Probing the Regulation of Ubc9 Activity Using a Mass Spectrometry-Based In Vitro SUMOylation Assay

摘要

Mass spectrometry provides complementary information to Western blot analysis for in vitro SUMOylation assays. Western blots can indicate the percentage of SUMOylated protein while mass spectrometry analyses highlighted in this work can indicate site-specific changes in SUMOylation. Moreover, this mass spectrometry approach not only allows for the determination of SUMO site occupancy on target proteins, but it can also be used to determine the extent of SUMO branching sites and autoSUMOylation on UBC9 E2 protein. SUMO site occupancy determined from in vitro assay provides valuable insights into the enzymatic process of protein SUMOylation.