Una sonda hidrofílica polimérica zwitteriónica para mapear el glicopéptidoma endógeno del suero humano

Endogenous glycopeptides in human serum are valuable candidates for disease biomarker discovery, but their low abundance and structural complexity pose significant analytical challenges. In this study, we developed and optimized a robust workflow for enriching and in-depth characterizing the endogenous glycopeptidome using a novel hydrophilic interaction liquid chromatography (HILIC) sorbent. The HILIC-based strategy effectively enriched glycopeptides from mono-glycosylated IgG and avidin digests, as well as multi-glycosylated horseradish peroxidase (HRP), demonstrating high selectivity, femtomolar-level sensitivity (down to 1 fmol), reproducibility (RSD > 1), and reusability for up to four cycles. Comprehensive analysis by mass spectrometry (MS) identified 334 endogenous intact N-linked glycopeptides, including 318 N-glycosites from 289 glycoproteins. In addition, 242 endogenous intact O-linked glycopeptides from 38 glycoproteins are identified. These represent one of the most extensive endogenous glycopeptide datasets to date. N-glycans were primarily complex biantennary types, while Core 1 structures dominated O-glycans. Motif analysis revealed a strong preference for canonical N-glycosylation motifs (NXT/NXS) and a nearly equal distribution of O-glycosylation on serine and threonine residues. Gene Ontology analysis indicated functional enrichment in membrane-associated, catalytic, and immune-related processes. This optimized HILIC-MS platform enables sensitive and reproducible glycoproteomic profiling, providing a valuable tool for biomarker discovery.