GPSeeker Enables Quantitative Structural N‑Glycoproteomics for Site- and Structure-Specific Characterization of Differentially Expressed N‑Glycosylation in Hepatocellular Carcinoma

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.