Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, United States.
Anal Chem. 2012 Nov 6;84(21):9601-8. doi: 10.1021/ac302488h. Epub 2012 Oct 19.
Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.
酰胺氢/氘交换是一种常用于研究蛋白质及其与其他蛋白质或配体相互作用的动力学的技术。当与液相色谱和质谱联用时，氢/氘交换相对于其他结构表征技术具有几个独特的优势，包括非常高的灵敏度、在复杂环境中分析蛋白质的能力以及大的质量范围。该技术的一个基本限制是在分析过程中氘标记（回交换）的损失。本文提出了一种在亚零温度反相色谱分析的分离阶段限制标记损失的方法。该方法通过使用缓冲剂修饰剂来防止冻结，从而实现了这种方法。我们评估了乙二醇、二甲基甲酰胺、甲酰胺和甲醇的冰点抑制能力、对肽保留的影响以及它们与电喷雾电离的相容性。由于其良好的电喷雾电离相容性，乙二醇被广泛使用；然而，如果可以克服对电离的不利影响，甲酰胺有可能成为更好的修饰剂。通过使用合适的缓冲剂修饰剂，即使在长时间的色谱分离过程中，也可以在低至-30°C 的温度下进行分离，而氘标记的损失可以忽略不计。回交换的减少表明，氢/氘交换质谱的动态范围可以增加，无论是混合物的复杂性还是氘化水平变化的幅度都可以被定量。
