A systems-level approach to decipher the protein interactome.

摘要 蛋白质相互作用，如蛋白质-蛋白质、蛋白质-核酸、蛋白质-代谢物和蛋白质-异源生物， 在定义生物有机体的独特性和复杂性方面发挥关键作用。了解地点和位置 相互作用发生的时间是描述相互作用组功能的关键一步。然而，尽管 在计算和蛋白质组学技术方面取得了显著的进步，但要准确地确定 以高吞吐量的方式精确定位整个蜂窝范围内的联系人站点。在这里，我们提出了一种新方法，称为快速 铃木光化学氧化和捕获(FPICS)，以高分辨率绘制蛋白质相互作用位点图。这个 我们方法的关键创新，代表着前所未有的技术进步，是使用单一的 卤素原子既是光活化分子的名片，指示相互作用发生的地方，也是 捕获手柄，用于基于质谱学的每个相互作用部位的蛋白质组检测。带FPICS、卤素 首先使用准分子激光照射从卤化小分子(例如药物)转移取代基 或天然产物)或卤化生物分子(例如蛋白质、脂类、多糖、寡核苷酸或代谢物) 相互作用的蛋白质。然后使用生物正交铃木-宫浦杂交捕获和识别标记的位置。 本课题组首创的联用化学蛋白质组学方法学。FPICS具有开创性，因为它消除了 与去卷积相关联的挑战与交联肽以及频繁和不需要的 生物大分子的碎裂。展示了这种方法的广泛应用，在这里我们将应用 FPICS绘制了小分子、脂类和核酸的蛋白质相互作用部位图，旨在识别新的 全蛋白质组的功能和治疗相关的结合位点。总而言之，这项研究将产生一个 对蛋白质相互作用组的系统水平的描述，这将为改善全球 了解发生在每个细胞内的数百万种相互作用的功能意义。其影响 我们的方法将是广泛的，跨越化学生物学、分析化学和系统的领域。 生物学。

Abstract Protein interactions, such as protein-protein, protein-nucleic acid, protein-metabolite, and protein-xenobiotic, play a critical role in defining the uniqueness and complexity of biological organisms. Understanding where and when interactions occur is an essential step to functionally characterize the interactome. However, and despite remarkable advances in computational and proteomic technologies, it remains surprisingly difficult to precisely pinpoint contact sites cell-wide in a high-throughput manner. Here we present a new approach, termed Fast Photochemical Oxidation and Capture by Suzuki (FPICS), to map protein interaction sites at high resolution. The key innovation of our method, which represents an unprecedented technical advance, is the use of a single halogen atom as both a photoactivatable molecular 'calling card,' to indicate where interactions occur, and a capture handle, for mass spectrometry-based proteomic detection of each interaction site. With FPICS, halogen substituents are first transferred, using excimer laser irradiation, from halogenated small molecules (e.g. drugs or natural products) or halogenated biomolecules (e.g. proteins, lipids, glycans, oligonucleotides, or metabolites) to interacting proteins. Labeled sites are then captured and identified using bioorthogonal Suzuki–Miyaura cross- coupling chemoproteomic methodology pioneered by our group. FPICS is groundbreaking because it eliminates challenges associated with deconvolving the spectra of crosslinked peptides and the frequent and unwanted fragmentation of large biomolecules. Showcasing the method's wide-ranging applications, here we will apply FPICS map the protein interaction sites for small molecules, lipids, and nucleic acids, aiming to identify new functional and therapeutically relevant binding sites proteome-wide. Taken together, this study will yield a systems-level portrait of the protein interactome, which will lay the foundation for an improved global understanding of the functional significance of the millions of interactions occurring within every cell. The impact of our methods will be wide ranging, spanning the fields of chemical biology, analytical chemistry, and systems biology.