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.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.