Extending the Mass Spectrometry-Detectable Landscape of MHC Peptides by Use of Restricted Access Material

Mass spectrometry-based immunopeptidomics enables the comprehensive identification of major histocompatibility complex (MHC) peptides from a cell culture as well as from tissue or tumor samples and is applied for the identification of tumor-specific and viral T-cell epitopes. Although mass spectrometry is generally considered an unbiased method for MHC peptide identification, the physicochemical properties of MHC peptides can greatly influence their detectability. Here, we demonstrate that highly hydrophobic peptides are lost 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 involving restricted access material (RAM). Compared to C18-SPE, RAM-SPE improved the overall MHC peptide recovery and extended the landscape of mass spectrometry-detectable MHC peptides toward more hydrophobic peptides.

Automated summary

Mass spectrometry-based immunopeptidomics allows for comprehensive identification of MHC peptides, but their detectability can be influenced by their physicochemical properties. Highly hydrophobic peptides are lost during sample preparation using C18 solid-phase extraction (SPE). To overcome this limitation, an optimized protocol involving restricted access material (RAM) was established, which improved overall MHC peptide recovery and expanded the range of mass spectrometry-detectable MHC peptides to include more hydrophobic peptides.