39–62 (1993).

    CAS  PubMed  Google Scholar 

  53. Cox, A. L. et al. Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines. Science 264, 716–719 (1994).

    CAS  PubMed  Google Scholar 

  54. Slingluff, C. L. Jr, Hunt, D. F. & Engelhard, V. H. Direct analysis of tumor-associated peptide antigens. Curr. Opin. Immunol. 6, 733–740 (1994).

    CAS  PubMed  Google Scholar 

  55. Skipper, J. C. et al. 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. 183, 527–534 (1996).

    CAS  PubMed  Google Scholar 

  56. Meadows, L. et al. The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity 6, 273–281 (1997).

    CAS  PubMed  Google Scholar 

  57. Crotzer, V. L. et al. Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines. J. Immunol. 164, 6120–6129 (2000).

    CAS  PubMed  Google Scholar 

  58. Zarling, A. L. et al. Phosphorylated peptides are naturally processed and presented by major histocompatibility complex class I molecules in vivo. J. Exp. Med. 192, 1755–1762 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Mohammed, F. et al. Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self. Nat. Immunol. 9, 1236–1243 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Van Bleek, G. M. & Nathenson, S. G. Isolation of an endogenously processed immunodominant viral peptide from the class I H-2Kb molecule. Nature 348, 213–216 (1990).

    PubMed  Google Scholar 

  61. Rotzschke, O. et al. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature 348, 252–254 (1990).

    CAS  PubMed  Google Scholar 

  62. Falk, K. et al. Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast. J. Exp. Med. 174, 425–434 (1991).

    CAS  PubMed  Google Scholar 

  63. Falk, K., Rötzschke, O., Stevanovic, S., Jung, G. & Rammensee, H.-G. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351, 290–296 (1991).

    CAS  PubMed  Google Scholar 

  64. Falk, K., Rötzschke, O. & Rammensee, H.-G. A self peptide naturally presented by both H-2Kb and H-2Kbm1 molecules demonstrates MHC restriction of self tolerance at the molecular level. Int. Immunol. 4, 321–325 (1992).

    CAS  PubMed  Google Scholar 

  65. Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. & Rammensee, H. G. Pool sequencing of natural HLA-DR, DQ, and DP ligands reveals detailed peptide motifs, constraints of processing, and general rules. Immunogenetics 39, 230–242 (1994).

    CAS  PubMed  Google Scholar 

  66. Rammensee, H.-G. Chemistry of peptide associated with class I and class II molecules. Curr. Opin. Immunol. 7, 85–95 (1995).

    CAS  PubMed  Google Scholar 

  67. Rammensee, H., Bachmann, J., Emmerich, N. P., Bachor, O. A. & Stevanovic, S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50, 213–219 (1999).

    CAS  PubMed  Google Scholar 

  68. Schittenhelm, R. B., Sivaneswaran, S., Lim Kam Sian, T. C., Croft, N. P. & Purcell, A. W. Human leukocyte antigen (HLA) B27 allotype-specific binding and candidate arthritogenic peptides revealed through heuristic clustering of data-independent acquisition mass spectrometry (DIA-MS) data. Mol. Cell. Proteomics 15, 1867–1876 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Trujillo, J. A. et al. The cellular redox environment alters antigen presentation. J. Biol. Chem. 289, 27979–27991 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Scally, S. W. et al. A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis. J. Exp. Med. 210, 2569–2582 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Illing, P. T. et al. Immune self-reactivity triggered by drug-modified HLA-peptide repertoire. Nature 486, 554–558 (2012).

    CAS  PubMed  Google Scholar 

  72. Wynne, J. W. et al. Characterization of the antigen processing machinery and endogenous peptide presentation of a bat MHC class I molecule. J. Immunol. 196, 4468–4476 (2016).

    CAS  PubMed  Google Scholar 

  73. Edwards, P. A., Smith, C. M., Neville, A. M. & O’Hare, M. J. A human-hybridoma system based on a fast-growing mutant of the ARH-77 plasma cell leukemia-derived line. Eur. J. Immunol. 12, 641–648 (1982).

    CAS  PubMed  Google Scholar 

  74. Alexander, J., Payne, J. A., Murray, R., Frelinger, J. A. & Cresswell, P. Differential transport requirements of HLA and H-2 class I glycoproteins. Immunogenetics 29, 380–388 (1989).

    CAS  PubMed  Google Scholar 

  75. Kavathas, P., Bach, F. H. & DeMars, R. Gamma ray-induced loss of expression of HLA and glyoxalase I alleles in lymphoblastoid cells. Proc. Natl Acad. Sci. USA 77, 4251–4255 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Shimizu, Y., Geraghty, D. E., Koller, B. H., Orr, H. T. & DeMars, R. Transfer and expression of three cloned human non-HLA-A,B,C class I major histocompatibility complex genes in mutant lymphoblastoid cells. Proc. Natl Acad. Sci. USA 85, 227–231 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Partridge, T. et al. Discrimination between human leukocyte antigen class I–bound and co-purified HIV-derived peptides in immunopeptidomics workflows. Front. Immunol. 9, 912 (2018).

    PubMed  PubMed Central  Google Scholar 

  78. Riberdy, J. M., Newcomb, J. R., Surman, M. J., Barbosa, J. A. & Cresswell, P. HLA-DR molecules from an antigen-processing mutant cell line are associated with invariant chain peptides. Nature 360, 474–477 (1992).

    CAS  PubMed  Google Scholar 

  79. Nelson, C. A., Roof, R. W., McCourt, D. W. & Unanue, E. R. Identification of the naturally processed form of hen egg white lysozyme bound to the murine major histocompatibility complex class II molecule I-Ak. Proc. Natl Acad. Sci. USA 89, 7380–7383 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    CAS  PubMed  Google Scholar 

  81. Eng, J. K., McCormack, A. L. & Yates, J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    CAS  PubMed  Google Scholar 

  82. Craig, R. & Beavis, R. C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004).

    CAS  PubMed  Google Scholar 

  83. Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).

    CAS  PubMed  Google Scholar 

  84. Shilov, I. V. et al. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Mol. Cell. Proteomics 6, 1638–1655 (2007).

    CAS  PubMed  Google Scholar 

  85. Zhang, J. et al. PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification. Mol. Cell. Proteomics 11, M111.010587 (2012).

    Google Scholar 

  86. Giansanti, P., Tsiatsiani, L., Low, T. Y. & Heck, A. J. Six alternative proteases for mass spectrometry–based proteomics beyond trypsin. Nat. Protoc. 11, 993–1006 (2016).

    CAS  PubMed  Google Scholar 

  87. Meyer, J. G. et al. Expanding proteome coverage with orthogonal-specificity alpha-lytic proteases. Mol. Cell. Proteomics 13, 823–835 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Faridi, P., Purcell, A. W. & Croft, N. P. In immunopeptidomics we need a sniper instead of a shotgun. Proteomics 18, e1700464 (2018).

    PubMed  Google Scholar 

  89. Nielsen, M. et al. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein. Sci. 12, 1007–1017 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Andreatta, M. & Nielsen, M. Gapped sequence alignment using artificial neural networks: application to the MHC class I system. Bioinformatics 32, 511–517 (2016).

    CAS  PubMed  Google Scholar 

  91. Schneider, T. D. & Stephens, R. M. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18, 6097–6100 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Thomsen, M. C. F. & Nielsen, M. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion. Nucleic Acids Res. 40, W281–W287 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Andreatta, M., Lund, O. & Nielsen, M. Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach. Bioinformatics 29, 8–14 (2013).

    CAS  PubMed  Google Scholar 

  94. Bassani-Sternberg, M. et al. Deciphering HLA-I motifs across HLA peptidomes improves neo-antigen predictions and identifies allostery regulating HLA specificity. PLoS Comput. Biol. 13, e1005725 (2017).

    PubMed  PubMed Central  Google Scholar 

  95. Nepom, B. S., Nepom, G. T., Coleman, M. & Kwok, W. W. Critical contribution of beta chain residue 57 in peptide binding ability of both HLA-DR and -DQ molecules. Proc. Natl Acad. Sci. USA 93, 7202–7206 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Lampson, L. A. & Levy, R. Two populations of Ia-like molecules on a human B cell line. J. Immunol. 125, 293–299 (1980).

    CAS  PubMed  Google Scholar 

  97. Gorga, J. C., Knudsen, P. J., Foran, J. A., Strominger, J. L. & Burakoff, S. J. Immunochemically purified DR antigens in liposomes stimulate xenogeneic cytolytic T cells in secondary in vitro cultures. Cell. Immunol. 103, 160–173 (1986).

    CAS  PubMed  Google Scholar 

  98. Kolstad, A., Johansen, B. & Hannestad, K. Two HLA-DQ-specific human-human hybridoma antibodies (TrG6;TrC5) define epitopes also expressed by a transcomplementing hybrid DQ molecule (DQw7 alpha/DQw4 beta). Hum. Immunol. 24, 15–29 (1989).

    CAS  PubMed  Google Scholar 

  99. Watson, A. J., DeMars, R., Trowbridge, I. S. & Bach, F. H. Detection of a novel human class II HLA antigen. Nature 304, 358–361 (1983).

    CAS  PubMed  Google Scholar 

  100. Ellis, S. A., Taylor, C. & McMichael, A. Recognition of HLA-B27 and related antigen by a monoclonal antibody. Hum. Immunol. 5, 49–59 (1982).

    CAS  PubMed  Google Scholar 

  101. Parham, P. & Brodsky, F. M. Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28. Hum. Immunol. 3, 277–299 (1981).

    CAS  PubMed  Google Scholar 

  102. Schittenhelm, R. B., Dudek, N. L., Croft, N. P., Ramarathinam, S. H. & Purcell, A. W. A comprehensive analysis of constitutive naturally processed and presented HLA-C*04:01 (Cw4)-specific peptides. Tissue Antigens 83, 174–179 (2014).

    CAS  PubMed  Google Scholar 

  103. Braud, V. M., Allan, D. S., Wilson, D. & McMichael, A. J. TAP- and tapasin-dependent HLA-E surface expression correlates with the binding of an MHC class I leader peptide. Curr. Biol. 8, 1–10 (1998).

    CAS  PubMed  Google Scholar 

  104. Thomas, R. et al. HLA-C cell surface expression and control of HIV/AIDS correlate with a variant upstream of HLA-C. Nat. Genet. 41, 1290–1294 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the following funding sources for supporting this work: Australian National Health and Medical Research Council (NHMRC) project grants APP1085018 and APP1084283, and Australian Research Council (ARC) project grant DP150104503. A.W.P. was supported by an NHMRC Principal Research Fellowship (APP 1137739).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia

    Anthony W. Purcell & Sri H. Ramarathinam

  2. The Jenner Institute, Mass Spectrometry Laboratory, _target Discovery Institute, University of Oxford, Oxford, UK

    Nicola Ternette

Authors
  1. Anthony W. Purcell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sri H. Ramarathinam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicola Ternette
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.W.P., S.H.R. and N.T. wrote the manuscript and collated data and figures.

Corresponding authors

Correspondence to Anthony W. Purcell or Nicola Ternette.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference(s) using this protocol

Schittenhelm, R. B., Dudek, N. L., Croft, N. P., Ramarathinam, S. H. & Purcell, A. W. Mol. Cell. Proteomics 15, 1867–1876 (2016): https://doi.org/10.1074/mcp.M115.056358

Scally, S. W. et al. J. Exp. Med. 210, 2569–2582 (2013): https://doi.org/10.1084/jem.20131241

Illing, P. T. et al. Nature 486, 554–558 (2012): https://doi.org/10.1038/nature11147

Wynne, J. W. et al. J. Immunol. 196, 4468–4476 (2016): https://doi.org/10.4049/jimmunol.1502062

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Purcell, A.W., Ramarathinam, S.H. & Ternette, N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc 14, 1687–1707 (2019). https://doi.org/10.1038/s41596-019-0133-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0133-y

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing
  NODES
Association 1
INTERN 2
Note 1
Project 3
twitter 1