Hydrogen exchange-mass spectrometry - Optimization of digestion conditions
Hydrogen exchange-mass spectrometry - Optimization of digestion conditions
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DOI：
10.1074/mcp.m100009-mcp200
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发表时间：
2002-02-01
影响因子：
7
通讯作者：
Smith, DL
中科院分区：
文献类型：
--
作者：
Wang, LT;Pan, H;Smith, DL
蛋白质折叠结构与其功能之间的直接联系增加了对高分辨率结构的需求。此外，还需要在各种条件下检测和定位蛋白质折叠结构变化的分析方法。位于肽酰胺键上的氢进行同位素交换的速率已成为检测这种结构变化的重要方法的基础。当通过质谱检测时，氢交换可以用于研究大蛋白质和蛋白质复合物的稀释溶液，具有非常高的灵敏度。为了定位结构变化，标记的蛋白质通常被酸性蛋白酶消化形成肽，其氢/氘水平通过质谱测定。这种方法只有在同位素交换缓慢的条件下蛋白质能被快速消化时才会成功。本研究描述了如何用固定胃蛋白酶填充的色谱柱来减少消化时间，并提供了一种有效的方法来从同位素标记的片段中分离胃蛋白酶。这些列是一个在线系统的一部分，有利于快速消化低浓度的蛋白质和肽的浓度。中国生物医学工程学报（英文版），2002。
The direct linkage between folded structures of proteins and their function has increased the need for high resolution structures. In addition, there is a need for analytical methods for detecting and locating changes in the folded structures of proteins under a wide variety of conditions. The rates at which hydrogens located at peptide amide linkages undergo isotopic exchange has become the basis for an important method for detecting such structural changes. When detected by mass spectrometry, hydrogen exchange can be used to study dilute solutions of large proteins and protein complexes with very high sensitivity. To locate structural changes, labeled proteins are often digested with acid proteases to form peptides whose hydrogen/deuterium levels are determined by mass spectrometry. This approach is successful only when the protein can be digested rapidly under conditions where isotope exchange is slow. This study describes how columns packed with immobilized pepsin can be used to reduce the digestion time and to provide an effective means for separating the pepsin from the isotopically labeled fragments. These columns are part of an on-line system that facilitates both rapid digestion of low concentrations of protein and concentration of the peptides. Molecular & Cellular Proteomics 1:132-138,2002.