Integrated Strategy for Large-Scale Investigation on Protein Core Fucosylation Stoichiometry Based on Glycan-Simplification and Paired-Peaks-Extraction
datasetposted on 2020-01-30, 17:35 authored by Xinyuan Zhao, Zixiang Yu, Yi Huang, Chang Liu, Mingchao Wang, Xiaoyu Li, Xiaohong Qian, Wantao Ying
Core fucosylation (CF) is a special form of N-glycosylation and plays an important role in pathological and biological processes. Increasing efforts in this area are focused on the identification of CF glycosites, whereas evidence showed that the stoichiometry of CF occupancy is functionally important. Here, an integrated strategy based on “Glycan-Simplification and Paired-Peaks-extraction” (GSPPE) for detecting large-scale stoichiometries of CF was developed. After HILIC enrichment of intact glycopeptides, sequential cleavages by endoglycosidases H and endoglycosidases F3 were performed to generate simplified glycopeptide forms (SGFs), i.e., peptide-GlcNAc (pep-HN) and peptide-GlcNAc-Fucose (pep-CF). These paired SGFs were found to be eluted consecutively on a reversed-phase chromatography column, which allowed us to obtain peak areas of SGF pairs, even if only one of the peaks was captured by the mass spectrometer (MS), by introducing the Paired-Peaks-Extraction algorithm. Thus, the missing value dilemma of random data-dependent MS/MS acquisition was reduced and the stoichiometry of site-specific CF could be calculated. We systematically evaluated the feasibility of this strategy using standard glycoproteins and then explored urinary samples from healthy individuals and hepatocellular carcinoma (HCC) patients. In total, 1449 highly reliable core fucose glycosites and their corresponding CF stoichiometries were obtained. Dozens of glycosites that differed significantly in the urine of healthy individuals and HCC patients were disclosed. The developed approach and program presented here may promote studies on core fucosylation and lead to a deeper understanding of their dysregulation in physiological- or pathological processes.
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GSPPEIntegrated Strategyreversed-phase chromatography columnglycopeptide formsendoglycosidases F 3CF stoichiometriesHCC patientsvalue dilemmasequential cleavagesProtein Core Fucosylation Stoichiometrycore fucosylationsite-specific CFendoglycosidases HLarge-Scale InvestigationMSHILIC enrichmentCF occupancyhepatocellular carcinomapeak areasCF glycositesSGF pairscore fucose glycositesmass spectrometerPaired-Peaks-Extraction Core fucosylationPaired-Peaks-Extraction algorithm