Identification of intact N- and O-glycopeptides of Ganoderma lucidum using ZIC-HILIC enrichment and high-resolution mass spectrometry (HRMS)

In this study, a comprehensive identification of protein glycosylation of G. lucidum polysaccharide peptide (GL-PPSQ₂) was investigated. With ZIC-HILIC enrichment, intact N- and O-glycopeptides were analyzed by higher-energy collisional dissociation (HCD) fragmentation on nanoRPLC-ESI-MS/MS. A total of 48 proteins and 62 peptides were identified, including 57 unique peptide segments. Glycoproteomic profiling using pGlyco revealed 42 intact N-glycopeptides from 35 N-glycosites of 32 N-glycoproteins, whereas GPSeeker identified four intact N-glycopeptides with regular N-glycan core structures. For O-glycosylation, pGlyco annotated 338 intact O-glycopeptides from 287 O-glycosites of 160 O-glycoproteins, revealing a marked predominance and heterogeneity of O-glycosylation. These glycopeptides exhibited diverse glycan compositions, including high mannose, complex, and hybrid types, as well as macro- and microheterogeneity. This study presents direct MS analysis of GL-PPSQ₂ glycosylation, enhancing the understanding of glycosylation modifications and glycoprotein research. The findings elucidate the glycosylation patterns and structural characteristics of GL-PPSQ₂, providing scientific evidence for understanding its biological effects and supporting the development of functional foods based on G. lucidum polysaccharide peptide components.