Phosphorylation events are critical post-translational events that are tightly controlled by a series of counteracting enzymes, kinases and phosphatases which transfer phosphate groups to the target protein or remove phosphates to alter the signaling cascade. The low stoichiometry of phosphorylated targets requires significant time investment for sample preparation to ensure that the analytes are sufficiently enriched to be analyzed by mass spectrometry. Previous works required extensive fractionation steps and other complex workflows that limits the application to select research groups. Here, we present the integrated workflow that incorporates cell lysis, reduction/alkylation, trypsin digestion, reverse phase desalting, and phosphopeptide enrichment. The entire process is automated from start to finish, with samples ready for injection, including sample identification that is directly exported to the mass spectrometry, allowing for higher throughput testing. An example of this workflow has been expanded to drug dosing effects and potential therapeutic effects by monitoring specific phosphopeptides by liquid chromatography-mass spectrometry, multiple-reaction monitoring.

Robust integrated workflow of phosphopeptide enrichment for monitoring kinase and phosphatase activities in drug efficacy modeling

2019 Spring National Meeting

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