Multiplexed proteomics-based kinase assay development

项目摘要 本补充申请要求CEM Corporation提供MultiPep2肽库合成仪， 用于制备合成肽候选物库，作为我们在优化激酶 底物肽设计。R01项目的主要总体目标是开发细胞可交付的面板 激酶的肽底物，其将使得能够使用 高通量靶向质谱法。这一进展有两个组成部分： ·激酶底物偏好图谱，以及基于这些偏好的肽底物设计 ·重点选择在质谱检测中表现良好的底物 这两个目标都需要筛选许多肽，以确定最佳候选物来定义给定的肽。 激酶的活性，我们最终将建立作为使用靶向质谱法的测定方法 我们和Cedars Sinai的合作者正在开发中。MultiPep2仪器是一种库级仪器， 肽合成仪，具有每次运行合成多达384个肽的多功能能力，在小型多孔- 板规模，这是远远更多的资源和试剂效率比我们目前的生产规模， 非文库合成器，其一次只能以约10倍的规模制备多达24个，因此试剂 和溶剂的使用。这将提高该项目筛选候选肽的效率，减少溶剂 使用、相关试剂成本和环境影响，并减少研究人员在文库合成上的时间， 把它保留给工作中更需要智力的方面。

Project Summary This supplement application is to request a MultiPep2 peptide library synthesizer from CEM Corporation, to be used for preparing libraries of synthetic peptide candidates as a critical part of our efforts in optimal kinase substrate peptide design. The major, overarching goal of this R01 project is to develop panels of cell-deliverable peptide substrates for kinases that will enable identification of signatures of signaling activity in live cells using high-throughput targeted mass spectrometry. There are two components of this development in progress: · Kinase substrate preference mapping, and peptide substrate design based on those preferences · Focused selection of substrates that perform well in mass spectrometry detection Both of these goals require screening of many peptides to determine the best candidates to define a given kinase's activity, which we will eventually establish as assays using the targeted mass spectrometry approaches that are in development with our collaborator at Cedars Sinai. The MultiPep2 instrument is a library-scale peptide synthesizer, with versatile capabilities to synthesize up to 384 peptides per run, at a small multiwell- plate scale that is far more resource and reagent-efficient for this purpose than our current production-scale, non-library synthesizer, which can only make up to 24 at a time at about 10X greater scale and thus reagent and solvent usage. This will enhance the project's efficiency in screening peptide candidates, reduce solvent usage, associated reagent costs, and environmental impact, and reduce researcher time on library synthesis, reserving it for more intellectually demanding aspects of the work.