Identification of MHC-Associated Peptides and Phosphopeptides Presented in Various Diseased Cells by Tandem Mass Spectrometry

Major histocompatibility complex (MHC) class I and II molecules are involved in the presentation of antigens to T cells, which can initiate an immune response upon recognition of abnormal peptide sequences and modifications. Specifically, phosphopeptides resulting from aberrantly increased protein phosphorylation in cancer are proposed as potential cancer-specific targets for developing novel immunotherapeutic treatments. However, it remains as a challenge to accurately identify these peptide sequences and to localize modification sites due to factors including lack of donors for certain tissue types, tissue-based variations in MHC expression, sample loss during preparation, and interfering species. We believe that mass spectrometry (MS) is a valuable tool for immunopeptide discovery. This work summarizes our approach for the identification of MHC class I phosphopeptides and MHC class II peptides by combining high-performance liquid chromatography and electrospray ionization with tandem mass spectrometry (HPLC-ESI-MS/MS). MHC class I peptides were isolated from tumor samples and their non-cancerous surrounding tissues collected from hepatocellular carcinoma (HCC) patients. Due to the low abundance of MHC class I phosphopeptides compared to unmodified peptides, we utilized MS-compatible immobilized metal affinity chromatography as an enrichment technique for phosphopeptides. This approach for MHC class I phosphopeptidomics analysis was further applied to other clinical samples including primary tumor tissues collected from head and neck squamous cell carcinoma patients, as well as to cell lines. Interestingly, the majority of the identified phosphopeptides have been previously found in other tumor samples analyzed in our lab. With our optimized sample preparation methods, we expanded our analysis to both MHC class I and II peptides isolated from tissues (on the scale of a few hundred milligrams) that are potentially involved in immune surveillance of the brain. A comparison of the presented peptides in samples from patients at different neurological states (no known condition/neurodegenerative diseases/brain tumors) provided mass spectrometric evidence for several dysregulated proteins associated with these diseases. Our analysis has revealed the presence of hundreds of naturally presented MHC peptides in various diseased cells. We believe that these disease-specific MHC-associated peptides hold enormous potential for the development of immunotherapeutic treatments that are applicable to multiple diseases.