Presentation description

The 518 kinases in the human genome, i.e., the kinome, control most cellular signaling networks and biological processes by protein phosphorylation. Kinases recognize their substrates through protein-protein interactions (PPI), and protein phosphorylation often alters PPIs. Therefore, mapping kinase-mediated interaction networks can be used to quantify cellular network states in health and disease. PPIs are often transient and challenging to capture using traditional affinity purification-mass spectrometry (AP-MS) methods. This project aims to develop an in-cell chemoproteomic platform for mapping kinase-protein interactions in their native context. As part of this effort, we evaluated the effectiveness of the amine-reactive crosslinker 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) in stabilizing kinase-protein interactions for downstream AP-MS analysis.

We used our kinobead-based chemoproteomic platform to map kinase PPIs in K562 cell lysates that were incubated with the ATP-competitive multi-kinase inhibitor staurosporine or a DMSO control. Crosslinking was performed at varying DTSSP concentrations to determine optimal conditions for preserving kinase-associated PPIs and to adapt an existing DSP protocol for DTSSP. After affinity enrichment, samples were washed, denatured, digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting protein profiles were assessed for the presence of kinase interactors; competition with staurosporine was used to distinguish unspecific precipitation of kinase complexes from specific binding to the kinobeads. Our preliminary results demonstrated that DTSSP effectively stabilizes PPIs and allows detection of kinase-interacting proteins that may otherwise be lost during purification. These findings support the utility of DTSSP-assisted AP-MS as a complementary strategy in refining in-cell methods for mapping kinase interaction networks, with broad potential applications in disease research, including cancer.