A new chemical and enzymatic approach to analyze protein sumoylation site.

(A) Schematic of the conventional method using intact SUMO remnant as a signature for MS analysis. (B) Schematic of our new method (see text for details). (C) In vitro sumoylation reaction was performed using purified E1, E2 and Mms21 enzymes to generate poly-SUMO chains. After acetylation, treatment by Ulp1 disassembled poly-SUMO chains. (D) Method to evaluate acetylation efficiency of known SUMO targets purified from yeast cells. Ulp1 was used prior to acetylation reaction. (E) Comparison of acetylated versus unmodified lysine identified for known SUMO targets after treated by increasing concentration of acetic anhydride. The numbers of peptides containing unmodified lysine and acetylated lysine (in parenthesis) are indicated.