Top-down detection of oxidative protein footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation and Electron-Capture Dissociation

Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.

蛋白质快速光化学氧化足迹法（Fast photochemical oxidation of proteins, FPOP）是一类通过快速不可逆化学反应实现蛋白质图谱绘制的结构质谱（mass spectrometry）分析方法。氧化修饰的位点可反映蛋白质的溶剂可及性与位点反应性，进而为蛋白质构象、结构动态及分子相互作用提供关键信息。自下而上质谱（bottom-up mass spectrometry）是目前分析FPOP样品的成熟标准方法。在自下而上的实验流程中，所有形式的蛋白质会被选定的蛋白酶共同酶解，最终得到包含不同光化学氧化程度的蛋白质亚群所产生的肽段混合物。但一旦蛋白质发生至少一次氧化，后续任意连续氧化事件的空间分布将无法再真实反映蛋白质初始的溶剂可及性与残基反应性，因为后续氧化的位点偏好性也会受到此前氧化所引发的结构变化的影响。因此，仅通过分析单次氧化的蛋白质亚群来基于FPOP数据评估蛋白质结构，将是极具研究价值的探索方向。这一目标需要采用更为特异性的自上而下质谱（top-down mass spectrometry）分析策略。任何自上而下质谱实验的核心环节均为选择合适的离子分离、激发与碎裂方法。本研究采用并对比了碰撞诱导解离（collision-induced dissociation, CID）、电子转移解离（electron-transfer dissociation, ETD）、电子捕获解离（electron-capture dissociation, ECD）与选择性离子多阶连续累积（multi continuous accumulation of selected ions, CASI）联用的分析方案。本研究以FPOP标记的泛素（ubiquitin）单次氧化亚群为对象对该方法进行了优化。随后，通过该方法可视化全血红素/脱血红素肌红蛋白（holo/apo myoglobin）体系中辅因子去除所诱导的结构变化，进一步验证了该方法的实用性。研究将自上而下质谱所得数据与已有文献报道数据及同一样本的自下而上质谱数据集进行了对比。结果显示，自上而下质谱的分析结果与二者均具有良好的一致性，这表明通过自上而下质谱监测单次FPOP氧化的离子亚群，可作为氧化蛋白质足迹法的可行工作流程。