Reduced-representation Phosphoprofiling

There are something like >100,000 distinct phosphorylation sites in the human proteome, yet <1000 kinases and phosphotases that govern their regulation. This begs the question: can a proxy set of readily observable phosphopeptides serve as a lens into the larger world of phosphorylation and signaling?

We believe the answer is yes, and we have taken a data-driven approach to identify a set of 96 phosphopeptide probes that we have configured as analytes in a targeted phosphoproteomic assay that we call the P100 assay. We have demonstrated that these probes are easily observable and quantifiable in diverse cell types ranging from embryonic stem cells to human leukemia cells, and that they behave in interesting and orthogonal ways in response to varied chemical and genetic pertrubations. The assay enables us to draw Connectivity Maps in the space of phosphosignaling. These intuitively visualized structures allow us to see both expected and novel associations among perturbations and cells.

P100-based Connectivity Map of Chemical Perturbagens

A.Connectivity Map of chemical perturbations in MCF7 (breast cancer) and PC3 (prostate cancer) cells. Note the tight clustering of the cardiac glycosides and the unexpected connection to the antibiotic anisomycin. B.Connectivity Map of chemical perturbations in HL60 (human leukemia) cells. Note the interesting connection of the cardiac glycosides to the chemotherapeutic agents.