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Anal Chem. 2011 Dec 1;83(23):9092-9. doi: 10.1021/ac202154r. Epub 2011 Oct 31.

Ratiometric pulse-chase amidination mass spectrometry as a probe of biomolecular complex formation.

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1
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405-7102, USA.

Abstract

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) (Δ = 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.

PMID:
22007758
PMCID:
PMC3246313
DOI:
10.1021/ac202154r
[Indexed for MEDLINE]
Free PMC Article
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