Site‐ and structure‐specific characterization of the human urinary N‐glycoproteome with site‐determining and structure‐diagnostic product ions

Abstract

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.

The glycosylation in SARS-CoV-2 and its receptor ACE2

“… 252 This kind of ions are in fact the fragmented N-glycan A/B/C/X/Y/Z ions which can independently distinguish a specific structure from the structural isomers. 253 , 254 N-glycan structures can be discriminate by detecting theoretical structure-diagnostic ions which are generated in silico relying on the theoretical N-glycan structure database created by the retrosynthetic strategy, 78 and structures of intact N-glycopeptides are figured out by assigning N-glycan structures to peptide backbones. 255 …”

Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity

“…Glycosylation plays critical roles in protein functions and cellular processes, and its aberration is closely associated with human diseases, including cancers [111], neurodegenerative diseases [112,113], immunology disorders [49] and virus infection [114]. Quantitative glycoproteomics studies characterize disease-related micro-and macro-heterogeneous glycosylation changes in purified proteins (e.g., immunoglobulins and therapeutic proteins) [49,[115][116][117][118][119][120][121][122] and complex samples, including serum [52,66,67], urine [51,123], cerebrospinal fluid [124], milk [125], tissues, cells and exosomes [126,127]. Altered protein glycosylation is a hallmark of various cancers and has become a promising target for disease biomarkers [128].…”

Progress of proteomics‐driven precision medicine: From a glycosylation view

“…The final important step for glycoproteomics analysis is data interpretation with the help of diverse bioinformatics tools, such as Byonic, 43 GP Finder, 44 GPQuest, 45 pMatchGlyco, 46 GPSeeker, 47,48 MSFragger‐Glyco, 49 as well as recently published pGlyco3 50 and StrucGP 51 . Different from the identification of unmodified peptides, it is quite difficult in regard to glycopeptides which requires simultaneous identification of peptides, glycans, and glycosylation sites.…”