System-wide identification of enzyme substrates by thermal analysis (SIESTA)

Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying the molecular components participating in these reactions on a cellular level. Here we developed a universal method called System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that post-translational modification of the substrate protein changes its thermal stability, and applies the concept of specificity to reveal the potential substrates. For selenoprotein thioredoxin reductase 1, SIESTA confirmed several known protein substrates and suggested novel candidates. For poly-(ADP-ribose) polymerase-10, SIESTA revealed a number of PARP10 putative substrates, which were confirmed by targeted mass spectrometry and functional assays. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, and facilitate drug discovery.