Site-specific amide hydrogen/deuterium exchange in E-coli thioredoxins measured by electrospray ionization mass spectrometry
Site-specific amide hydrogen/deuterium exchange in E-coli thioredoxins measured by electrospray ionization mass spectrometry
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DOI：
10.1021/ja010901n
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发表时间：
2001-10-10
影响因子：
15
通讯作者：
Deinzer, ML
中科院分区：
文献类型：
--
作者：
Kim, MY;Maier, CS;Deinzer, ML
质谱法作为一种分析工具，通过氢/氘交换来研究蛋白质的折叠和结构是一种相对较新的方法。本研究在D2O磷酸盐缓冲溶液（pH 5.7）中培养20 s后，采用b(n)离子电喷雾电离CID MS/MS实验，测定氧化和还原大肠杆菌硫氧还毒素中位点特异性酰胺氘含量。氘水平与报道的核磁共振测定的H/D汇率常数相关良好。然而，用y(n)离子测量的氘与速率交换数据的相关性要低得多。一般来说，当用b(n)离子测量α螺旋和β片上的残基时，氘的掺入量较低，而环和环的氘含量较高。两种蛋白质形式中大多数酰胺位点的氘含量与预期的结构相似性一致，但也存在一些差异。特别是由残基18-22组成的回合，氘含量的变化更大，这与报道的两种形式的结构差异一致。由S-（2-氯乙基）谷胱甘肽和S-（2-氯乙基）半胱氨酸在Cys-32位点烷基化的硫氧还蛋白对氘的摄取，在肽1-24和45-58中，与氧化和还原硫氧还蛋白所观察到的相似，但一些残基，特别是Leu-53和Thr-54显示出轻微的氘水平升高，这表明蛋白质在Cys-32位点的烷基化发生了结构变化。结果表明，b(n)离子对确定位点特异性酰胺氢同位素交换的程度是可靠的，质谱法可以作为核磁共振和其他分析方法的补充技术，用于探测蛋白质的区域结构特征。
Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach. In this study, site-specific amide deuterium content was measured in oxidized and reduced E coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D2O phosphate-buffered solution (pH 5.7). The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants. The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data. In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels, Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences. The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms. The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.