Discovery and Interrogation of Functional Protein Modifications by Hotspot Thermal Profiling

Abstract Hundreds of protein posttranslational modification types have been reported across diverse organisms, however we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function under specific cellular contexts. This protocol describes a method to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This mass spectrometry-based protocol measures shifts in overall protein stability in response to site-specific phosphorylation sites. The resulting dataset can enable discovery of intrinsic changes to protein structure as well as extrinsic changes to protein-protein, and protein-metabolite interactions, and can help prioritize site-specific study in a high-throughput and unbiased fashion. This approach takes several days complete, can be performed with multiple samples in parallel and is applicable to diverse organisms, cell types and posttranslational modifications.