The Expedit-Isotopomeric CrossLinking Mass Spectrometry (Expedit-ICLMS) technology for mapping global and dynamic protein-protein interaction networks

摘要 该项目寻求开发新技术，以实现蛋白质-蛋白质相互作用(PPI)的全球测绘 以具体情况、及时和负担得起的方式，由对特定生物学感兴趣的个人研究人员提供 系统。这项技术的成功开发有望对所有领域产生革命性的影响 生物医学研究，直接解决重点技术研究的目的和 开发R01 FOA(PAR-19-253)。全球PPI网络不仅是 了解正常和异常生物过程背后的分子机制，以及 了解遗传相互作用网络、构建基因调控网络和定量 生物过程的建模。当前用于绘制全球PPI网络的技术是劳动密集型的， 耗时、昂贵、受假阳性/阴性的困扰，并且不能为个人提供一种方法 研究人员有效地绘制了特定生物系统的全球PPI网络图。建议的技术 发展的目标是解决交联质谱学面临的两个最重要的挑战 基于化学发光光谱(CLMS)的全球PPI图谱研究：1)交联肽混合物的复杂性 蛋白质数量多、丰度动态范围大的样品；2)高效、 通过整个蛋白质组数据库搜索确定交联肽。我们试图克服 通过开发一种名为Expedit的新的交联肽浓缩策略来应对这些挑战，以及 将其与ICL交联剂的强大功能相结合，形成了一类新型的MS可裂解、同分异构体、双... 用于交联肽鉴定的功能交联剂。ICL的独特功能允许1)高效 从MS2谱确定每个交联物中的单个多肽质量，以及2)鉴定 利用每个交联物的单一MS2谱通过整个蛋白质组数据库搜索交联肽 多肽。Expedit与ICLMS的结合有望解决当前CLMS的主要局限性 首次启用常规大规模PPI研究的方法。 在AIMS中，我们将首先合成新型Expedit试剂，并评估它们对 使用日益复杂的混合物富集交联肽。优化后，我们将集成Expedit 与ICLMS合作，评估在酵母中建立全球PPI网络的技术的有效性。我们会 根据确定的交联链/PPI的数量和重复性、丰度来评价该方法 识别的蛋白质、它们的定位、亲和力和复合体成员(如果有)。我们会比较一下 我们的PPI网络与之前描述的酵母PPI网络类似。这项技术对 将通过与最先进的基于CLMS的方法进行比较来评估交联肽的鉴定 接近了。如果成功，该项目将为研究全球PPI提供一种通用和可靠的方法 以及它们的动态，可以应用于任何可以获得测序基因组的有机体。

SUMMARY This project seeks to develop new technology to enable global mapping of protein-protein interactions (PPIs) in a condition-specific, timely and affordable manner, by individual researchers interested in specific biological systems. Successful development of this technology is expected to have a transformative effect on all fields of biomedical research, directly addressing the purpose of the Focused Technology Research and Development R01 FOA (PAR-19-253). Global PPI networks are not only the most important resources for understanding the molecular mechanisms underlying normal and aberrant biological processes, but also the bases for understanding genetic interaction networks, constructing gene regulatory networks, and quantitative modeling of biological processes. Current technologies for mapping global PPI networks are labor intensive, time consuming, costly, plagued by false positives/negatives, and do not provide a way for individual researchers to efficiently map global PPI networks for particular biological systems. The proposed technology development is targeted to solve the two most important challenges facing crosslinking-mass spectrometry (CLMS)-based global PPI mapping studies: 1) the complexity of peptide mixtures derived from crosslinking samples with a large number of proteins and a large dynamic range of abundances, and 2) efficient and confident identification of crosslinked peptides by whole proteome database searches. We seek to overcome these challenges by developing a novel crosslinked peptide enrichment strategy, called Expedit, and combining it with the powerful capabilities of ICL crosslinkers, a new class of MS-cleavable, isotopomeric, bi- functional crosslinkers for crosslinked peptide identification. Unique features of ICLs permit 1) efficient determination of individual peptide masses in each crosslink from MS2 spectra, and 2) identification of crosslinked peptides by whole proteome database searching using a single MS2 spectrum per crosslinked peptide. The combination of Expedit with ICLMS is expected to address the major limitations of current CLMS approaches to enable routine large scale PPI studies for the first time. In the Aims, we will first synthesize novel Expedit reagents and evaluate their effectiveness for crosslinked peptide enrichment using increasingly complex mixtures. Once optimized, we will integrate Expedit with ICLMS and evaluate the effectiveness of the technology for building global PPI networks in yeast. We will evaluate the method in terms of the quantity and reproducibility of identified crosslinks/PPIs, the abundances of the identified proteins, their localization, affinities and complex membership (if available). We will compare our PPI networks to previously described yeast PPI networks. The effectiveness of the technology for crosslinked peptide identification will be evaluated by comparing it to state-of-the-art CLMS-based approaches. If successful, this project would provide a general and robust method for studying global PPIs and their dynamics that can be applied to any organism for which a sequenced genome is available.