Resolving isotopic fine structure to detect and quantify natural abundance- and hydrogen/deuterium exchange-derived isotopomers.
Resolving isotopic fine structure to detect and quantify natural abundance- and hydrogen/deuterium exchange-derived isotopomers.
复制标题
解析同位素精细结构以检测和量化天然丰度和氢/氘交换衍生的同位素异构体。
DOI：
10.1021/ac403365g
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发表时间：
2014
影响因子：
7.4
通讯作者：
Agar,JeffreyN
中科院分区：
文献类型：
--
作者：
Liu,Qian;Easterling,MichaelL;Agar,JeffreyN
氢/氘交换（HDX）质谱（MS）用于分析蛋白质动力学、蛋白质折叠/解折叠和分子相互作用。在这项研究之前，HDX MS实验采用质谱分辨率，在给定肽的同位素分布中每个标称质量仅提供一个峰，并相应地开发了HDX MS数据分析方法。HDX MS固有的复杂性水平仍未得到解决，即天然丰度重同位素和交换氘的各种组合共享相同的标称质量，并且在先前的分辨能力上重叠。例如，A + 2峰由（除其他同位素异构体之外）两个2 H交换/零个13 C同位素异构体、一个2 H交换/一个13 C同位素异构体和零个2 H交换/两个13 C同位素异构体组成。值得注意的是，由于2 H和13 C原子之间的质量亏损为103 mDa，因此这些同位素异构体的质量略有不同。以前的HDX MS方法没有解决这些同位素异构体，需要一个自然丰度只有（HDX或“时间零”之前）的光谱和数据处理，以消除其贡献。这里证明了高分辨率质谱法可用于检测同位素精细结构，例如在上述A + 2分布图示例中，对由氘掺入产生的同位素异构体物质进行去卷积。因此，在HDX MS期间解析同位素精细结构允许直接监测HDX，其可以计算为氘交换的同位素异构体的分数峰幅度的总和。这避免了对时间零点谱的需要以及用于解释天然丰度重同位素的数据处理，从而节省了仪器和分析时间。
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-2H-exchanged/zero-13C isotopomer, a one-2H-exchanged/one-13C isotopomer, and a zero-2H-exchanged/two-13C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ∼3 mDa mass defect between2H and13C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before HDX or “time zero”) spectrum and data processing to remove its contribution. It is demonstrated here that high-resolution mass spectrometry can be used to detect isotopic fine structure, such as in the A + 2 profile example above, deconvolving the isotopomer species resulting from deuterium incorporation. Resolving isotopic fine structure during HDX MS therefore permits direct monitoring of HDX, which can be calculated as the sum of the fractional peak magnitudes of the deuterium-exchanged isotopomers. This obviates both the need for a time zero spectrum as well as data processing to account for natural abundance heavy isotopes, saving instrument and analysis time.