SUMO-Activated Target Traps (SATTs) for the Identification of Specific SUMOylated Proteins for an E3 Ligase of Interest.

SUMOylation is a dynamic and reversible post-translational modification that occurs on acceptor lysines of substrate proteins. SUMO is conjugated via a dedicated enzymatic cascade of E1-E2-E3 enzymes, where the E3 confers substrate specificity. More than 6500 SUMO2/3 target proteins have been identified by mass spectrometry-based proteomics with important regulatory roles, predominantly in nuclear processes. However, although there are fewer than a dozen bona fide E3 ligases, matching an E3 ligase to the modification of specific SUMOylated proteins remains challenging. Here, we introduce SATTs (SUMO-activated target traps) to directly trap substrates of a SUMO E3 ligase of interest (E3OI) and subsequently identify them by LC-MS/MS analysis. The SATT methodology consists of a linear fusion of your E3OI with either SUMO1 or SUMO2/3 flanked by two 10xHis tags. This strategy allows the E3OI to conjugate the fused SUMO moiety to target proteins, forming E3OI-SUMO-target complexes, which can be Ni-NTA purified and detected by LC-MS/MS. A detailed description of the methodology is provided in this chapter, including a discussion of potential pitfalls and specific recommendations.