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This protocol describes the proteomics technique called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis or SIESTA 1,2. SIESTA can be used for universal discovery of enzyme substrates that shift in thermal stability or solubility upon post-translational modification (PTM). Experimental design, proteomics sample preparation and data analysis are the key stages of this protocol. Data analysis can be performed using our SIESTA package hosted on GitHub 3. Whendoi:10.21203/rs.3.pex-1327/v1 fatcat:vmlz3wlscvfpjaduzsmebo7vlu