Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.
Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.
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DOI：
10.1021/ac801417d
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发表时间：
2008-12-01
影响因子：
7.4
通讯作者：
Marshall, Alan G.
中科院分区：
文献类型：
--
作者：
Zhang, Hui-Min;Kazazic, Sasa;Schaub, Tanner M.;Tipton, Jeremiah D.;Emmett, Mark R.;Marshall, Alan G.
液相氢/氘交换（HDX）高分辨率FT-ICR质谱监测提供了一种快速的方法来研究蛋白质构象和蛋白质-蛋白质相互作用。胃蛋白酶通常用于消化HDX中的蛋白质，并且已知缺乏切割特异性。为了提高消化效率和特异性，我们优化了来自斋藤曲霉的胃蛋白酶和蛋白酶XIII型的消化条件和切割偏好。使用蛋白酶的稀释系列来确定几种测试蛋白的消化效率。XIII型蛋白酶倾向于在碱性氨基酸的C-末端上切割，并且与来自Rhizhopus的胃蛋白酶或XVIII型蛋白酶相比产生最高数量的片段和最佳的序列覆盖度。此外，蛋白酶XIII型表现出比胃蛋白酶少得多的自消化，因此对于HDX实验是上级的。来自蛋白酶XIII型和胃蛋白酶消化的许多高度重叠的片段，结合高分辨率FT-ICR质谱，为酰胺氢交换速率的分配提供了高序列分辨率（少至一个或两个氨基酸）。我们的H/D交换结果与肌红蛋白的二级和三级结构相关。这种高度重叠片段的分配有望大大提高用于确定由配体结合或蛋白质-蛋白质相互作用引起的构象差异的准确性和序列分辨率。
Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution FT-ICR mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known as lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin, and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FT-ICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions.
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DOI：
10.1016/s1044-0305(02)00384-7
发表时间：
2002-06-01
影响因子：
3.2
作者：
Schwartz, JC;Senko, MW;Syka, JEP
通讯作者：
Syka, JEP
影响因子：
4.1
作者：
Lam, TT;Lanman, JK;Prevelige, PE
通讯作者：
Prevelige, PE
影响因子：
5.6
作者：
Lanman, J;Lam, TT;Prevelige, PE
通讯作者：
Prevelige, PE
DOI：
10.1016/s1044-0305(97)00126-8
发表时间：
1997-09-01
影响因子：
3.2
作者：
Senko, MW;Hendrickson, CL;Marshall, AG
通讯作者：
Marshall, AG
DOI：
10.1016/1044-0305(94)85001-1
发表时间：
1994-07-01
影响因子：
3.2
作者：
EMMETT, MR;CAPRIOLI, RM
通讯作者：
CAPRIOLI, RM