Simultaneous Kinetic Characterization of Multiple Protein Forms by Top Down Mass Spectrometry

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 Ϯ 0.004 min Ϫ1 agree well with the 0.027 Ϯ 0.003 min Ϫ1 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 ϭ 26803.9) and its E98A (M r ϭ 26745.9) or D182A (M r ϭ 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 SOS 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. (J Am Soc Mass Spectrom 2005, 16, 1052-1059

Unraveling Molecular Complexity of Phosphorylated Human Cardiac Troponin I by Top Down Electron Capture Dissociation/Electron Transfer Dissociation Mass Spectrometry

“…A "top down" mass spectrometry (MS) approach has been shown to be uniquely suited for characterization of proteins (10 -200 kDa) with complex post-translational modifications (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). In the traditional "bottom up" approach, proteins of interest are digested with an enzyme (e.g.…”

“…However, bottom up analysis has intrinsic limitations for characterizing protein modifications because the peptide sequences recovered from the digestion typically represent only partial coverage of the protein sequence, and most of the tryptic peptides are relatively small resulting in a loss of correlation between modifications on disparate portions of the protein (34,35). The top down strategy measures molecular weights of intact proteins and dissociates these intact molecular ions directly in the gas phase providing highly reliable detection and characterization of sequence alterations and post-translational modifications (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)36). Use of the newly developed MS/MS techniques of electron capture dissociation (ECD) (37,38) and electron transfer dissociation (ETD) (39) greatly improves both the efficiency and sequence coverage in top down analyses.…”

Observation of pronounced b•,y cleavages in the electron capture dissociation mass spectrometry of polyamidoamine (PAMAM) dendrimer ions with amide functionalities

“…(J Am Soc Mass Spectrom 2006, 17, 536 -543) © 2006 American Society for Mass Spectrometry E lectron capture dissociation (ECD) offers a very powerful tandem mass spectrometry analysis of peptide/protein ions by exhibiting extensive backbone fragmentations [1][2][3][4][5][6]. In particular, ECD has made possible the detailed top-down characterizations of intact proteins [7,8], especially useful for the analysis of posttranslational modifications (PTMs) [8,9]. Such a powerful potential of ECD applications has led to numerous experimental and theoretical investigations [1-5, 10 -17].…”

Evaluation of Direct Infusion-Multiple Reaction Monitoring Mass Spectrometry for Quantification of Heat Shock Proteins

“…23 In addition, “top-down” proteomics approaches often use direct infusion nanospray ion sources. 24–26 Applications have also been demonstrated in “bottom-up” or peptide-based proteomics. 27,28 The use of infusion nanospray will complement matrix assisted laser desorption ionization (MALDI) MRM assay development for quantification of small molecules 29–31 and proteins.…”