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Download fileIntegrated Strategy for Large-Scale Investigation on Protein Core Fucosylation Stoichiometry Based on Glycan-Simplification and Paired-Peaks-Extraction
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posted on 2020-01-30, 17:35 authored by Xinyuan Zhao, Zixiang Yu, Yi Huang, Chang Liu, Mingchao Wang, Xiaoyu Li, Xiaohong Qian, Wantao YingCore
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