Ratiometric pulse-chase amidination mass spectrometry as a probe of biomolecular complex formation.
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.
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405-7102, USA.
Feng-Ming James
FM
Chang
Matthew A
MA
Lauber
William E
WE
Running
James P
JP
Reilly
David P
DP
Giedroc
D000577:Amides / Q000737:chemistry*; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D010455:Peptides / Q000032:analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000577:Amides; D001426:Bacterial Proteins; D010455:Peptides; D003300:Copper; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D008239:Lysine