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Updated: Jan 24, 2026
Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments
Published on: February 7, 2025
GPSeeker Enables Quantitative Structural N-Glycoproteomics for Site- and Structure-Specific Characterization of
1School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability , Tongji University , Shanghai 200092 , China.
Abstract:
N-Glycosylation, being one of the most common and complex protein post-translational modifications (PTMs), is known to have microheterogeneity with the presence of different N-glycan structures at a single specific glycosite. These different structures may have exactly the same monosaccharide composition but totally different differential expressions and pathological relevance. Mass spectrometry-based N-glycoproteomics has so far been successful in large-scale characterization of these N-glycans at the composition level, and structure-level identification and quantitation is urgently needed. Here we report our development of the intact N-glycopeptide search engine GPSeeker and the GPSeeker-centered quantitative structural N-glycoproteomics pipeline. In benchmark characterization of differentially expressed N-glycosylation in hepatocellular carcinoma HepG2 cells relative to LO2 cells, 5 405 and 1 081 intact N-glycopeptides with putative linkage structures were identified and quantified with isotopic dimethyl labeling and 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Among the 5 405 IDs, 837 were identified with no less than one structure diagnostic fragment ion for the N-glycan moieties. Besides double isomers of sialic acid linkages and fucose sequences, quadruple isomers from combination of two linages and two sequences were chromatographically separated and confidently identified; microheterogeneity with different differentially expressions were observed on 183 out of the 231 quantified N-glycosites. This GPSeeker-centered quantitative structural N-glycoproteomics pipeline can be widely applied to precise qualitative and quantitative characterization of N-glycosylation with physiological and pathological relevance.
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