Oral Presentation 23rd Annual Lorne Proteomics Symposium 2018

High Resolution - Mass Spectrometry Cellular Thermal Shift Assay (HR-MS-CETSA)- post-translational modifications impact on thermal protein stability (#66)

Lingyun Dai 1 , Tianyun Zhao 1 , Liyan Chen 1 , Yanting Lim 1 , Chris Soon Heng Tan 2 , Nayana Prabhu 1 , Par Nordlund 1 2 3 , Radoslaw Sobota 2
  1. School of Biological Sciences, Nanyang Technological University, Singapore
  2. Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A-STAR), Singapore
  3. Department of Oncology-Pathology , Karolinska Institutet, Stockholm, Sweden

Observation of the proteins thermal stability towards increasing temperature forms the foundation for methods that explore thermally induced protein unfolding. Heat treatment induced protein unfolding and aggregation can be graphically presented as a sigmoidal melting curve which allows estimation of melting temperatures (Tm). It was shown that ligand engagement changes the functional state of the protein and induces thermal shift (Tm). This is the principle of thermal shift assays (TSA) and recent extension of this method - Cellular Thermal Shift Assay (CETSA). CETSA principle takes into account the fact that biophysical thermal stability (thermal induced unfolding) of individual proteins can be monitored and quantified in lysates/intact cells/tissue samples. MS based CETSA tackles thermal shifts in whole proteome using quantitative mass spectrometry. To further extend CETSA application in drug target deconvolution, here we proposed high resolution (HR) MS-CETSA to increase a target specificity.
Accordingly, we investigated further impact of post-translational modifications on the protein stability.  Following the data analysis, we were able to detect in the sample with limited input more than 5000 distinct phosphorylation residues in the single experiment. It corresponds to more than 5000 unique melting profiles and or ITDR (isothermal dose responses). We have confirmed our hypothesis that phosphorylated proteins exhibit different stability towards increasing temperature. Overall the data points towards destabilizing effect of the phosphorylation, although detailed mechanism has to be further determined. Furthermore, presented method could help to identify functional phosphorylation sites in the protein which occupancy would be reflected in protein stability.

The Cellular PTM resolved MS-CETSA has potential to add a very valuable dimension to current pre-clinical and clinical drug development defining drug target engagement and off target effects in relevant cell and tissue systems.