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Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.* Contributions of these authors were equivalent and their order should be considered arbitrary. HHS Public AccessAuthor manuscript J Immunol. Author manuscript; available in PMC 2015 October 28. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptCytotoxic T lymphocytes (CTLs) are a part of the immune system concerned with recognition of host cells that express new antigens as a result of viral infection or transformation. CTLs do not recognize new antigens directly, but only as short peptides bound to a deep cleft in class I molecules of the MHC (1-3). Newly synthesized viral and cellular proteins are degraded into peptides in the cytoplasm, transported to the endoplasmic reticulum where they bind to class I molecules, and then expressed on the cell surface (4-7). Each of the allelic forms of the class I MHC molecule binds to a complex mixture of structurally distinct peptides (8,9). Information on the nature of these peptides has been obtained from studies with synthetic peptides (10-12) and from Edman degradation applied to unfractionated mixtures of peptides extracted from five different class I MHC molecules (8). Sequences of 11 peptides extracted from HLA-B27 were identified after highperformance liquid chromatography (HPLC) fractionation and Edman degradation (9). Because HPLC was unable to completely resolve the complex mixture, this analysis could only be applied to the few fractions that contained one or two dominant peptides. Declining PTH (phenylthiohydantoin)-amino acid yields made it difficult to determine the exact number of residues in several peptides.We have applied microcapillary HPLC-electrospray ionization-tandem mass spectrometry to circumvent the above problems. In a matter of hours, this technique determines the molecular mass and therefore maximum length of each peptide component, and the approximate number and quantity of individual peptides. Sequence information can be also obtained on subpicomolar amounts of peptides. We analyzed the naturally processed peptides bound to HLA-A2.1, one of the most widely distributed class I molecules within the human population. The three-dimensional structure of this molecule allows modeling of the complex (2).HLA-A2.1 molecules were purified by immunoprecipitation from the human B lymphoblastoid cell line C1R-A2.1. The associated p...

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.