Multiplexed proteomics-based kinase assay development

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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.