Identification of SUMO Target Proteins by Quantitative Proteomics

The identification of target proteins for small ubiquitin-like modifiers (SUMOs) is a critical step towards a detailed understanding of the cellular functions of SUMOs. Substrate protein identification for SUMOs is hampered by the low abundance of SUMO targets, the finding that only a small fraction of these target proteins is sumoylated, and the high activity of deconjugating enzymes. Quantitative proteomics is a powerful tool to overcome these challenges, because it allows discrimination between contaminating proteins in SUMO-enriched preparations and true target proteins. In this chapter, the methodological details of the application of stable isotope labeling of amino acids in cell culture (SILAC) for the identification of target proteins for SUMOs are described. In addition to steady state sumoylation, the sumoylated proteome undergoes dynamic rearrangements in response to a diverse array of stimuli. SILAC also allows the study of sumoylation dynamics in response to these stimuli.