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学科主题物理化学
Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion
Wu, Shuaibin1,2; Yang, Kaiguang1; Liang, Zhen1; Zhang, Lihua1; Zhang, Yukui1; Zhang LH(张丽华)
关键词Acid Cleavage Mass Spectrometry Protein Identification Proteomics Sample Preparation
刊名TALANTA
2011-10-30
ISSN待补充
DOI10.1016/j.talanta.2011.08.052
86期:待补充页:429-435
收录类别SCI
文章类型Article
部门归属18
项目归属1810
产权排名1,1
WOS标题词Science & Technology ; Physical Sciences
类目[WOS]Chemistry, Analytical
研究领域[WOS]Chemistry
关键词[WOS]LC-ESI-MS/MS ; HYDROLYSIS ; THROUGHPUT ; MIXTURES ; RESIDUES
英文摘要Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion; A formic acid (FA)-assisted sample preparation method was presented for protein identification via mass spectrometry (MS). Detailedly, an aqueous solution containing 2% FA and dithiothreitol was selected to perform protein denaturation, aspartic acid (D) sites cleavage and disulfide linkages reduction simultaneously at 108 degrees C for 2h. Subsequently. FA wiped off via vacuum concentration. Finally, iodoacetamide (IAA) alkylation and trypsin digestion could be performed ordinally. A series of model proteins (BSA. beta-lactoglobulin and apo-Transferrin) were treated respectively using such method, followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The identified peptide number was increased by similar to 80% in comparison with the conventional urea-assisted sample preparation method. Moreover, BSA identification was achieved efficiently down to femtomole (25 +/- 0 sequence coverage and 16 +/- 1 peptides) via such method. In contrast, there were not peptides identified confidently via the urea-assisted method before desalination via the C18 zip tip. The absence of urea in this sample preparation method was an advantage for the more favorable digestion and MALDI-TOF MS analysis. The performances of two methods for the real sample (rat liver proteome) were also compared, followed by a nanoflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry system analysis. As a result, 1335 +/- 43 peptides were identified confidently (false discovery rate <1%) via FA-assisted method, corresponding to 295 +/- 12 proteins (of top match = 1 and requiring 2 unique peptides at least). In contrast, there were only 1107 +/- 16 peptides (corresponding to 231 +/- 10 proteins) obtained from the conventional urea-assisted method. It was serving as a more efficient protein sample preparation method for researching specific proteomes better, and providing assistance to develop other proteomics analysis methods, such as, peptide quantitative analysis. (C) 2011 Elsevier B.V. All rights reserved.
语种英语
WOS记录号WOS:000298126300057
引用统计
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/115860
专题中国科学院大连化学物理研究所
通讯作者Zhang LH(张丽华)
作者单位1.Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China
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GB/T 7714
Wu, Shuaibin,Yang, Kaiguang,Liang, Zhen,et al. Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion[J]. TALANTA,2011,86(待补充):429-435.
APA Wu, Shuaibin,Yang, Kaiguang,Liang, Zhen,Zhang, Lihua,Zhang, Yukui,&张丽华.(2011).Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion.TALANTA,86(待补充),429-435.
MLA Wu, Shuaibin,et al."Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion".TALANTA 86.待补充(2011):429-435.
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