使用质谱法对全长膜蛋白进行蛋白质组学研究。

Proteomics on full-length membrane proteins using mass spectrometry.

作者信息

le Coutre J, Whitelegge J P, Gross A, Turk E, Wright E M, Kaback H R, Faull K F

机构信息

Howard Hughes Medical Institute, Departments of Physiology and of Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles 90095, USA.

出版信息

Biochemistry. 2000 Apr 18;39(15):4237-42. doi: 10.1021/bi000150m.

DOI:10.1021/bi000150m

PMID:10757971

Abstract

A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.

摘要

已开发出一种通用技术，可对全长膜蛋白进行快速质谱分析[怀特利奇，J.P.，勒库特尔，J.等人（1999年）《美国国家科学院院刊》96，10695 - 10698]。使用在线高效液相色谱电喷雾电离质谱法（LC - MS），对分子量高达61 kDa的不同天然和重组细菌膜蛋白进行了表征。给出了来自三种细菌生物体的四种完全不同的膜蛋白、两种转运蛋白、一种通道蛋白和一种孔蛋白的质谱数据。除了以±0.01%的精度测定分子量外，该技术还可监测单个半胱氨酸残基的烷基化或氧化以及推导氨基酸序列中的错误。最后，使用在线LC - MS，无需事先纯化即可从溶解的大肠杆菌膜中鉴定出未知蛋白质。