Charlebois Jay P, Patrie Steven M, Kelleher Neil L
Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Anal Chem. 2003 Jul 1;75(13):3263-6. doi: 10.1021/ac020690k.
For localization of deuterium atoms after solution-phase exchange with D2O, intact proteins are often digested prior to analysis by mass spectrometry (MS) and tandem MS (MS/MS). Amelioration of limitations associated with this approach (e.g., <70% sequence coverage and some D atom scrambling during MS/MS) were sought using intact proteins and two newer methods applied to tracking H/D exchange dynamics for the first time. Using 2-4-fold signal enhancements through depletion of 13C and 15N isotopes and implementing the new MS/MS technique of electron capture dissociation (ECD) yielded an increased number of c and z* ions observed (43 vs 25) for recombinant yeast ubiquitin (9.3 kDa). Initial determination of D atom content in consecutive c ion series (c4-c7, c28, c31, c32, and c33) was demonstrated. The improved ion signal and experiment speed combined with narrower isotopic distributions markedly increases the degree of localization and feasibility of ECD-based MS/MS after solution-phase H/D exchange.
为了在与重水进行溶液相交换后定位氘原子，完整蛋白质在通过质谱（MS）和串联质谱（MS/MS）分析之前通常要进行消化。人们首次使用完整蛋白质和两种更新的方法来跟踪氢/氘交换动力学，以改善与该方法相关的局限性（例如，序列覆盖率<70%以及MS/MS过程中的一些氘原子重排）。通过去除¹³C和¹⁵N同位素实现2至4倍的信号增强，并采用新的电子捕获解离（ECD）MS/MS技术，重组酵母泛素（9.3 kDa）中观察到的c离子和z*离子数量增加（43个对25个）。展示了在连续的c离子系列（c4 - c7、c28、c31、c32和c33）中对氘原子含量的初步测定。改进后的离子信号和实验速度，再加上更窄的同位素分布，显著提高了溶液相氢/氘交换后基于ECD的MS/MS的定位程度和可行性。
