Improved Identification of SUMO Attachment Sites Using C-Terminal SUMO Mutants and Tailored Protease Digestion Strategies

A growing number of biological processes have been found to be regulated by the covalent attachment
of the ubiquitin-like protein SUMO to key cellular targets. A critical step in the process of analyzing the
role of SUMO in regulating the activity of these proteins is the identification of the lysine residues that
are targeted by this modification. Unfortunately, current methods aimed at mapping these attachmentsites are laborious and often ineffective. We report here the development of a platform that combines
the use of different C-terminal SUMO mutants with different protease digestion strategies to enable
the rapid and efficient identification of SUMO attachment sites. We successfully apply this approach
to several model SUMO substrates as well as to a mixture of SUMO conjugates purified from
Saccharomyces cerevisiae. Although we specifically employ this strategy for the identification of SUMO
attachment sites in yeast, this general approach can easily be adapted to map the sites of conjugation
for other ubiquitin-like proteins from a wide range of organisms.
Keywords: SUMO • Smt3p • mass spectrometry • site mapping • in vitro conjugation system