FIGURE 4.

LC-MS/MS identification of Cys-39 as a prominent site of succination of adiponectin. Adiponectin from 3T3 adipocytes cultured in either 5 or 30 mm glucose was immunoprecipitated and electrophoresed on one-dimensional gels. The isolated adiponectin bands were excised from the gel, alkylated with iodoacetamide, and digested with trypsin, and the peptides were analyzed by high pressure liquid chromatography-ESI-MS/MS. A–C, extracted ion chromatograms for either the carbamidomethylated cysteine-containing peptide (residues 37–58; GTCAGWMAGIPGHPGHNGTPGR) of adiponectin from cells grown in 5 or 30 mm glucose, respectively (A and B), or the succinated peptide from cells grown in 30 mm glucose (C). The masses indicated in each frame correspond to the +4 charge state for the peptide. D, MS/MS sequencing data obtained from fragment ions derived from m/z 574.87 [M + 4H]⁴⁺, demonstrating that Cys-39 is the site of succination on adiponectin adipocytes grown in 30 mm glucose. OH-P, hydroxyproline residues in the peptide.