Purification of HLA Immunopeptidomes from Human Thymus

Mass spectrometry has become an essential technique for the analysis of peptide repertoires presented by MHC molecules to T lymphocytes. Years ago, analyses of MHC peptidomes were performed using a great number of cells, and cell lines were chosen as the main peptide source. Mass spectrometry devices have been improved in terms of sensitivity and resolution, making feasible the analysis of samples with relatively small amounts of cells. Thus, analyses of MHC peptide repertoires from different tissue samples are now available. Here, I describe a protocol to process human thymus samples to purify HLA class I- or HLA-DR-associated peptidomes. For that, cells are lysed using a nonionic detergent together with a mechanical cell rupture. Immunopeptidomes are purified by immunoaffinity chromatography. The peptide pool is fractionated by ionic chromatography. Finally, peptide fragmentation and identification are conducted by LC-MS/MS and the use of MASCOT search engine.