Simultaneous kinetic characterization of multiple protein forms by top down mass spectrometry.
Top down mass spectrometry, using a Fourier transform instrument, has unique capabilities for biomolecule kinetic studies, in that the concentration of large molecules in a reaction mixture can be monitored simultaneously from its mass spectrum produced by electrospray ionization. This is demonstrated with enzyme modifications occurring in the biosynthesis of the thiazole moiety of thiamin phosphate. The formation rate of ThiS-thiocarboxylate from ThiS was determined from the relative abundance of the corresponding m/z 10162 and 10146 isotopic peak clusters for all the observable charge states in the mass spectra measured at different reaction times. Even without measuring standard ionization efficiencies, the rate and precision of 0.018 +/- 0.004 min(-1) agree well with the 0.027 +/- 0.003 min(-1) obtained with a radiochemical assay, which requires a separate derivatization step. To illustrate the simultaneous characterization of the reaction kinetics of a native enzyme and its mutant, the imine formation rate of ThiG and its substrate DXP was compared between the native protein (M(r) = 26803.9) and its E98A (M(r) = 26745.9) or D182A (M(r) = 26759.9) mutant in the same reaction mixture. The kinetic data show clearly that neither the E98 nor the D182 residues participate in the imine formation. The high resolution and MS/MS capabilities of FTMS should make possible the extension of this kinetics approach to far more complicated systems, such as simultaneous monitoring of 24 native, intermediate, and reduced forms in the reductive unfolding of a mixture of ribonuclease A and the five isoforms of ribonuclease B. Stable intermediates with different SS bonding (same molecular weight) can be differentiated by MS/MS, while molecular ions differing by only 2 Da are distinguished clearly by synthesizing isotopically depleted proteins.
Zhai
Huili
H
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301, USA.
Dorrestein
Pieter C
PC
Chatterjee
Abhishek
A
Begley
Tadhg P
TP
McLafferty
Fred W
FW
D001412:Bacillus subtilis / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013838:Thiamine Triphosphate / Q000737:chemistry / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism
D007527:Isoenzymes; D013844:Thiazoles; D013838:Thiamine Triphosphate; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic