Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography
Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography
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DOI：
10.1016/s0021-9673(01)00961-x
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发表时间：
2001-07-27
影响因子：
4.1
通讯作者：
Regnier, FE
中科院分区：
文献类型：
--
作者：
Wang, SH;Regnier, FE
本文介绍了一种程序，其中半胱氨酸含有肽从胰蛋白酶消化的复杂的蛋白质混合物中选择共价色谱法的基础上硫醇-二硫键交换，鉴定质谱，并通过差分同位素标记定量。在用2，2 ' -联吡啶二硫化物破坏二硫键后，用胰蛋白酶消化所有蛋白质并用琥珀酸酐酰化。然后通过在硫丙基琼脂糖凝胶上与巯基进行二硫键交换，从酰化消化物中选择含半胱氨酸的肽。用含有1 mM（亚乙基二氮基）四乙酸二钠盐的25 mM二硫苏糖醇（pH 7.5）从凝胶中释放捕获的含半胱氨酸的肽，并用碘乙酸烷基化，随后通过反相液相色谱（RPLC）分级。通过基质辅助激光解吸电离质谱法分析从RPLC柱收集的级分。基于来自分别用琥珀酸和氘代琥珀酸酐标记的实验和对照样品的肽的同位素比，可以确定两种样品之间每种肽物质的相对浓度。从实验样品中上调的蛋白质获得的肽容易通过氘代肽的相对量的增加来鉴定。这些研究的结果表明，通过选择含半胱氨酸的肽，可以降低蛋白质消化的复杂性，并大大简化数据库搜索。当结合同位素标记策略进行定量时，可以确定当诱导质粒蛋白表达时在携带质粒的大肠杆菌中上调的蛋白。E.还注意到大肠杆菌来源。(C)出版社：Elsevier Science B. V.
This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange, identified by mass spectrometry, and quantified by differential isotope labeling. Following disruption of disulfide bridges with 2,2 ' -dipyridyl disulfide, all proteins were digested with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide interchange with sulfhydryl groups on a thiopropyl Sepharose gel. Captured cysteine containing peptides were released from the gel with 25 mM dithiothreitol (pH 7.5) containing 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt and alkylated with iodoacetic acid subsequent to fractionation by reversed-phase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Based on isotope ratios of peptides from experimental and control samples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide species between the two samples. Peptides obtained from proteins that were up-regulated in the experimental sample were easily identified by an increase of the relative amount of the deuterated peptide. The results of these studies indicate that by selecting cysteine containing peptides, the complexity of protein digest could be reduced and database searches greatly simplified. When coupled with the isotope labeling strategy for quantification it was possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regulation of several proteins of E. coli origin was also noted. (C) 2001 Published by Elsevier Science B.V.