Extending the Mass Spectrometry-Detectable Landscape of the Immunopeptidome by use of Restricted access Material

Mass spectrometry-based immunopeptidomics enables the comprehensive identification of major histocompatibility complex (MHC) peptides from cell culture as well as from tissue or tumor samples and is increasingly applied for the identification of tumor-specific and viral T-cell epitopes. Although mass spectrometry is generally accounted as an unbiased method for MHC peptide identification, the physicochemical properties of MHC peptides can greatly influence their detectability. For instance, highly hydrophobic peptides get easily lost already during sample preparation when C18 solid phase extraction (SPE) is used for separating MHC peptides from proteins. To overcome this limitation, we established an optimized protocol for this sample preparation step applying restricted access material (RAM). Compared to C18-SPE, RAM-SPE improves the overall MHC peptide recovery and extends the landscape of mass spectrometry-detectable MHC peptides towards more hydrophobic peptides.