Identification of protein O-glycosylation site and corresponding glycans using liquid chromatography-tandem mass spectrometry via mapping accurate mass and retention time shift

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Title:	Identification of protein O-glycosylation site and corresponding glycans using liquid chromatography-tandem mass spectrometry via mapping accurate mass and retention time shift
Authors:	陳彥文;Huang, Li-Juan;Lin, Jen-Hui;Tsai, Jung-Heng;Chu, Yen-Yin;Chen, Yen-Wen;Chen, Shun-Li;Chen, Shu-Hui
Contributors:	資訊電機學院通訊工程學系
Keywords:	Amino Acid Sequence;Analysis;Animals;Biological and medical sciences;Caseins - chemistry;Cattle;Chromatography;Chromatography, High Pressure Liquid - methods;dephosphorylation;dissociation;Enbrel;enzymes;fetuins;Filtering;Filtration;General pharmacology;Glycan;Glycopeptides;Glycopeptides - chemistry;Glycosylation;kappa-casein;LC–MS;liquid chromatography;Mathematical models;Medical sciences;Molecular Sequence Data;Molecular Weight;O-glycosylation;O-glycosylation site;Pharmacology. Drug treatments;Phosphorylation;polysaccharides;Polysaccharides - chemistry;protein tagging;serine;tandem mass spectrometry;Tandem Mass Spectrometry - methods;therapeutics;threonine;β-Elimination
Date:	2014-01-01
Issue Date:	2026-04-23 13:02:30 (UTC+8)
Publisher:	Elsevier;Amsterdam: Elsevier B.V
Abstract:	摘要： •O-glycosylation sites were identified from cleaved digests made by a feasible kit.•Intact glycopeptides of each site were mapped based on in-house glycan database.•Retention time filtering reduced 90% of FDR associated with the O-glycan mapping.•Site-specific O-glycosylations of Enbrol® and Factor IX therapeutics were reported. We reported an improved combinatorial approach for identifying site-specific O-glycosylation using both glycan cleaved and non-cleaved methods. In this approach, a non-reducing β-elimination kit coupled with non-specific enzymes performed efficient digestion, O-glycan cleavage, and partial dephosphorylation without significant side reactions, thus enabling an automatic database search for the cleaved O-glycosylation or serine/threonine (S/T) phosphorylation sites. From the same sample concurrently prepared without β-elimination, the corresponding intact O-glycopeptides were mapped by accurate precursor ion mass using an in-house glycan database majorly composed of GalNAc (mucin-type) core and the retention-time shift (ΔRt). Each glycopeptide assignment was verified by the detection of glycan-specific fragments using collision-induced dissociation (CID) to estimate False Discovery Rate (FDR). Using fetuin as a model, all identified S/T elimination sites were matched to multiple intact glycopeptides with a 31% FDR. This considerably reduced to 0% FDR by ΔRt filtering. This approach was then applied to a protein mixture composed of therapeutic Factor IX and Enbrel® mixed with fetuin and kappa-casein. A total of 26 glycosylation sites each of which corresponds to 1–4 glycans were positively mapped and confirmed. The FDR decreased from 33% to 3.3% by ΔRt filtering and exclusion of repeated peptide tags that covered the same glycosylation sites. Moreover, the phosphorylation and O-glycosylation on the same site such as T159 of Factor IX and T170 of kappa-casein were able to be unambiguously differentiated. Thus, our approach is useful for in-depth characterization of site-specific O-glycosylation of a simple mixture such as protein-based therapeutics. 其他題名： J Chromatogr A 出版者： Amsterdam: Elsevier B.V 出版日期： 2014-12-05 出處： Journal of Chromatography A, 2014-12, Vol.1371, p.136-145 版權： 2014 Elsevier B.V. 版權： 2015 INIST-CNRS 版權： Copyright © 2014 Elsevier B.V. All rights reserved. 識別號： ISSN: 0021-9673 識別號： ISSN: 1873-3778 識別號： EISSN: 1873-3778 識別號： DOI: 10.1016/j.chroma.2014.10.046 識別號： PMID: 25456591 識別號： CODEN: JOCRAM
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