Motif-Targeting Phosphoproteome Analysis of Cancer Cells for Profiling Kinase Inhibitors

Simple Summary Phosphoproteomics is essential for basic understanding of cell biology. However, analysis of the phosphoproteome remains far from comprehensive. Therefore, we proposed a new method to enrich a subset of the phosphoproteome and expand the scope of analysis by using in vitro kinase reactions. We showed our novel workflow identified and quantified the phosphopeptides which have not been observed in the conventional workflow. We also demonstrated that this method is effective for profiling kinase inhibitors. We envision our workflow could easily be adapted to target different subsets of the phosphoproteome by utilizing different sets of kinases, and should be applicable to variety of samples. We present a motif-targeting phosphoproteome analysis workflow utilizing in vitro kinase reaction to enrich a subset of peptides with specific primary sequence motifs. Phosphopeptides are enriched and dephosphorylated with alkaline phosphatase, followed by in vitro kinase reaction to phosphorylate substrate peptides with specific primary-sequence motifs. These phosphopeptides are enriched again, TMT-labeled, dephosphorylated to enhance MS-detectability, and analyzed by LC/MS/MS. We applied this approach to inhibitor-treated cancer cells, and successfully profiled the inhibitory spectra of multiple kinase inhibitors. We anticipate this approach will be applicable to target specific subsets of the phosphoproteome using the wide variety of available recombinant protein kinases.