LC-MS analysis of in vitro SUMOylated SRFR1 protein.

FLAG-SRFR1 was expressed in reconstituted E. coli system (Okada el al 2009) in the presence of SUMO1 T91R SUMOylation-competent (containing diglycine, GG) or -deficient (diglycine replaced by dialanine, AA) isoforms, SCE1a, and SAE1/2. After induction of expressions, total extracts were probed with anti-FLAG antibodies. A slower migrating band than FLAG-SRFR1 was detected in the presence of only GG, but not AA forms of co- expressed SUMO1. We then subjected the slower migrating protein bands of SRFR1 expressed with SUMO1 T91R for trypsin digestion and LC-MS/MS analysis. SUMO1, but not SUMO2 or SUMO3 footprints on selective lysine residues of SRFR1 was detected in the identified peptides. Remarkably, lysine residue present in the previously predicted LK325EE SUMOylation motif was identified with SUMO1-modifications. In addition, the K229 residue of SRFR1 was also covalently modified by SUMO1 even though conserved features of a SUMOylation motif was absent for this lysine.