期刊
ANALYTICAL CHEMISTRY
卷 83, 期 23, 页码 9092-9099出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac202154r
关键词
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资金
- U.S. National Institutes of Health [GM042569]
- U.S. National Science Foundation [CHE-1012855]
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [1012855] Funding Source: National Science Foundation
Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse-chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from Bacillus subtilis as a model system. CsoR in various allosteric states was reacted with S-methyl thioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methyl thiopropionimidate (SMTP) (Delta = 14 amu), quenched and digested with chymotrypsin or Glu-C protease, and peptides were quantified by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and/or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We show that the reactivities of individual lysines from peptides containing up to three Lys residues are readily quantified using this method. New insights into operator DNA binding and the Cu(I)-mediated structural transition in the tetrameric copper sensor CsoR are also obtained.
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