Journal of Proteome Research · 2019 · 102 citations · 35 references
Life SciencesBiochemistryOncologyGlycobiologyMolecular OncologyProteomicsCancer GlycobiologyGlycan AnalysisGlycan StructureGlycoproteomicsN-linked GlycosylationProtein GlycosylationGlycan Structure AnalysisGlycopeptide AnalysisGlycoproteomics QuantificationMass Spectrometry-based GlycoproteomicsN-glycan AnalysisN-glycan HeterogeneityN-glycan ProfilingN-glycosylation In Cancer
N‑glycosylation is a common, highly heterogeneous post‑translational modification whose diverse structures can have identical monosaccharide compositions yet distinct expression patterns and pathological roles, and while mass spectrometry has enabled large‑scale compositional profiling, structure‑level identification and quantitation remain urgently needed. The authors developed GPSeeker, an intact N‑glycopeptide search engine, and a GPSeeker‑centered quantitative structural N‑glycoproteomics pipeline to address this gap. They benchmarked the pipeline on HepG2 hepatocellular carcinoma cells versus normal LO2 cells, identifying 5,405 and 1,081 intact N‑glycopeptides with putative linkage structures and quantifying them via isotopic dimethyl labeling and 2‑D LC‑MS/MS. The analysis revealed 837 glycopeptides with diagnostic fragment ions, chromatographic separation of double and quadruple isomeric sialic acid and fucose linkages, and microheterogeneity across 183 of 231 quantified sites, demonstrating the pipeline’s capacity for precise qualitative and quantitative N‑glycosylation profiling with physiological and pathological relevance.
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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