LAUSR

N-Glycosylation, being one of the most common and complex protein post-translational modifications (PTMs), is known to have microheterogeneity with 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 sofar been successfully in large-scale characterization of these N-glycan at the composition level, and structure-level identification and quantitation is urgently needed. Here we report our development of intact N-glycopeptide search engine GPSeeker and GPSeeker-centered quantitative structural N-glycoproteomics pipeline. In benchmark characterization of differentially expressed N-glycosylation in hepatocellular carcinoma HepG2 cells relative to LO2 cells, 5,584 and 781 intact N-glycopeptides with putative linkage structures were identified and quantified with isotopic dimethyl labelling and 2DLC-MS/MS analysis. Among 5,584 IDs, 911 were identified with more than structure diagnostic fragment ions for the N-glycan moiety. 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; microhegerogeneity with different differentially expressions were observed on 183 out of 231 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.