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

总结 该项目旨在开发新技术，以实现蛋白质-蛋白质相互作用（PPI）的全球映射 由对特定生物学领域感兴趣的研究人员以特定条件、及时和负担得起的方式 系统.这项技术的成功开发预计将对所有领域产生变革性影响 生物医学研究，直接解决重点技术研究的目的， 开发R 01 FOA（PAR-19-253）。全球PPI网络不仅是最重要的资源， 了解正常和异常生物过程的分子机制，但也 为理解遗传相互作用网络，构建基因调控网络和定量 模拟生物过程。目前用于绘制全球PPI网络的技术是劳动密集型的， 耗时、昂贵、受到假阳性/阴性的困扰，并且不提供用于个体检测的方法。 研究人员可以有效地绘制特定生物系统的全球PPI网络。所提出的技术 发展的目标是解决面临的两个最重要的挑战，交联-质谱 （CLMS）为基础的全球PPI映射研究：1）复杂的肽混合物衍生自交联 具有大量蛋白质和大的丰度动态范围的样品，以及2）高效且 通过全蛋白质组数据库检索可靠鉴定交联肽。我们力求克服 通过开发一种新的交联肽富集策略，称为Expedit， 将其与ICL交联剂的强大功能相结合，一类新的MS-可裂解，同位素，双- 用于交联肽鉴定的功能性交联剂。ICL的独特功能允许1）高效 从MS 2光谱确定每个交联中的单个肽质量，和2）鉴定 通过全蛋白质组数据库搜索，使用单个MS 2谱图， 肽。Expedit与ICLMS的结合有望解决当前CLMS的主要局限性 首次实现常规大规模PPI研究的方法。 在目标中，我们将首先合成新型Expedit试剂，并评估其对 使用日益复杂的混合物进行交联肽富集。一旦优化，我们将整合Expedit 与ICLMS和评估的有效性的技术，建立全球PPI网络的酵母。我们将 根据已鉴别交联/PPI的数量和重现性、丰度和 鉴定的蛋白质，它们的定位，亲和力和复杂的成员关系（如果可用）。我们将比较 将我们的PPI网络与先前描述的酵母PPI网络进行比较。该技术的有效性， 将通过将其与最先进的基于CLMS的 接近。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 以及它们的动力学，可以应用于任何有测序基因组的生物体。

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.