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

IDBR：用于探测蛋白质-蛋白质相互作用的片上电化学交联质谱法的 A 型开发

• 批准号: 1455554
• 负责人: Shiyong Wu
• 金额: $ 32.27万
• 依托单位: Ohio University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2019-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1455554&HistoricalAwards=false
• 关键词: IDBR; TYPE; Development; Chip; Electrochemical

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.

授予俄亥俄州大学开发一种芯片上电化学交联质谱法的奖项，该质谱法将用于鉴定体内蛋白质-蛋白质相互作用（PPI）。PPI的研究对于了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通过与其他分子的特异性非共价相互作用提供的。蛋白质复合物或聚集体的相互作用伴侣和相互作用位点的鉴定将有助于阐明生物过程机制。这个仪器项目将为学生提供机会，在跨学科的研究，涉及分析化学，生物化学和仪器进行培训。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，俄亥俄州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所开发的仪器。该仪器还将作为商业化的原型仪器，以便向生物研究界传播。预计拟议的研究将有利于结构生物学，分子生物学和药物发现的研究社区。 交联质谱（MS）已成为一种有用的技术，用于绘制蛋白质-蛋白质相互作用和阐明活细胞中的蛋白质网络。然而，几个实验障碍限制了交联MS的有用性，包括难以识别复杂混合物中的交联肽、交联肽离子的碎片模式的复杂性以及无法量化交联。该项目提出了一种新的方法，使用电化学（EC）辅助的交联MS探测体内蛋白质-蛋白质相互作用，基于电化学交联剂，二硒[琥珀酰亚胺丙酸酯]（SSP）。 在线消化后，交联的蛋白质复合物将经历毛细管电解（CE）分离、在线EC还原和在线MS检测。EC还原提供了一种基于还原电流（也基于MS信号）快速识别和量化交联的新方法，从而将数据分析所需的时间显著减少一个数量级以上。在线EC还原只需几秒钟，这比化学还原所需的几小时快得多。此外，EC还原产生容易识别的线性肽，便于MS/MS鉴定的交联结构，以查明在蛋白质复合物中的相互作用位点。