, Volume 61, Issue 3, pp 241–246 | Cite as

Identification of naturally processed ligands in the C57BL/6 mouse using large-scale mass spectrometric peptide sequencing and bioinformatics prediction

  • Julio C. Delgado
  • Hernando Escobar
  • David K. Crockett
  • Eduardo Reyes-Vargas
  • Peter E. Jensen
Brief Communication


Most major histocompatibility complex (MHC) class I–peptide-binding motifs are currently defined on the basis of quantitative in vitro MHC–peptide-binding assays. This information is used to develop bioinformatics-based tools to predict the binding of peptides to MHC class I molecules. To date few studies have analyzed the performance of these bioinformatics tools to predict the binding of peptides determined by sequencing of naturally processed peptides eluted directly from MHC class I molecules. In this study, we performed large-scale sequencing of endogenous peptides eluted from H2Kb and H2Db molecules expressed in spleens of C57BL/6 mice. Using sequence data from 281 peptides, we identified novel preferred anchor residues located in H2Kb and H2Db-associated peptides that refine our knowledge of these H2 class I peptide-binding motifs. The analysis comparing the performance of three bioinformatics methods to predict the binding of these peptides, including artificial neural network, stabilized matrix method, and average relative binding, revealed that 61% to 94% of peptides eluted from H2Kb and H2Db molecules were correctly classified as binders by the three algorithms. These results suggest that bioinformatics tools are reliable and efficient methods for binding prediction of naturally processed MHC class I ligands.


MHC Motifs C57BL/6 Mass spectrometry Bioinformatics 



This work was supported by the ARUP Institute for Clinical and Experimental Pathology® (to J.C.D., D.K.C., and P.E.J.).

Supplementary material

251_2009_360_MOESM1_ESM.doc (362 kb)
ESM 1 (DOC 370 kb)


  1. Achour A, Michaëlsson J, Harris RA, Odeberg J, Grufman P, Sandberg JK, Levitsky V, Kärre K, Sandalova T, Schneider G (2002) A structural basis for LCMV immune evasion: subversion of H-2D(b) and H-2K(b) presentation of gp33 revealed by comparative crystal structure analyses. Immunity 17:757–768 doi: 10.1016/S1074-7613(02)00478-8 PubMedCrossRefGoogle Scholar
  2. Bertoni R, Sidney J, Fowler P, Chesnut RW, Chisari FV, Sette A (1997) Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis. J Clin Invest 100:503–513 doi: 10.1172/JCI119559 PubMedCrossRefGoogle Scholar
  3. Bui HH, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton KA, Mothe BR, Chisari FV, Watkins DI, Sette A (2005) Automated generation and evaluation of specific MHC binding predictive tools: ARB matrix applications. Immunogenetics 57:304–314 doi: 10.1007/s00251-005-0798-y PubMedCrossRefGoogle Scholar
  4. Buus S, Lauemoller SL, Worning P, Kesmir C, Frimurer T, Corbet S, Fomsgaard A, Hilden J, Holm A, Brunak S (2003) Sensitive quantitative predictions of peptide-MHC binding by a ‘Query by Committee’ artificial neural network approach. Tissue Antigens 62:378–384 doi: 10.1034/j.1399-0039.2003.00112.x PubMedCrossRefGoogle Scholar
  5. Doolan DL, Hoffman SL, Southwood S, Wentworth PA, Sidney J, Chesnut RW, Keogh E, Appella E, Nutman TB, Lal AA, Gordon DM, Oloo A, Sette A (1997) Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles. Immunity 7:97–112 doi: 10.1016/S1074-7613(00)80513-0 PubMedCrossRefGoogle Scholar
  6. Escobar H, Crockett DK, Reyes-Vargas E, Baena A, Rockwood AL, Jensen PE, Delgado JC (2008) Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs. J Immunol 181:4874–4882PubMedGoogle Scholar
  7. Falk K, Rötzschke O, Rammensee HG (1990) Cellular peptide composition governed by major histocompatibility complex class I molecules. Nature 348:248–251 doi: 10.1038/348248a0 PubMedCrossRefGoogle Scholar
  8. Falk K, Rötzschke O, Stevanović S, Jung G, Rammensee HG (1991) Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351:290–296 doi: 10.1038/351290a0 PubMedCrossRefGoogle Scholar
  9. Fortier MH, Caron E, Hardy MP, Voisin G, Lemieux S, Perreault C, Thibault P (2008) The MHC class I peptide repertoire is molded by the transcriptome. J Exp Med 205:595–610 doi: 10.1084/jem.20071985 PubMedCrossRefGoogle Scholar
  10. Fremont DH, Matsumura M, Stura EA, Peterson PA, Wilson IA (1992) Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science 257:919–927 doi: 10.1126/science.1323877 PubMedCrossRefGoogle Scholar
  11. Hammerling GJ, Rusch E, Tada N, Kimura S, Hammerling U (1982) Localization of allodeterminants on H-2Kb antigens determined with monoclonal antibodies and H-2 mutant mice. Proc Natl Acad Sci U S A 79:4737–4741 doi: 10.1073/pnas.79.15.4737 PubMedCrossRefGoogle Scholar
  12. Karunakaran KP, Rey-Ladino J, Stoynov N, Berg K, Shen C, Jiang X, Gabel BR, Yu H, Foster LJ, Brunham RC (2008) Immunoproteomic discovery of novel T cell antigens from the obligate intracellular pathogen Chlamydia. J Immunol 180:2459–2465PubMedGoogle Scholar
  13. Kisselev AF, Akopian TN, Woo KM, Goldberg AL (1999) The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 274:3363–3371 doi: 10.1074/jbc.274.6.3363 PubMedCrossRefGoogle Scholar
  14. Lundegaard C, Nielsen M, Lund O (2006) The validity of predicted T-cell epitopes. Trends Biotechnol 24:537–538 doi: 10.1016/j.tibtech.2006.10.001 PubMedCrossRefGoogle Scholar
  15. Matsueda S, Takedatsu H, Sasada T, Azuma K, Ishihara Y, Komohara Y, Noguchi M, Shichijo S, Itoh K, Harada M (2007) New peptide vaccine candidates for epithelial cancer patients with HLA-A3 supertype alleles. J Immunother 30:274–281 doi: 10.1097/01.cji.0000211340.88835.e7 PubMedCrossRefGoogle Scholar
  16. Minami T, Matsueda S, Takedatsu H, Tanaka M, Noguchi M, Uemura H, Itoh K, Harada M (2007) Identification of SART3-derived peptides having the potential to induce cancer-reactive cytotoxic T lymphocytes from prostate cancer patients with HLA-A3 supertype alleles. Cancer Immunol Immunother 56:689–698 doi: 10.1007/s00262-006-0216-9 PubMedCrossRefGoogle Scholar
  17. Molano A, Erdjument-Bromage H, Fremont DH, Messaoudi I, Tempst P, Nikolić-Zugić J (1998) Peptide selection by an MHC H-2Kb class I molecule devoid of the central anchor (“C”) pocket. J Immunol 160:2815–2823PubMedGoogle Scholar
  18. Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, Brunak S, Lund O (2003) Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12:1007–1017 doi: 10.1110/ps.0239403 PubMedCrossRefGoogle Scholar
  19. Peters B, Sette A (2005) Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method. BMC Bioinformatics 6:132 doi: 10.1186/1471–2105–6–132 PubMedCrossRefGoogle Scholar
  20. Peters B, Bui HH, Frankild S, Nielson M, Lundegaard C, Kostem E, Basch D, Lamberth K, Harndahl M, Fleri W, Wilson SS, Sidney J, Lund O, Buus S, Sette A (2006) A community resource benchmarking predictions of peptide binding to MHC-I molecules. PLOS Comput Biol 2:e65 doi: 10.1371/journal.pcbi.0020065 PubMedCrossRefGoogle Scholar
  21. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanović S (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50:213–219 doi: 10.1007/s002510050595 PubMedCrossRefGoogle Scholar
  22. Ressing ME, de Jong JH, Brandt RM, Drijfhout JW, Benckhuijsen WE, Schreuder GM, Offringa R, Kast WM, Melief CJ (1999) Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma. Eur J Immunol 29:1292–1303 doi: 10.1002/(SICI)1521-4141(199904)29:04<1292::AID-IMMU1292>3.0.CO;2-6 PubMedCrossRefGoogle Scholar
  23. Saric T, Chang S, Hattori A, York I, Markant S, Rock K, Tsujimoto M, Goldberg A (2002) An IFN-γ–induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I–presented peptides. Nat Immunol 3:1169–1176 doi: 10.1038/ni859 PubMedCrossRefGoogle Scholar
  24. Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18:6097–6100 doi: 10.1093/nar/18.20.6097 PubMedCrossRefGoogle Scholar
  25. Serwold T, Gonzalez F, Kim J, Jacob R, Shastri N (2002) ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum. Nature 419:480–483 doi: 10.1038/nature01074 PubMedCrossRefGoogle Scholar
  26. Stern LJ (2007) Characterizing MHC-associated peptides by mass spectrometry. J Immunol 179:2667–2668PubMedGoogle Scholar
  27. Toes RE, Nussbaum AK, Degermann S, Schirle M, Emmerich NP, Kraft M, Laplace C, Zwinderman A, Dick TP, Muller J, Schonfisch B, Schmid C, Fehling HJ, Stevanovic S, Rammensee HG, Schild H (2001) Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products. J Exp Med 194:1–12 doi: 10.1084/jem.194.1.1 PubMedCrossRefGoogle Scholar
  28. Trachtenberg E, Korber B, Sollars C, Kepler TB, Hraber PT, Hayes E, Funkhouser R, Fugate M, Theiler J, Hsu YS, Kunstman K, Wu S, Phair J, Erlich H, Wolinsky S (2003) Advantage of rare HLA supertype in HIV disease progression. Nat Med 9:928–935 doi: 10.1038/nm893 PubMedCrossRefGoogle Scholar
  29. York I, Chang S, Saric T, Keys J, Favreau J, Goldberg A, Rock K (2002) The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8–9 residues. Nat Immunol 3:1177–1184 doi: 10.1038/ni860 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Julio C. Delgado
    • 1
    • 2
  • Hernando Escobar
    • 1
  • David K. Crockett
    • 2
  • Eduardo Reyes-Vargas
    • 1
  • Peter E. Jensen
    • 1
    • 2
  1. 1.Department of PathologyUniversity of UtahSalt Lake CityUSA
  2. 2.ARUP Institute for Clinical & Experimental Pathology®Salt Lake CityUSA

Personalised recommendations