Extensive Characterization of N-Glycosylation from Major Histocompatibility Complex with Hydrophilic Interaction Chromatography and Mass Spectrometry.

The major histocompatibility complex (MHC) is an essential part of the adaptive immune systems that present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes, which is vital for our body to recognize and respond to foreign pathogens, including viruses, bacteria, and cancerous cells. Human leukocyte antigen (HLA) genes encode proteins located on the surface of most cells in the body, and studies have shown that protein glycosylation plays a significant role in the folding of human leukocyte antigen (HLA) proteins, loading of peptides, and forming of HLA-peptide complex. In the meantime, some of those peptides presented by the MHC system are also glycosylated. Thus, glycosylation has a huge impact on cellular uptake, proteolytic processing, presentation by MHC, and subsequent T-cell priming. However, glycosylation characterization of the whole MHC system has often been neglected during conventional immunopeptidomics analysis due to the lower abundance of glycosylated peptides and increased complexity of the mass spectrum. To tackle this problem, we utilized hydrophilic interaction chromatography to enrich glycopeptides from both peptides bound to HLA and digest of HLA proteins that obtained from immunoprecipitation of MHC complex from cell lysate. Enriched glycopeptides are analyzed by mass spectrometry that enables the characterization of glycosylation of MHC by database search.