用质谱法监测含二硫键的蛋白质的同时还原和酶解进行氢/氘交换。
Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry.
机构信息
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.
出版信息
Anal Chem. 2010 Feb 15;82(4):1450-4. doi: 10.1021/ac902550n.
Abstract
Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent-exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.
摘要
蛋白水解肽为各种溶液相蛋白质片段的溶剂可及性的质量分析氢/氘交换（HDX）提供了基础。米曲霉 XIII 型蛋白酶和猪胃蛋白酶可以生成重叠序列和高序列覆盖率的肽。然而，如果存在二硫键，蛋白水解可能会受到严重限制，特别是在二硫键连接（s）附近。在不影响蛋白质高级结构的情况下，在 H/D 交换之前或期间不能还原二硫键。在这里，我们展示了 H/D 交换后进行的同时淬灭/消化/还原，以便随后进行质量分析。在三（2-羧乙基）膦盐酸盐（TCEP·HCl）和脲的存在下进行蛋白水解，并且保持 H/D 交换和分析的所有其他步骤。对于含有二硫键的溶液相 H/D 交换的蛋白质的质量分析，该方法显著增加了溶剂暴露片段的序列覆盖率和定位。
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