Nano-HPLC assay for determination of the deacylated peptides of CobB
This protocol is extracted from research article:
Protein lysine de-2-hydroxyisobutyrylation by CobB in prokaryotes
Sci Adv, Jul 17, 2019; DOI: 10.1126/sciadv.aaw6703

2-Hydroxyisobutyrylated, acetylated, or succinylated peptides (50 μM) with 1 μM CobB were incubated in a 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)] for 2 hours at 37°C. The reaction was then quenched with 1 volume of 10% (v/v) trifluoroacetic acid and spun for 10 min at 18,000g (Beckman Coulter Microfuge) to separate the enzyme from the reaction. The sample was separated by a 50-min HPLC gradient [linear gradient from 2 to 35% HPLC buffer B (0.1% formic acid in acetonitrile) for 35 min and then to 90% buffer B for 15 min]. The supernatant was then analyzed using nano-HPLC–MS/MS. The HPLC elute was electrosprayed directly into an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA).

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