WT and mutated (Y55F and R58M) CobB were incubated with peptides in the reaction buffer [50 mM tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM NAD+ (pH 8.5)] at 37°C for 2 min. The concentrations of the 2-hydroxysiobutyrylated peptide were 2, 5, 8, 10, 12, 16, 32, 40, 60, 142, and 285 μM. One volume of 10% (v/v) trifluoroacetic acid was added to quench the reaction. After cleaning with C18 ZipTips, the resulting peptides were analyzed with an Autoflex III TOF/TOF mass spectrometer (Bruker Daltonics). The measurements were carried out in a reflex positive-ion mode with delayed ion extraction. Before analysis, the instrument was externally calibrated with a mixture of peptide standards. 2,5-dihydroxybenzoic acid (DHB) was used as the matrix for the analysis of labeled/unlabeled peptides. The sample aliquots (1.0 μl) were placed onto a MALDI plate, and then 1.0 μl of the DHB matrix was added and dried at room temperature before MS analysis. An acceleration voltage of 20 kV was used. MS data were analyzed using FlexAnalysis software for spectral processing and peak detection.

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