Rand Kasper D, Adams Christopher M, Zubarev Roman A, Jørgensen Thomas J D
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
J Am Chem Soc. 2008 Jan 30;130(4):1341-9. doi: 10.1021/ja076448i. Epub 2008 Jan 3.
Hydrogen (1H/2H) exchange combined with mass spectrometry (HX-MS) has become a recognized method for the analysis of protein structural dynamics. Presently, the incorporated deuterons are typically localized by enzymatic cleavage of the labeled proteins and single residue resolution is normally only obtained for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation in tandem mass spectrometers would greatly increase the applicability of the HX-MS method. The biggest obstacle in achieving this goal is the intramolecular hydrogen migration (i.e., hydrogen scrambling) that occurs during vibrational excitation of gas-phase ions. Unlike traditional collisional ion activation, electron capture dissociation (ECD) is not associated with substantial vibrational excitation. We investigated the extent of intramolecular backbone amide hydrogen (1H/2H) migration upon ECD using peptides with a unique selective deuterium incorporation. Our results show that only limited amide hydrogen migration occurs upon ECD, provided that vibrational excitation prior to the electron capture event is minimized. Peptide ions that are excessively vibrationally excited in the electrospray ion source by, e.g., high declustering potentials or during precursor ion selection (via sideband excitation) in the external linear quadrupole ion trap undergo nearly complete hydrogen (1H/2H) scrambling. Similarly, collision-induced dissociation (CID) in the external linear quadrupole ion trap results in complete or extensive hydrogen (1H/2H) scrambling. This precludes the use of CID as a method to obtain site-specific information from proteins that are labeled in solution-phase 1H/2H exchange experiments. In contrast, the deuteration levels of the c- and z-fragment ions generated from ECD closely mimic the known solution deuteration pattern of the selectively labeled peptides. This excellent correlation between the results obtained from gas phase and solution suggests that ECD holds great promise as a general method to obtain single residue resolution in proteins from solution 1H/2H exchange experiments.
氢（1H/2H）交换结合质谱法（HX-MS）已成为一种公认的蛋白质结构动力学分析方法。目前，掺入的氘核通常通过对标记蛋白质进行酶切来定位，通常仅能对少数几个残基获得单残基分辨率。通过串联质谱仪中的气相碎裂来测定位点特异性氘水平将大大提高HX-MS方法的适用性。实现这一目标的最大障碍是气相离子振动激发过程中发生的分子内氢迁移（即氢重排）。与传统的碰撞离子活化不同，电子捕获解离（ECD）与大量的振动激发无关。我们使用具有独特选择性氘掺入的肽研究了ECD过程中分子内主链酰胺氢（1H/2H）迁移的程度。我们的结果表明，只要电子捕获事件之前的振动激发最小化，ECD过程中仅发生有限的酰胺氢迁移。例如，在电喷雾离子源中因高去簇电位或在外部线性四极杆离子阱中进行前体离子选择（通过边带激发）而过度振动激发的肽离子会发生几乎完全的氢（1H/2H）重排。同样，外部线性四极杆离子阱中的碰撞诱导解离（CID）会导致完全或广泛的氢（1H/2H）重排。这排除了将CID用作从溶液相1H/2H交换实验中标记的蛋白质获取位点特异性信息的方法。相比之下，ECD产生的c-和z-碎片离子的氘化水平紧密模拟了选择性标记肽的已知溶液氘化模式。气相和溶液中获得的结果之间的这种出色相关性表明，ECD作为从溶液1H/2H交换实验中获得蛋白质单残基分辨率的通用方法具有很大的前景。
