The Cellular Thermal Shift Assay (CETSA) is based on the discovery that protein melting curves can also be generated in intact cells and that drug binding leads to very significant thermal stabilization of proteins. It quantifies the changes in the thermal stability of proteins upon ligand binding in intact cells. (Martinez et. al., Science, 2013). IP2 has thermal proteome profiling temperature range (TPPTR) module to analyze changes in protein thermal stability across the proteome using quantitative mass spectrometry (Franken, et. al., Nat Protoc. 2015). Currently, IP2 supports isobaric labeling (TMT, iTRAQ).
1. In the experiment page, click the “TPPTR” link.
Figure 13.7.1 Experiment page
2. It opens Thermal Proteome Profile (TPP) page. Select “New TPPTR” link to run an analysis.
Figure 13.7.2 Thermal Proteome Profile page
3. Type a number of temperature points. In this example, we typed in 10. Click next.
Figure 13.7.3 a number of temperature points
4. Fill in temperatures. Typically temperatures of vehicle and treatment are same. Click next.
Figure 13.7.4 Temperature fill-in page
7. After reviewing the vehicle and the treatment, click next button.
Figure 13.7.7 Review temperature selection
8. Select either raw intensity or normalized intensity. Give an analysis name to save. Click Run button.
Figure 13.7.8 Run page
9. Analysis results in text format can be downloaded by clicking the download link. The result file can be loaded in Excel. To view results, click an analysis name in the name column.
Figure 13.7.9 TPPTR main page with a list of the analysis
10. It opens the result page. User can sort the data by the adjusted p-value column. Click the results to display a graph.