Measuring Protein Thermal Stability and Phosphorylation Changes to Capture Early Cellular Responses to Small Molecule Drugs

Simultaneous CETSA® And Phosphoproteomics Profiling

CETSA® experiments have been performed in compressed format (also known as one-pot or PISA) After short (60 minutes) incubation of cells with different compounds, cell suspensions were divided into aliquots, followed by heat shock treatment (44–66 °C) for 3 minutes. After heat treatment, the samples were pooled back together followed by centrifugation to remove aggregated proteins. Proteins were digested and labelled with TMTpro (16plex). For the Phosphoproteomics analysis phosphopeptides were enriched using titanium dioxide resin.

Key Learnings

  • Both target engagement (CETSA®) and cellular signalling (phosphoproteomics) capture early cellular responses to treatment with small molecule drugs
  • Changes in binding specificity translates into change in downstream phosphorylation events
  • Parallel CETSA® and Phosphoproteomics analysis provides most comprehensive data on specificity and MoA of kinase inhibitors

Download our CETSA® and Phosphoproteomics analysis poster.


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