IDBR: TYPE A Development of On-Chip Electrochemical Cross-Linking Mass Spectrometry for Probing Protein-Protein Interactions

俄亥俄大学被授予开发一种芯片上电化学交联质谱学的奖项，该质谱学将用于识别活体中的蛋白质-蛋白质相互作用(PPI)。PPI的研究对于理解蛋白质的生物学功能非常重要，因为几乎所有蛋白质的生物学功能都是由特定的、非共价的与其他分子的相互作用提供的。识别蛋白质复合体或聚集体的相互作用伙伴和相互作用部位将有助于阐明生物过程的机制。这个仪器项目将为学生提供在涉及分析化学、生物化学和仪器的跨学科研究方面进行培训的机会。拟议的生物研究仪器将首先用于研究紫外线诱导的细胞凋亡信号通路。在仪器建造和软件培训之后，俄亥俄大学结构和分子生物学的许多其他生物研究小组将可以使用开发的仪器。该仪器还将作为商业化的原型仪器，使其能够传播到生物研究界。预计拟议的研究将使结构生物学、分子生物学和药物发现的研究界受益。交联质谱(MS)已经成为绘制蛋白质-蛋白质相互作用图和阐明活细胞中蛋白质网络的有用技术。然而，一些实验障碍限制了交联型MS的应用，包括难以识别复杂混合物中的交联肽、交联肽离子碎片模式的复杂性以及无法对交联链进行量化。本项目基于电化学交联剂丁二胺丙酸酯(SSP)，提出了一种利用电化学辅助的交联质谱来探测体内蛋白质-蛋白质相互作用的新方法。在线消化后，交联的蛋白质复合体将经过毛细管电解(CE)分离、在线EC还原和在线MS检测。EC缩减提供了一种基于还原电流(也基于MS信号)快速识别和量化交叉链接的新方法，大大减少了数据分析所需的时间一个数量级以上。在线EC减少只需几秒钟，比化学减少所需的几个小时要快得多。此外，EC还原生成易于识别的线状肽，便于MS/MS鉴定交联物结构，以确定蛋白质复合体中的相互作用部位。

An Award is made to Ohio University to develop an on-chip electrochemical cross-linking mass spectrometry which will be used to identify protein-protein interactions (PPIs) in vivo. The study of PPIs is very important to understand protein biological functions as the biological functions of almost all proteins are provided by specific, non-covalent interactions with other molecules. The identification of interacting partners and interaction sites of protein complexes or aggregates will shed light on the elucidation of biological process mechanisms. This instrumentation project will provide opportunities for students to be trained in interdisciplinary research involving analytical chemistry, biochemistry, and instrumentation. The proposed instrument for biological research will first be used for the study of UV-induced apoptotic signaling pathways. Following instrument construction and software training, many other biological research groups in structural and molecular biology at Ohio University will have access to the developed instrument. The instrument will also serve as a prototype instrument for commercialization, enabling dissemination to the biological research community. It is expected that the proposed research will benefit the research communities of structural biology, molecular biology, and drug discovery. Cross-linking mass spectrometry (MS) has become a useful technique for mapping protein-protein interactions and elucidating protein networks in living cells. However, several experimental obstacles limit the usefulness of cross-linking MS, including difficulty in the identification of cross-linked peptides in the complex mixture, the complexity of the fragmentation patterns of cross-linked peptide ions and the inability to quantify cross-links. This project proposes a new approach using electrochemistry (EC)-assisted cross-linking MS for probing protein-protein interactions in vivo, based on an electrochemical cross-linking reagent, diselenide [succinimidyl propionate] (SSP). Following online digestion, the cross-linked protein complexes will undergo capillary electrolysis (CE) separation, online EC reduction and online MS detection. The EC reduction provides a novel way to quickly identify and quantify cross-links based on the reduction current (also on MS signal), significantly reducing time required for data analysis by more than one order of magnitude. The online EC reduction takes seconds, which is much faster than hours required in chemical reduction. Furthermore, EC reduction yields easily identifiable linear peptides, facilitating MS/MS identification of cross-link structures for pinpointing out the interaction sites in the protein complexes.