Fig. 3

Site-specific modification of proteins with GDL. a Deconvoluted ESI-TOF spectra of the reaction of GDL with GH₆-tagged EGFP, MBP, and SUMO. The GH₆-tagged proteins were reacted in 200 mM HEPES buffer at pH 7.5 and room temperature. GH₆-EGFP (350 µM) was treated with 200 mM lactone for 1 h. GH₆-MBP (350 µM) was treated with 350 mM lactone for 4 h. GH₆-SUMO (42 µM) was treated with 189 mM lactone for 1 h. b Detection of N-terminal selective gluconoylation of GH₆-EGFP. MS spectra of non-reacted and gluconoylated GH₆-EGFP after digestion with trypsin or chymotrypsin are depicted. MALDI-TOF spectra of the N-terminal fragment consisting of amino acids #1–14, deconvoluted ESI-TOF spectra of tryptic fragment #29–276, and ESI-TOF spectra of chymotryptic fragment #20–29 containing Lys21 (the only Lys residue in the first 28 amino acids of the protein) are depicted. The peak at 1595.219 Da detected in the MALDI-TOF spectrum of the gluconoylated protein was attributed to the partial reversibility of this reaction, which was also observed for peptide 15 (Supplementary Fig. 9 and 10). c Deconvoluted ESI-TOF spectra of the reaction of three EGFP variants with GDL. The proteins were reacted at a concentration of 35 µM with 200 mM GDL for 1 h in 200 mM HEPES buffer at pH 7.5 and room temperature. The degrees of conversion, based on the deconvoluted MS data, were 92%, 60%, and 66% for GH₆-EGFP, GSSH₆-EGFP, and GSH-EGFP, respectively. Unmodified proteins are labeled SM, and species corresponding to the correct product mass are labeled P