Multi-SUMO3 mass spectrometry
To identify novel multi-SUMO–binding proteins, a pull down assay was carried out with HeLa nuclear extracts, using GST-COMP-SUMO1 (multi-SUMO1), GST-COMP-SUMO3 (multi-SUMO3) and mutated GST-COMP-SUMO3AAA (multi-SUMO3AAA) as baits. Multi-SUMO3AAA, a multi-SUMO3 mutated on its SIM recognition site, was employed in order to find proteins binding to alternative sites on the SUMO3 protein. Specific interactions with multi-SUMOs were identified by first removing nonspecific binding proteins. Bound protein identities were then determined by MS with positive set to at least 2 unique peptides, screened for background proteins using the Contaminant Repository for Affinity Purification - CRAPome. We chose a threshold of 100/411 experiments represented in CRAPome, and plotted specifically binding proteins. In two separate mass spectrometry repeats a total of 369 proteins were identified as bound to multi-SUMO3, out of which 206 were specifically binding, and 40 of those proteins appeared in both repeats. 362 proteins were identified as bound to multi-SUMO1, out of which 177 proteins were specifically binding (CRAPome), but only 2 proteins appeared in both experiments. Proteins that potentially bind to an alternative binding site on SUMO3 - 395 proteins were identified as bound to multi-SUMO3AAA out of which 224 were high confidence and 50 appeared in both repeats.