Identification of Substrates of Protein-Group SUMOylation

Protein modification by conjugation to the ubiquitin-related protein SUMO (SUMOylation) regulates numerous cellular functions and is reversible. However, unlike typical posttranslational modifications, SUMOylation often targets and regulates proteins of functionally and physically linked protein groups, rather than individual proteins. Functional studies of protein-group SUMOylation are thus particularly challenging, as they require the identification of ideally all members of a modified protein group. Here, we describe mass spectrometric approaches to detect SUMOylated protein groups in Saccharomyces cerevisiae, yet the protocols can be readily adapted for studies of SUMOylation in mammalian cells.