Abstract
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 (Mr = 26803.9) and its E98A (Mr = 26745.9) or D182A (Mr = 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 S—S 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.
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Hsieh, F. Y. L.; Tong, X.; Wachs, T.; Ganem, B.; Henion J. Kinetic Monitoring of Enzymatic Reactions in Real Time by Quantitative High-Performance Liquid Chromatography-Mass Spectrometry. Anal. Biochem 1995, 229, 20–25.
Bothner, B.; Chavez, R.; Wei, J.; Strupp, C.; Phung, Q.; Schneemann, A.; Siuzdak G. Monitoring Enzyme Catalysis with Mass Spectrometry. J. Biol. Chem. 2000, 275, 13455–13459.
Ge, X.; Sirich, T. L.; Beyer, M. K.; Desaire, H.; Leary J. A. A Strategy for Determination of Enzyme Kinetics Using Electrospray Ionization with an Ion Trap Mass Spectrometer. Anal. Chem. 2001, 73, 5078–5082, and references cited therein.
Gerber, S. A.; Scott, C. R.; Turecek, F.; Gelb M. H. Direct Profiling of Multiple Enzyme Activities in Human Cell Lysates by Affinity Chromatography/Electrospray Ionization Mass Spectrometry: Application to Clinical Enzymology. Anal. Chem. 2001, 73, 1651–1657.
Zechel, D. L.; Konermann, L.; Withers, S. G.; Douglas D. J. Pre-steady State Kinetic Analysis of an Enzymatic Reaction Monitored by Time-Resolved Electrospray Ionization Mass Spectrometry. Biochemistry 1998, 37, 7664–7669.
Houston, C. T.; Taylor, W. P.; Widlanski, T. S.; Reilly J. P. Investigation of Enzyme Kinetics Using Quench-Flow Techniques with MALDI-TOF Mass Spectrometry. Anal. Chem. 2000, 72, 3311–3319.
Comisarow, M. B.; Marshall A. G. Fourier Transform Ion Cyclotron Resonance Spectroscopy. Chem. Phys. Lett. 1974, 25, 282–283.
Amster I. J. Fourier Transform Mass Spectrometry. J. Mass Spectrom. 1996, 31, 1325–1337.
Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty F. W. Top Down Versus Bottom Up Protein Characterization by Tandem High-Resolution Mass Spectrometry. J. Am. Chem. Soc. 1999, 121, 806–812.
Ge, Y.; Lawhorn, B. G.; ElNaggar, M.; Strauss, E.; Park, J.; Begley, T. P.; McLafferty F. W. Top Down Characterization of Larger Proteins (45 kDa) by Electron Capture Dissociation Mass Spectrometry. J. Am. Chem. Soc. 2002, 124, 672–678.
Ge, Y.; ElNaggar, M.; Sze, S. K.; Oh, H.; Begley, T. P.; McLafferty, F. W.; Boshoff, H.; Barry C. E. Top Down Characterization of Secreted Proteins from Mycobacterium tuberculosis by Electron Capture Dissociation Mass Spectrometry. J. Am. Soc. Mass Spectrum. 2003, 14, 253–261.
Zabrouskov, V.; Giacomelli, L.; van Wijk, K. J.; McLafferty F. W. A New Approach for Plant Proteomics: Characterization of Chloroplast Proteins of Arabidopsis thaliana by Top Down Mass Spectrometry. Mol. Cell. Proteom. 2003, 2, 1253–1260.
Valaskovic, G. A.; Kelleher, N. L.; McLafferty F. W. Attomole Protein Characterization by Capillary Electrophoresis/Mass Spectrometry. Science 1996, 273, 1199–1202.
Xu, G.; Zhai, H.; Narayan, M.; McLafferty, F. W.; Scheraga H. A. Simultaneous Characterization of the Reductive Unfolding Pathways of RNase B Isoforms by Top Down Mass Spectrometry. Chem. Biol. 2004, 11, 517–524.
Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M.; McLafferty F. W. Fourier-Transform Electrospray Instrumentation for Tandem High-resolution Mass Spectrometry of Large Molecules. J. Am. Soc. Mass Spectrom. 1993, 4, 557–565.
Horn, D. M.; Zubarev, R. A.; McLafferty F. W. Automated Reduction and Interpretation of High Resolution Electrospray Mass Spectra of Large Molecules. J. Am. Soc. Mass. Spectrom. 2000, 11, 320–332.
Jordan F. Current Mechanistic Understanding of Thiamin Diphosphate-Dependant Enzymatic Reactions. Nat. Prod. Rep. 2003, 20, 184–201.
Park, J.; Dorrestein, P. C.; Zhai, H.; Kinsland, C.; McLafferty, F. W.; Begley T. P. Biosynthesis of the Thiazole Moiety of Thiamin Pyrophosphate (Vitamin B1). Biochemistry 2003, 42, 12430–12438.
Dorrestein, P. C.; Zhai, H.; Taylor, S. V.; McLafferty, F. W.; Begley T. P. The Biosynthesis of the Thiazole Phosphate Moiety of Thiamin (Vitamin B1): The Early Steps Catalyzed by Thiazole Synthase. J. Am. Chem. Soc. 2004, 126, 3091–3096.
Settembre, E. C.; Dorrestein, P. C.; Zhai, H.; Chatterjee, A.; McLafferty, F. W.; Begley, T. P.; Ealick S. E. Thiamin Biosynthesis in Bacillus subtilis: Structure of the Thiazole Synthase/Sulfur Carrier Protein Complex. Biochemstry 2004, 43, 11647–11657.
Cerda, B. A.; Horn, D. M.; Breuker, K.; McLafferty F. W. Sequencing of Specific Copolymer Oligomers by Electron-Capture-Dissociation Mass Spectrometry. J. Am. Chem. Soc. 2002, 124, 9287–9291.
Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S.; Guan, S.; Logan T. M. Protein Molecular Weight to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433–434.
Dorrestein, P. C.; Zhai, H.; McLafferty, F. W.; Begley T. P. The Biosynthesis of the Thiazole Phosphate Moiety of Thiamin: The Sulfur Transfer Mediated by the Sulfur Carrier Protein ThiS. Chem. Biol. 2004, 11, 1373.
Pesavento, J. J.; Kim, Y.; Taylor, G. K.; Kelleher N. L. Shotgun Annotation of Histone Modifications. A New Approach for Streamlined Characterization of Proteins by Top Down Mass Spectrometry. J. Am. Chem. Soc. 2004, 126, 3386–3387.
Hicks, L. M.; O’Conner, S. E.; Mazur, M. T.; Walsh, C. T.; Kelleher N. L. Mass Spectrometric Interrogation of Thioester-Bound Intermediates in the Initial Stages of Epothilone Biosynthesis. Chem. Biol. 2004, 11, 327–335.
Mazur, M. T.; Walsh, C. T.; Kelleher N. L. Site-Specific Observation of Acyl Intermediate Processing in Thiotemplate Biosynthesis by Fourier Transform Mass Spectrometry. Biochemistry 2003, 42, 13393–13400.
Narayan, M.; Xu, G.; Ripoll, D.; Zhai, H.; Breuker, K.; Wanjalla, C.; Ceung, H. J.; Navon, A.; Welker, E.; McLafferty, F. W.; Scheraga H. A. Dissimilarity in the Reductive Unfolding Pathways of Two Ribonuclease Homologues. J. Mol. Biol. 2004, 338, 795–809.
Xi, J.; Ge, Y.; Kinsland, C.; McLafferty, F. W.; Begley T. P. Biosynthesis of the Thiazole Moiety of Thiamin in Escherichia coli: Identification of an Acyldisulfide-Linked Protein-Protein Conjugate that is Functionally Analogous to the Ubiquitin/E1 Complex. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8513–8518.
McLoughlin, S. M.; Kelleher N. L. Kinetic and Regiospecific Interrogation of Covalent Intermediates in the Biosynthesis of Yersiniabactin. J. Am. Chem. Soc. 2004, 126, 13265–13275.
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Published online May 23, 2005
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Zhai, H., Dorrestein, P.C., Chatterjee, A. et al. Simultaneous Kinetic Characterization of Multiple Protein Forms by Top Down Mass Spectrometry. J Am Soc Mass Spectrom 16, 1052–1059 (2005). https://doi.org/10.1016/j.jasms.2005.02.016
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DOI: https://doi.org/10.1016/j.jasms.2005.02.016