Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a powerful tool that allows the characterization of MHC-associated peptidomes. In addition to conventional nonmodified peptides, these complex mixtures also contain phosphorylated species, which may be of great interest for personalized cancer immunotherapy. Here, we provide a detailed protocol to identify phosphopeptides displayed by human HLA class I molecules consisting of four main steps: (1) immunopurification of MHC-I molecules, (2) phosphopeptide enrichment, (3) LC-MS/MS analysis, and (4) MS/MS ion search.
Keywords: Affinity chromatography; HLA; IMAC; LC–MS/MS; MHC; MS/MS ion search; W6/32.
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.