Department of Chemistry, Washington University in St. Louis, St. Louis, MO, USA.
Nat Protoc. 2020 Dec;15(12):3942-3970. doi: 10.1038/s41596-020-0396-3. Epub 2020 Nov 9.
The higher-order structure (HOS) of proteins plays a critical role in their function; therefore, it is important to our understanding of their function that we have as much information as possible about their three-dimensional structure and how it changes with time. Mass spectrometry (MS) has become an important tool for determining protein HOS owing to its high throughput, mid-to-high spatial resolution, low sample amount requirement and broad compatibility with various protein systems. Modern MS-based protein HOS analysis relies, in part, on footprinting, where a reagent reacts 'to mark' the solvent-accessible surface of the protein, and MS-enabled proteomic analysis locates the modifications to afford a footprint. Fast photochemical oxidation of proteins (FPOP), first introduced in 2005, has become a powerful approach for protein footprinting. Laser-induced hydrogen peroxide photolysis generates hydroxyl radicals that react with solvent-accessible side chains (14 out of 20 amino acid side chains) to fulfill the footprinting. The reaction takes place at sub-milliseconds, faster than most of labeling-induced protein conformational changes, thus enabling a 'snapshot' of protein HOS in solution. As a result, FPOP has been employed in solving several important problems, including mapping epitopes, following protein aggregation, locating small molecule binding, measuring ligand-binding affinity, monitoring protein folding and unfolding and determining hidden conformational changes invisible to other methods. Broader adoption will be promoted by dissemination of the technical details for assembling the FPOP platform and for dealing with the complexities of analyzing FPOP data. In this protocol, we describe the FPOP platform, the conditions for successful footprinting and its examination by mass measurements of the intact protein, the post-labeling sample handling and digestion, the liquid chromatography-tandem MS analysis of the digested sample and the data analysis with Protein Metrics Suite. This protocol is intended not only as a guide for investigators trying to establish an FPOP platform in their own lab but also for those willing to incorporate FPOP as an additional tool in addressing their questions of interest.
蛋白质的高级结构 (HOS) 在其功能中起着关键作用；因此，了解其三维结构及其随时间的变化对我们理解其功能非常重要。质谱 (MS) 已成为确定蛋白质 HOS 的重要工具，因为它具有高通量、中高空间分辨率、低样品量要求和与各种蛋白质系统广泛兼容的特点。现代基于 MS 的蛋白质 HOS 分析部分依赖于足迹分析，其中试剂反应“标记”蛋白质的溶剂可及表面，而 MS 支持的蛋白质组学分析定位修饰以提供足迹。快速光化学氧化蛋白质 (FPOP) 于 2005 年首次引入，已成为蛋白质足迹分析的强大方法。激光诱导过氧化氢光解产生羟基自由基，与溶剂可及的侧链（20 种氨基酸侧链中的 14 种）反应以完成足迹分析。反应发生在亚毫秒级，比大多数标记诱导的蛋白质构象变化快，从而能够在溶液中获得蛋白质 HOS 的“快照”。因此，FPOP 已被用于解决包括映射表位、跟踪蛋白质聚集、定位小分子结合、测量配体结合亲和力、监测蛋白质折叠和展开以及确定其他方法无法识别的隐藏构象变化等几个重要问题。通过传播组装 FPOP 平台的技术细节以及处理分析 FPOP 数据的复杂性，将促进更广泛的采用。在本方案中，我们描述了 FPOP 平台、成功足迹分析的条件及其通过完整蛋白质的质量测量进行检查、标记后样品处理和消化、消化样品的液相色谱-串联质谱分析以及使用 Protein Metrics Suite 进行数据分析。本方案不仅旨在为试图在自己的实验室中建立 FPOP 平台的研究人员提供指南，也旨在为那些愿意将 FPOP 作为解决其感兴趣问题的附加工具的人员提供指南。
