Abstract
Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of α-lactalbumin homodimers with 15+ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8+/7+. In contrast, dissociation of the 15+ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11+ charges, and the oxidized molecule carries away 4+ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17+ charge state undergoes symmetric charge partitioning whereas dissociation of the 13+ is asymmetric. Reduction of the charge state of dimers with 17+ charges to 13+ via gas-phase proton transfer and subsequent dissociation of the mass selected 13+ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13+ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Kaleta, D. T.; Jarrold, M. F. Peptide Pinwheels. J. Am. Chem. Soc. 2002, 124, 1154–1155.
Kaleta, D. T.; Jarrold, M. F. Noncovalent Interactions Between Unsolvated Peptides. J. Phys. Chem. A. 2002, 106, 9655–9664.
Clemmer, D. E.; Jarrold, M. F. Ion Mobility Measurements and Their Applications to Clusters and Biomolecules. J. Mass Spectrom. 1997, 32, 577–592.
Counterman, A. E.; Valentine, S. J.; Srebalus, C. A.; Henderson, S. C.; Hoaglund, C. S.; Clemmer, D. E. High-Order Structure and Dissociation of Gaseous Peptide Aggregates That are Hidden in Mass Spectra. J. Am. Soc. Mass Spectrom. 1998, 9, 743–759.
Counterman, A. E.; Hilderbrand, A. E.; Barnes, C. A. S.; Clemmer, D. E. Formation of Peptide Aggregates During ESI: Size, Charge, Composition, and Contributions to Noise. J. Am. Soc. Mass Spectrom. 2001, 12, 1020–1035.
Gidden, J.; Wyttenbach, T.; Batka, J. J.; Weis, P.; Jackson, A. T.; Scrivens, J. H.; Bowers, M. T. Folding Energetics and Dynamics of Macromolecules in the Gas Phase: Alkali Ion-Cationized Poly(Ethylene Terephthalate) Oligomers. J. Am. Chem. Soc. 1999, 121, 1421–1422.
Guevremont, R.; Purves, R. W. High Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry: An Investigation of Leucine Enkephalin Ions Produced by Electrospray Ionization. J. Am. Soc. Mass Spectrom. 1999, 10, 492–501.
Purves, R. W.; Barnett, D. A.; Guevremont, R. Separation of Protein Conformers Using Electrospray-High Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry. Int. J. Mass Spectrom. 2000, 197, 163–177.
Deng, Y. Z.; Zhang, Z. Q.; Smith, D. L. Comparison of Continuous and Pulsed Labeling Amide Hydrogen Exchange/Mass Spectrometry for Studies of Protein Dynamics. J. Am. Soc. Mass Spectrom. 1999, 10, 675–684.
Zhu, M. M.; Rempel, D. L.; Gross, M. L. Modeling Data from Titration, Amide H/D Exchange, and Mass Spectrometry to Obtain Protein-Ligand Binding Constants. J. Am. Soc. Mass Spectrom. 2004, 15, 388–397.
Gard, E.; Willard, D.; Bregar, J.; Green, M. K.; Lebrilla, C. B. Site-Specificity in the H-D Exchange-Reactions of Gas-Phase Protonated Amino-Acids with CH3OD. Org. Mass Spectrom. 1993, 28, 1632–1639.
Campbell, S.; Rodgers, M. T.; Marzluff, E. M.; Beauchamp, J. L. Structural and Energetic Constraints on Gas-Phase Hydrogen-Deuterium Exchange Reactions of Protonated Peptides with D2O, CD3OD, CD3CO2D, and ND3. J. Am. Chem. Soc. 1994, 116, 9765–9766.
Gard, E.; Green, M. K.; Bregar, J.; Lebrilla, C. B. Gas-Phase Hydrogen-Deuterium Exchange as a Molecular Probe for the Interaction of Methanol and Protonated Peptides. J. Am. Soc. Mass Spectrom. 1994, 5, 623–631.
Campbell, S.; Rodgers, M. T.; Marzluff, E. M.; Beauchamp, J. L. Deuterium Exchange Reactions as a Probe of Biomolecule Structure. Fundamental Studies of Gas Phase H/D Exchange Reactions of Protonated Glycine Oligomers with D2O, CD3OD, CD3CO2D, and ND3. J. Am. Chem. Soc. 1995, 117, 12840–12854.
Green, M. K.; Penn, S. G.; Lebrilla, C. B. The Complexation of Protonated Peptides with Saccharides in the Gas Phase Decreases the Rates of Hydrogen/Deuterium Exchange Reactions. J. Am. Soc. Mass Spectrom. 1995, 6, 1247–1251.
Kaltashov, I. A.; Doroshenko, V. M.; Cotter, R. J. Gas Phase Hydrogen/Deuterium Exchange Reactions of Peptide Ions in a Quadrupole Ion Trap Mass Spectrometer. Proteins. 1997, 28, 53–58.
Heck, A. J. R.; Jorgensen, T. J. D.; O’Sullivan, M.; von Raumer, M.; Derrick, P. J. Gas-Phase Noncovalent Interactions Between Vancomycin-Group Antibiotics and Bacterial Cell-Wall Precursor Peptides Probed by Hydrogen/Deuterium Exchange. J. Am. Soc. Mass Spectrom. 1998, 9, 1255–1266.
Freitas, M. A.; Marshall, A. G. Rate and Extent of Gas-Phase Hydrogen/Deuterium Exchange of Bradykinins: Evidence for Peptide Zwitterions in the Gas Phase. Int. J. Mass Spectrom. 1999, 183, 221–231.
Reyzer, M. L.; Brodbelt, J. S. Gas-Phase H/D Exchange Reactions of Polyamine Complexes: [M + H)(+), (M plus alkali metal(+)], and (M + 2H)(2+). J. Am. Soc. Mass Spectrom. 2000, 11, 711–721.
Levy-Seri, E.; Koster, G.; Kogan, A.; Gutman, K.; Reuben, B. G.; Lifshitz, C. An Electrospray Ionization-Flow Tube Study of H/D Exchange in Protonated Bradykinin. J. Phys. Chem. A. 2001, 105, 5552–5559.
Solouki, T.; Fort, R. C.; Alomary, A.; Fattahi, A. Gas Phase Hydrogen-Deuterium Exchange Reactions of a Model Peptide: FT-ICR and Computational Analyses of Metal Induced Conformational Mutations. J. Am. Soc. Mass Spectrom. 2001, 12, 1272–1285.
Suckau, D.; Shi, Y.; Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M.; McLafferty, F. W. Coexisting Stable Conformations of Gaseous Protein Ions. Proc. Nat. Acad. Sci. U.S.A. 1993, 90, 790–793.
Wood, T. D.; Chorush, R. A.; Wampler, F. M.; Little, D. P.; O’Connor, P. B.; McLafferty, F. W. Gas-Phase Folding and Unfolding of Cytochrome-c Cations. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2451–2454.
McLafferty, F. W.; Guan, Z. Q.; Haupts, U.; Wood, T. D.; Kelleher, N. L. Gaseous Conformational Structures of Cytochrome c. J. Am. Chem. Soc. 1998, 120, 4732–4740.
Reid, G. E.; O’Hair, R. A. J.; Styles, M. L.; McFadyen, W. D.; Simpson, R. J. Gas Phase Ion-Molecule Reactions in a Modified Ion Trap: H/D Exchange of Noncovalent Complexes and Coordinatively Unsaturated Platinum Complexes. Rapid Commun. Mass Spectrom. 1998, 12, 1701–1708.
Kogan, A.; Ustyuzhanin, P.; Reuben, B. G.; Lifshitz, C. Hydrogen/Deuterium Exchange of Monomers and Dimers of Leucine Enkephalin. Int. J. Mass Spectrom. 2002, 213, 1–4.
Mao, D.; Douglas, D. J. H/D Exchange of Gas Phase Bradykinin Ions in a Linear Quadrupole Ion Trap. J. Am. Soc. Mass Spectrom. 2003, 14, 85–94.
Lee, S. W.; Lee, H. N.; Kim, H. S.; Beauchamp, J. L. Selective Binding of Crown Ethers to Protonated Peptides Can be Used to Probe Mechanisms of H/D Exchange and Collision-Induced Dissociation Reactions in the Gas Phase. J. Am. Chem. Soc. 1998, 120, 5800–5805.
Gross, D. S.; Schnier, P. D.; Rodriguezcruz, S. E.; Fagerquist, C. K.; Williams, E. R. Conformations and Folding of Lysozyme Ions in Vacuo. Proc. Nat. Acad. Sci. U.S.A. 1996, 93, 3143–3148.
Williams, E. R. Proton Transfer Reactivity of Large Multiply Charged Ions. J. Mass Spectrom. 1996, 31, 831–842.
Loo, J. A. Studying Noncovalent Protein Complexes by Electrospray Ionization Mass Spectrometry. Mass Spectrom. Rev. 1997, 16, 1–23.
Lim, H. K.; Hsieh, Y. L.; Ganem, B.; Henion, J. Recognition of Cell-Wall Peptide Ligands by Vancomycin Group Antibiotics—Studies Using Ion-Spray Mass-Spectrometry. J. Mass Spectrom. 1995, 30, 708–714.
Loo, J. A.; Hu, P. F.; McConnell, P.; Mueller, W. T.; Sawyer, T. K.; Thanabal, V. A Study of Src SH2 Domain Protein-Phosphopeptide Binding Interactions by Electrospray Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 234–243.
Jorgensen, T. J. D.; Roepstorff, P.; Heck, A. J. R. Direct Determination of Solution Binding Constants for Noncovalent Complexes Between Bacterial Cell Wall Peptide Analogues and Vancomycin Group Antibiotics by Electrospray Ionization Mass Spectrometry. Anal. Chem. 1998, 70, 4427–4432.
Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Quantitative Determination of Noncovalent Binding Interactions Using Soft Ionization Mass Spectrometry. Int. J. Mass Spectrom. 2002, 216, 1–27.
Gross, D. S.; Zhao, Y. X.; Williams, E. R. Dissociation of Heme-Globin Complexes by Blackbody Infrared Radiative Dissociation: Molecular Specificity in the Gas Phase?. J. Am. Soc. Mass Spectrom. 1997, 8, 519–524.
Schnier, P. D.; Klassen, J. S.; Strittmatter, E. E.; Williams, E. R. Activation Energies for Dissociation of Double Strand Oligonucleotide Anions: Evidence for Watson-Crick Base Pairing in Vacuo. J. Am. Chem. Soc. 1998, 120, 9605–9613.
Rogalewicz, F.; Hoppilliard, Y.; Ohanessian, G. Structures and Fragmentations of Zinc(II) Complexes of Amino Acids in the Gas Phase. IV. Solvent Effect on the Structure of Electrosprayed Ions. Int. J. Mass Spectrom. 2003, 227, 439–451.
Gabelica, V.; De Pauw, E. Comparison Between Solution-Phase Stability and Gas-Phase Kinetic Stability of Oligodeoxynucleotide Duplexes. J. Mass Spectrom. 2001, 36, 397–402.
Gabelica, V.; De Pauw, E. Comparison of the Collision-Induced Dissociation of Duplex DNA at Different Collision Regimes: Evidence for a Multistep Dissociation Mechanism. J. Am. Soc. Mass Spectrom. 2002, 13, 91–98.
Gabelica, V.; De Pauw, E. Collision-Induced Dissociation of 16-mer DNA Duplexes with Various Sequences: Evidence for Conservation of the Double Helix Conformation in the Gas Phase. Int. J. Mass Spectrom. 2002, 219, 151–159.
Wan, K. X.; Shibue, T.; Gross, M. L. Noncovalent Complexes Between DNA-Binding Drugs and Double-Stranded Oligodeoxynucleotides: A Study by ESI Ion-Trap Mass Spectrometry. J. Am. Chem. Soc. 2000, 122, 300–307.
Chen, Y. L.; Campbell, J. M.; Collings, B. A.; Konermann, L.; Douglas, D. J. Stability of a Highly Charged Noncovalent Complex in the Gas Phase: Holomyoglobin. Rapid Commun. Mass Spectrom. 1998, 12, 1003–1010.
Hunter, C. L.; Mauk, A. G.; Douglas, D. J. Dissociation of Heme from Myoglobin and Cytochrome b(5): Comparison of Behavior in Solution and the Gas Phase. Biochem. U.S.A. 1997, 36, 1018–1025.
Jorgensen, T. J. D.; Delforge, D.; Remacle, J.; Bojesen, G.; Roepstorff, P. Collision-Induced Dissociation of Noncovalent Complexes Between Vancomycin Antibiotics and Peptide Ligand Stereoisomers: Evidence for Molecular Recognition in the Gas Phase. Int. J. Mass Spectrom. 1999, 188, 63–85.
Loo, J. A.; He, J. X.; Cody, W. L. Higher Order Structure in the Gas Phase Reflects Solution Structure. J. Am. Chem. Soc. 1998, 120, 4542–4543.
Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Detection and Selective Dissociation of Intact Ribosomes in a Mass Spectrometer. Proc. Nat. Acad. Sci. U.S.A. 2000, 97, 5185–5190.
Pinkse, M. W. H.; Maier, C. S.; Kim, J. I.; Oh, B. H.; Heck, A. J. R. Macromolecular Asserably of Helicobacter pylori Urease Investigated by Mass Spectrometry. J. Mass Spectrom. 2003, 38, 315–320.
Light-Wahl, K. J.; Schwartz, B. L.; Smith, R. D. Observation of the Noncovalent Quaternary Associations of Proteins by Electrospray Ionization Mass Spectrometry. J. Am. Chem. Soc. 1994, 116, 5271–5278.
Schwartz, B. L.; Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Rockwood, A. L.; Smith, R. D.; Chilkoti, A.; Stayton, P. S. Dissociation of Tetrameric Ions of Noncovalent Streptavidin Complexes Formed By Electrospray Ionization. J. Am. Soc. Mass Spectrom. 1995, 6, 459–465.
Fitzgerald, M. C.; Chernushevich, I.; Standing, K. G.; Whitman, C. P.; Kent, S. B. H. Probing the Oligomeric Structure of an Enzyme By Electrospray Ionization Time-of-Flight Mass Spectrometry. Proc. Nat. Acad. Sci. U.S.A. 1996, 93, 6851–6856.
Felitsyn, N.; Kitova, E. N.; Klassen, J. S. Thermal Decomposition of a Gaseous Multiprotein Complex Studied by Blackbody Infrared Radiative Dissociation. Investigating the Origin of the Asymmetric Dissociation Behavior. Anal. Chem. 2001, 73, 4647–4661.
Benesch, J. L. P.; Sobott, F.; Robinson, C. V. Thermal Dissociation of Multimeric Protein Complexes by Using Nanoelectrospray Mass Spectrometry. Anal. Chem. 2003, 75, 2208–2214.
http://www.rcsb.org/pdb/entry2IZA
Versluis, C.; van der Staaij, A.; Stokvis, E.; Heck, A. J. R.; de Craene, B. Metastable Ion Formation and Disparate Charge Separation in the Gas-Phase Dissection of Protein Assemblies Studied by Orthogonal Time-of-Flight Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 329–336.
Jurchen, J. C.; Williams, E. R. Origin of Asymmetric Charge Partitioning in the Dissociation of Gas-Phase Protein Homodimers. J. Am. Chem. Soc. 2003, 125, 2817–2826.
Jurchen, J. C.; Garcia, D. E.; Williams, E. R. Gas-Phase Dissociation Pathways of Multiply Charged Peptide Clusters. J. Am. Soc. Mass Spectrom. 2003, 14, 1373–1386.
Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Sustained Off-Resonance Irradiation for Collision-Activated Dissociation Involving Fourier-Transform Mass-Spectrometry-Collision-Activated Dissociation Technique That Emulates Infrared Multiphoton Dissociation. Anal. Chim. Acta. 1991, 246, 211–225.
Winger, B. E.; Light-Wahl, K. J.; Rockwood, A. L.; Smith, R. D. Probing Qualitative Conformation Differences of Multiply Protonated Gas-Phase Proteins via H/D Isotopic Exchange With D2O. J. Am. Chem. Soc. 1992, 114, 5897–5898.
Felitsyn, N.; Kitova, E. N.; Klassen, J. S. Thermal Dissociation of the Protein Homodimer Ecotin in the Gas Phase. J. Am. Soc. Mass Spectrom. 2002, 13, 1432–1442.
Apostol, I. Assessing the Relative Stabilities of Engineered Hemoglobins Using Electrospray Mass Spectrometry. Anal. Biochem. 1999, 272, 8–18.
Versluis, C.; Heck, A. J. R. Gas-Phase Dissociation of Hemoglobin. Int. J. Mass Spectrom. 2001, 210, 637–649.
Price, W. D.; Williams, E. R. Activation of Peptide Ions by Blackbody Radiation: Factors That Lead to Dissociation Kinetics in the Rapid Energy Exchange Limit. J. Phys. Chem. A. 1997, 101, 8844–8852.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online September 3, 2004
Rights and permissions
About this article
Cite this article
Jurchen, J.C., Garcia, D.E. & Williams, E.R. Further studies on the origins of asymmetric charge partitioning in protein homodimers. J Am Soc Mass Spectrom 15, 1408–1415 (2004). https://doi.org/10.1016/j.jasms.2004.06.006
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2004.06.006