Evaluation and minimization of over-alkylation in proteomic sample preparation

doi:10.1016/j.ijms.2022.116919

	Evaluation and minimization of over-alkylation in proteomic sample preparation
	Ren, Yan 1; Shi, Zhaomei2 ; Zhang, Chengqian2 ; Liu, Siqi 3; Hao, Piliang2
	2022-11
发表期刊	INTERNATIONAL JOURNAL OF MASS SPECTROMETRY (IF:1.6[JCR-2023],1.6[5-Year])
ISSN	1387-3806
EISSN	1873-2798
卷号	481
发表状态	已发表
DOI	10.1016/j.ijms.2022.116919
摘要	Reduction and alkylation are essential steps in shotgun proteomic sample preparation, but over-alkylation can occur on peptide N-terminus and amino acid residues other than cysteine, which adversely affects protein identification and quantification. To date, different sample preparation protocols are used in different laboratories, but a systematic comparison of the published protocols has not been done yet in the aspect of over-alkylation. Here, we comprehensively evaluated various protocols using different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents. The suggested protocols from the manufacturers of RapiGest and ProteaseMAX induce the highest degree of over-alkylation compared with others. Carbamidomethylation at lysine increases over 4 times while using triethylammonium bicarbonate buffer compared with that using others, and it should be minimized for improving the labeling efficiency of stable isotope labeling reagents that label lysine residues. The use of digestion buffers at pH 6 reduces over-alkylation, but greatly introduces some other artificial modifications. Excessive iodoacetamide was proved to be the major cause of over-alkylation during trypsin digestion. Therefore, over-alkylation can be minimized by using low concen-tration of dithiothreitol and iodoacetamide for reduction and alkylation or quenching the excessive iodoacetamide before digestion.(c) 2022 Elsevier B.V. All rights reserved.
关键词	Artificial modification Over-alkylation Carbamidomethylation Carbamylation Mass spectrometry Sample preparation
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收录类别	SCI ; SCIE
语种	英语
资助项目	National Key R &D Program of China[2020YFE0202200]
WOS研究方向	Physics ; Spectroscopy
WOS类目	Physics, Atomic, Molecular & Chemical ; Spectroscopy
WOS记录号	WOS:000862868200008
出版者	ELSEVIER
引用统计	正在获取...
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/238165
专题	生命科学与技术学院_公共科研平台_组学分析平台
通讯作者	Ren, Yan; Liu, Siqi; Hao, Piliang
作者单位	1.Shanghai Univ Tradit Chinese Med, Expt Ctr Sci & Technol, Shanghai 201203, Peoples R China 2.ShanghaiTech Univ, Sch Life Sci & Technol, 393 Middle Huaxia Rd, Shanghai 201210, Peoples R China 3.BGI Shenzhen, Beishan Ind Zone 11th Bldg, Shenzhen 518083, Guangdong, Peoples R China
通讯作者单位	生命科学与技术学院
推荐引用方式 GB/T 7714	Ren, Yan,Shi, Zhaomei,Zhang, Chengqian,et al. Evaluation and minimization of over-alkylation in proteomic sample preparation[J]. INTERNATIONAL JOURNAL OF MASS SPECTROMETRY,2022,481.
APA	Ren, Yan,Shi, Zhaomei,Zhang, Chengqian,Liu, Siqi,&Hao, Piliang.(2022).Evaluation and minimization of over-alkylation in proteomic sample preparation.INTERNATIONAL JOURNAL OF MASS SPECTROMETRY,481.
MLA	Ren, Yan,et al."Evaluation and minimization of over-alkylation in proteomic sample preparation".INTERNATIONAL JOURNAL OF MASS SPECTROMETRY 481(2022).