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题名: Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC-MS/MS
作者: Chen, Rui1, 2;  Seebun, Deeptee1;  Ye, Mingliang2;  Zou, Hanfa2;  Figeys, Daniel1, 3
关键词: N-glycoproteome ;  Mass spectrometry ;  HEK 293T ;  Glycan composition
刊名: JOURNAL OF PROTEOMICS
发表日期: 2014-05-30
DOI: 10.1016/j.jprot.2014.03.040
卷: 103, 页:194-203
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Biochemical Research Methods
研究领域[WOS]: Biochemistry & Molecular Biology
英文摘要: Glycosylation of membrane proteins plays an important role in cellular behaviors such as cell-cell interaction, immunologic recognition and cell signaling. However, the effective extraction of membrane proteins, the selective isolation of glycopeptides and the mass spectrometric characterization of glycosylation are challenging with current analytical techniques. In this study, a systematic approach was developed which combined: an integrated hydrophilic interaction chromatography solid phase interaction (HILIC SPE) for simultaneous detergent removal and glycopeptide enrichment, and mass spectrometric identification of both protein N-glycosylation sites and site-specific glycan composition. The HILIC SPE conditions were optimized to enable the use of a high concentration of strong detergents, such as SDS and Triton X-100 and to dissolve highly hydrophobic membrane proteins, thus increasing the yield of membrane protein extraction. We illustrated the performance of this approach for the study of membrane protein glycosylation from human embryonic kidney cell lines (HEK 293T). 200 mu g total protein digest was processed using this approach, leading to the identification of 811 N-glycosylation sites from 567 proteins within two experimental replicates. Furthermore, 177 glycopeptides representing 82 N-glycosites with both glycan composition and peptide sequence were identified by high energy collision dissociation.
关键词[WOS]: MASS-SPECTROMETRIC IDENTIFICATION ;  LIQUID-CHROMATOGRAPHY ;  SAMPLE PREPARATION ;  GLYCOPROTEINS ;  QUANTIFICATION ;  GLYCOPEPTIDES ;  ENRICHMENT ;  PROTEOME
语种: 英语
WOS记录号: WOS:000337858700015
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/145596
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Univ Ottawa, Fac Med, Dept Biochem Microbiol & Immunol, Ottawa Inst Syst Biol, Ottawa, ON K1H 8M5, Canada
2.Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
3.Univ Ottawa, Fac Sci, Dept Chem, Ottawa, ON K1N 6N5, Canada

Recommended Citation:
Chen, Rui,Seebun, Deeptee,Ye, Mingliang,et al. Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC-MS/MS[J]. JOURNAL OF PROTEOMICS,2014,103:194-203.
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