Multiplexed proteomics-based kinase assay development

项目摘要 本补充申请请求CEM公司的MultiPep2多肽文库合成器， 用于制备合成多肽候选文库，作为我们在最佳激酶方面所做努力的关键部分 底物多肽设计。这个R01项目的主要总体目标是开发可交付电池的电池板 用于激酶的多肽底物，将能够识别活细胞中的信号活动的特征 高通量靶向质谱学。正在进行的这项发展有两个组成部分： ·酶底物偏好图谱，以及基于这些偏好的多肽底物设计 ·重点选择在质谱学检测中表现良好的底物 这两个目标都需要对许多肽进行筛选，以确定定义给定的 激酶的活性，我们最终将建立它作为使用靶向质谱学方法的分析方法 正在与我们在雪松西奈的合作者一起开发。MultiPep2仪器是一个图书馆规模的仪器 多肽合成器，具有多用途的能力，每次可合成多达384个多肽，在一个小的多孔- 这比我们目前的生产规模在资源和试剂效率上要高得多， 非文库合成器，一次最多只能合成24个，规模约为试剂的10倍 和溶剂的使用。这将提高项目在筛选候选多肽方面的效率，减少溶剂 使用、相关试剂成本和环境影响，并减少研究人员合成文库的时间， 把它留给工作中对智力要求更高的方面。

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.