Fig. 1: SIESTA workflow for unbiased proteome-wide identification and prioritization of enzyme substrates.

A master cell lysate is prepared by multiple freeze-thawing in a non-denaturing buffer. The cell lysate aliquots are treated with vehicle (control), cosubstrate, enzyme, or combination of enzyme with cosubstrate (both). After treatment, each aliquot is split into ten tubes, with each tube heated to a temperature point in the range from 37 to 67 °C. After removing unfolded proteins by ultracentrifugation, identical volumes of supernatants are digested with trypsin. The samples are then serially labeled with 10-plex tandem mass tag (TMT) reagents, pooled, cleaned, and fractionated by reversed-phase chromatography. After LC-MS/MS analyses of each fraction, protein IDs and abundances are determined, and sigmoid curves are fitted through an automated algorithm to determine the melting temperature (T_m) for each protein. For each non-vehicle treatment, the read-out is the protein’s ∆T_m shifts (of both signs) compared to control. Any protein shifting more upon addition of enzyme and cosubstrate compared to when they are added alone, are putative substrates of the enzyme under study. Such candidate protein substrates are subsequently confirmed by orthogonal verification methods, starting from the proteins exhibiting largest ∆T_m shifts and/or involved in relevant pathways.