Shi Liuqing, Gross Michael L
Department of Chemistry, Washington University, St. Louis, MO 63130, United States.
Protein Pept Lett. 2019;26(1):27-34. doi: 10.2174/0929866526666181128124554.
Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting.
We survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP's quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.
通过使用与质谱（MS）相结合的结构蛋白质组学方法，可以实现对大分子组成和一些结构特征的测定。一种方法是羟基自由基蛋白质足迹法，即使用反应性试剂修饰氨基酸侧链，从而不可逆地修饰蛋白质侧链。当用基于质谱的蛋白质组学方法解析这些结果时，可以增加我们对溶液中大分子的结构、组装和构象动力学的了解。通过激光照射产生羟基自由基，Hambly和Gross开发了蛋白质快速光化学氧化（FPOP）方法，该方法在亚毫秒时间尺度上标记蛋白质，并通过质谱分析更深入地了解蛋白质结构以及蛋白质-配体和蛋白质-蛋白质相互作用。本综述重点介绍了FPOP的基本原理，并描述了羟基自由基以及其他自由基和卡宾的产生、蛋白质的羟基标记以及蛋白质修饰位点的测定。我们还总结了FPOP与MS联用在蛋白质足迹分析中的一些最新应用。
我们考察了一些结果，这些结果表明FPOP能够在残基水平上定性测量蛋白质的溶剂可及性。为了使这些方法更有价值，我们描述了最近的方法进展，这些进展提高了FPOP的定量能力并增加了蛋白质序列的空间覆盖率。为了进一步改进FPOP，已经开发了几种新的标记试剂，包括卡宾和其他自由基。这些不断的改进将使与MS联用的氧化足迹法在确定大分子及其组装体的结构和动力学方面发挥越来越重要的作用。
