System-wide identification and prioritization of enzyme substrates by thermal analysis

Nat Commun. 2021 Feb 26;12(1):1296. doi: 10.1038/s41467-021-21540-6.


Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma
  • Drug Discovery
  • Enzymes / chemistry*
  • Enzymes / genetics
  • Enzymes / metabolism*
  • HCT116 Cells
  • Humans
  • Mass Spectrometry
  • Poly(ADP-ribose) Polymerases / chemistry
  • Poly(ADP-ribose) Polymerases / genetics
  • Poly(ADP-ribose) Polymerases / metabolism
  • Protein Processing, Post-Translational*
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / metabolism
  • Proteomics / methods
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt / chemistry
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism
  • Substrate Specificity
  • Thioredoxin Reductase 1 / chemistry
  • Thioredoxin Reductase 1 / genetics


  • Enzymes
  • Proteins
  • Proto-Oncogene Proteins
  • TXNRD1 protein, human
  • Thioredoxin Reductase 1
  • PARP10 protein, human
  • Poly(ADP-ribose) Polymerases
  • Proto-Oncogene Proteins c-akt