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Updated: Dec 8, 2025
Bioinformatics Resources for the Study of Glycan-Mediated Protein Interactions
Published on: January 20, 2022
Site- and structure-specific characterization of the human urinary N-glycoproteome with site-determining and
Yun Shen1, Kaijie Xiao1, Zhixin Tian1
1School of Chemical Science and Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai, 200092, China.
Rationale:
N-glycosylation is one of the most common protein post-translational modifications; it is extremely complex with multiple glycoforms from different monosaccharide compositions, sequences, glycosidic linkages, and anomeric positions. Each glycoform functions with a particular site- and structure-specific N-glycan that can be fully characterized using state-of-the-art tandem mass spectrometry (MS/MS) and the intact N-glycopeptide database search engine GPSeeker that we recently developed. Urine has recently gained increasing attention as a non-invasive source for disease marker discovery. In this study, we report our structure-specific N-glycoproteomics study of human urine.
Methods:
We performed trypsin digestion, Zwitterionic Hydrophilic Interaction chromatography (ZIC-HILIC) enrichment, C18-RPLC/nano-ESI-MS/MS using HCD with stepped normalized collisional energies, and GPSeeker database search for a comprehensive site- and structure-specific N-glycoproteomics characterization of the human urinary N-glycoproteome at the intact N-glycopeptide level. For this, we used b/y product ion pairs from the GlcNAc-containing site-determining peptide backbone and structure-diagnostic product ions from the N-glycan moieties, respectively.
Results:
We identified 2986 intact N-glycopeptides with comprehensive site and structure information for the peptide backbones (amino acid sequences and N-glycosites) and the N-glycan moieties (monosaccharide compositions, sequences/linkages). The 2986 intact N-glycopeptide IDs corresponded to 754 putative N-glycan linkage structures on 419 N-glycosites of 450 peptide backbones from 327 intact N-glycoproteins. Next, 146 linkage structures and 200 N-glycosites were confirmed with structure-diagnostic and GlcNAc-containing site-determining product ions, respectively.
Conclusions:
We found 106 new N-glycosites not annotated in the current UniProt database. The elution-abundance patterns of urinary intact N-glycopeptide oxonium ions (m/z 138 and 204) of the same subject were temporally stable during the day and over 6 months. These patterns are rather different among different subjects. The results implied an interesting possibility that glycopeptide oxonium ion patterns could serve as distinguishing markers between individuals and/or between physiological and pathological states.
