HLA-Glyco: A large-scale interrogation of the glycosylated immunopeptidome

MHC-associated peptides (MAPs) bearing post-translational modifications (PTMs) have raised intriguing questions regarding their attractiveness for targeted therapies. Here, we developed a novel computational glyco-immunopeptidomics workflow that integrates the ultrafast glycopeptide search of MSFragger with a glycopeptide-focused false discovery rate (FDR) control. We performed a harmonized analysis of 8 large-scale publicly available studies and found that glycosylated MAPs are predominantly presented by the MHC class II. We created HLA-Glyco, a resource containing over 3,400 human leukocyte antigen (HLA) class II N-glycopeptides from 1,049 distinct protein glycosylation sites. Our comprehensive resource reveals high levels of truncated glycans, conserved HLA-binding cores, and differences in glycosylation positional specificity between classical HLA class II allele groups. To support the nascent field of glyco-immunopeptidomics, we include the optimized workflow in the FragPipe suite and provide HLA-Glyco as a free web resource.