Detecting protein SUMOyaltion with mass spectrometry
Nadya Galeva, Wenqi Cui, Vinidhra Sridharan, Jeffrey Staudinger, Yoshiaki Azuma, Todd Williams
Small ubiquitin-like modifiers (SUMOs) belong to a family of proteins that covalently attach individually or in chain to other proteins to modify their function. The process of SUMOyaltion is similar to protein-ubiquitin conjugation and requires a series of enzymatic reactions that involve E1, E2 and E3 enzymes. As a result, an isopeptide bond is formed between SUMO C-terminal glycine and lysine side chain of a conjugated protein. Detecting and localizing SUMOylation sites in proteins represents a challenging analytical problem due to low abundance and an inconveniently long sequence that remains on a tryptic peptide after digestion with trypsin. SUMO3Q87R mutant was designed to shorten the tail on tryptic SUMOylated peptides. In this work we applied LC-MS/MS methodology for studying covalent attachment of SUMO3 and SUMO3Q87R to the Plk1-interacting checkpoint helicase, a member of the SNF2 ATPase family, and steroid and xenobiotic sensing nuclear receptor, Pregnane X receptor (PXR).