Ferguson Peter L, Konermann Lars
Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
Anal Chem. 2008 Jun 1;80(11):4078-86. doi: 10.1021/ac8001963. Epub 2008 May 7.
There is an ongoing debate whether collision-induced dissociation (CID) of electrosprayed proteins after solution-phase hydrogen/deuterium exchange (HDX) is a viable approach for determining spatially resolved deuteration patterns. This work explores the use of two methods, source-CID and hexapole tandem mass spectrometry (MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer, for measuring the fragment deuteration levels of regioselectively labeled ubiquitin. Both methods reveal that b-ions exhibit HDX levels significantly below that of the intact protein, whereas several y'' fragments are labeled to a much greater extent. These results are consistent with earlier source-CID data (Akashi, S.; Naito, Y.; Takio, K. Anal. Chem. 1999, 71, 4974-4980). However, the measured b-ion deuteration levels are in disagreement with the known solution-phase behavior of ubiquitin. Partial agreement is observed for y''-ions. Control experiments on homogeneously labeled ubiquitin (having the same average deuteration level at every exchangeable site) result in highly nonuniform fragment HDX levels. In particular, b-ions exhibit deuteration levels significantly below that of intact ubiquitin, thereby mimicking the behavior seen for the regioselectively labeled protein. This effect is likely caused by isotope fractionation during collisional activation, facilitated by the high mobility of charge carriers (scrambling) in the gas phase. The observation that the b-ion labeling behavior is largely independent of the spatial isotope distribution within solution-phase ubiquitin invalidates these ions as reporters of the protein deuteration pattern. This work questions the common practice of interpreting any nonuniformities in fragment deuteration as being indicative of regioselective solution-phase labeling. Artifactual deuterium enrichment or depletion during collisional activation may have contributed to the current lack of consensus as to whether HDX/CID represents a potentially viable tool for measuring solution-phase deuteration patterns.
关于溶液相氢/氘交换（HDX）后电喷雾蛋白质的碰撞诱导解离（CID）是否是确定空间分辨氘化模式的可行方法，目前仍存在争议。这项工作探索了在四极杆飞行时间（Q-TOF）质谱仪上使用两种方法，即源CID和六极杆串联质谱（MS/MS），来测量区域选择性标记泛素的片段氘化水平。两种方法均表明，b离子的HDX水平显著低于完整蛋白质，而几个y''片段的标记程度要高得多。这些结果与早期的源CID数据一致（赤ashi，S.；内藤，Y.；滝尾，K.分析化学，1999年，71卷，4974 - 4980页）。然而，测得的b离子氘化水平与泛素已知的溶液相行为不一致。对于y''离子，观察到部分一致。对均匀标记泛素（在每个可交换位点具有相同的平均氘化水平）的对照实验导致片段HDX水平高度不均匀。特别是，b离子的氘化水平显著低于完整泛素，从而模拟了区域选择性标记蛋白质的行为。这种效应可能是由碰撞激活期间的同位素分馏引起的，气相中电荷载流子的高迁移率（重排）促进了这种分馏。b离子标记行为在很大程度上与溶液相泛素内的空间同位素分布无关这一观察结果，使这些离子作为蛋白质氘化模式的报告分子无效。这项工作对将片段氘化中的任何不均匀性解释为区域选择性溶液相标记的常见做法提出了质疑。碰撞激活期间人为的氘富集或消耗可能导致了目前关于HDX/CID是否代表测量溶液相氘化模式的潜在可行工具缺乏共识。
