Thermal Proteome Profiling (TPP)

Thermal Proteome Profiling (TPP) or Cellular Thermal Shift Assay (CESTA) is an emerging label-free technique that can be used to prioritize the putative targets of the bait molecule by measuring the changes in thermal stability of the proteins after the ligand binding.
[Low False Positive Identifications]


Working Principle

This methodology relies on the fact that upon ligand binding, target proteins become more stable and less susceptible to thermal degradation. Proteins that display significant alteration in their thermal denaturation/degradation profile in presence and absence of the bait-molecule are prioritized as its targets.

Workflow

In a typical experiment cell-lysate or cells are incubated in presence and absence of the test-molecule. These biological samples are then divided into multiple fractions and subsequently each fraction is heated at different temperature. Degraded proteins are removed by centrifugation and soluble protein fraction is subjected to protein digestion and peptide labeling with tandem isobaric tags. The proteins are than identified and quantified using mass-spectrometry based method. Based on the relative amount of protein present at each temperature condition, melting temperature of each protein is calculated and the protein showing significant increase in melting temperature in the condition where molecule was present are treated as putative target.



Points of Consideration

  • This method is limited to soluble proteins only.
  • Target deconvolution takes more time due to analysis of huge data-set.