Abstract
Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Bernstein, S. L.; Wyttenbach, T.; Baumketner, A.; Shea, J. E.; Bitan, G.; Teplow, D. B.; Bowers, M. T. Amyloid β-Protein: Monomer Structure and Early Aggregation States of Aβ42 and Its Pro19 Alloform. J. Am. Chem. Soc. 2005, 127, 2075–2084.
Koeniger, S. L.; Clemmer, D. E. Resolution and Structural Transitions of Elongated States of Ubiquitin. J. Am. Soc. Mass Spectrom. 2007, 18, 322–331.
Loo, J. A.; Berhane, B.; Kaddis, C. S.; Wooding, K. M.; Xie, Y.; Kaufman, S. L.; Chernushevich, I. V. Electrospray Ionization Mass Spectrometry and Ion Mobility Analysis of the 20S Proteasome Complex. J. Am. Soc. Mass Spectrom. 2005, 16, 998–1008.
Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Evidence for Macromolecular Protein Rings in the Absence of Bulk Water. Science 2005, 310, 1658–1661.
Sharon, M.; Witt, S.; Felderer, K.; Rockel, B.; Baumeister, W.; Robinson, C. V. 20S Proteasomes Have the Potential to Keep Substrates in Store for Continual Degradation. J. Biol. Chem. 2006, 281, 9569–9575.
Loo, J. A. Studying Noncovalent Protein Complexes by Electrospray Ionization Mass Spectrometry. Mass Spectrom. Rev. 1997, 16, 1–23.
Kaufman, S. L.; Kuchumov, A. R.; Kazakevich, M.; Vinogradov, S. N. Analysis of a 3.6-MDa Hexagonal Bilayer Hemoglobin from Lumbricus terrestris Using a Gas-Phase Electrophoretic Mobility Molecular Analyzer. Anal. Biochem. 1998, 259, 195–202.
Bacher, G.; Szymanski, W. W.; Kaufman, S. L.; Zollner, P.; Blaas, D.; Allmaier, G. Charge-Reduced Nano-Electrospray Ionization Combined with Differential Mobility Analysis of Peptides, Proteins, Glycoproteins, Noncovalent Protein Complexes, and Viruses. J. Mass Spectrom. 2001, 36, 1038–1052.
de la Mora, J. F.; Ude, S.; Thomson, B. A. The Potential of Differential Mobility Analysis Coupled to MS for the Study of Very Large Singly and Multiply Charged Proteins and Protein Complexes in the Gas Phase. Biotechnol. J. 2006, 1, 988–997.
Kaufman, S. L.; Skogen, J. W.; Dorman, F. D.; Zarrin, F.; Lewis, K. C. Macromolecule Analysis Based on Electrophoretic Mobility in Air: Globular Proteins. Anal. Chem. 1996, 68, 1895–1904.
Kaufman, S. L. Analysis of Biomolecules Using Electrospray and Nanoparticle Methods: The Gas-Phase Electrophoretic Mobility Molecular Analyzer (GEMMA). J. Aerosol. Sci. 1998, 29, 537–552.
Scalf, M.; Westphall, M. S.; Krause, J.; Kaufman, S. L.; Smith, L. M. Controlling Charge States of Large Ions. Science 1999, 283, 194–197.
Thomas, J. J.; Bothner, B.; Traina, J.; Benner, W. H.; Siuzdak, G. Electrospray Ion Mobility Spectrometry of Intact Viruses. Spectroscopy 2004, 18, 31–36.
Myung, S.; Badman, E. R.; Lee, Y. J.; Clemmer, D. E. Structural Transitions of Electrosprayed Ubiquitin Ions Stored in an Ion Trap Over ∼10 ms to 30 s. J. Phys. Chem. A. 2002, 106, 9976–9982.
Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. Protein Structure in Vacuo: Gas-Phase Conformations of BPTI and Cytochrome c. J. Am. Chem. Soc. 1997, 119, 2240–2248.
Kedersha, N. L.; Heuser, J. E.; Chugani, D. C.; Rome, L. H. Vaults. III. Vault Ribonucleoprotein Particles Open into Flower-Like Structures with Octagonal Symmetry. J. Cell Biol. 1991, 112, 225–235.
Kedersha, N. L.; Miquel, M. C.; Bittner, D.; Rome, L. H. Vaults. II. Ribonucleoprotein Structures Are Highly Conserved among Higher and Lower Eukaryotes. J. Cell Biol. 1990, 110, 895–901.
van Zon, A.; Mossink, M. H.; Scheper, R. J.; Sonneveld, P.; Wiemer, E. A. The Vault Complex. Cell. Mol. Life Sci. 2003, 60, 1828–1837.
Kickhoefer, V. A.; Rajavel, K. S.; Scheffer, G. L.; Dalton, W. S.; Scheper, R. J.; Rome, L. H. Vaults are Up-Regulated in Multidrug-Resistant Cancer Cell Lines. J. Biol. Chem. 1998, 273, 8971–8974.
Poderycki, M. J.; Kickhoefer, V. A.; Kaddis, C. S.; Raval-Fernandes, S.; Johansson, E.; Zink, J. I.; Loo, J. A.; Rome, L. H. The Vault Exterior Shell is a Dynamic Structure That Allows Incorporation of Vault-Associated Proteins Into Its Interior. Biochemistry 2006, 45, 12184–12193.
Knutson, E. O.; Whitby, K. T. Aerosol Classification by Electric Mobility: Apparatus, Theory, and Applications. J. Aerosol Sci. 1975, 6, 443–451.
Knutson, E. O.; Whitby, K. T. Accurate Measurement of Aerosol Electric Mobility Moments. J. Aerosol Sci. 1975, 6, 453–460.
Hinds, W. C. Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles John Wiley and Sons, Inc: New York, N.Y, 1999 pp 42–72.
Tammet, H. Size and Mobility of Nanometer Particles, Clusters, and Ions. J. Aerosol Sci. 1995, 26, 459–475.
Wang, S. C.; Flagan, R. C. Scanning Electrical Mobility Spectrometer. Aerosol Sci. Technol. 1990, 13, 230–240.
Fuchs, N. A. On the Stationary Charge Distribution on Aerosol Particles in a Bipolar Ionic Atmosphere. Geofis. Pura. Appl. 1963, 56, 185–193.
Wiedensohler, A. An Approximation of the Bipolar Charge Distribution for Particles in the Submicron Size Range. J. Aerosol Sci. 1988, 19, 387–389.
Ude, S.; de la Mora, J. F.; Thomson, B. A. Charge-Induced Unfolding of Multiply Charged Polyethylene Glycol Ions. J. Am. Chem. Soc. 2004, 126, 12184–12190.
Hogan, C. J.; Kettleson, E. M.; Ramaswami, B.; Chen, D. R.; Biswas, P. Charge Reduced Electrospray Size Spectrometry of Mega- and Giga-Dalton Complexes: Whole Viruses and Virus Fragments. Anal. Chem. 2006, 78, 844–852.
Lebioda, L.; Stec, B.; Brewer, J. M. The Structure of Yeast Enolase at 2.2 -Å Resolution: An 8-fold β + α-Barrel with a Novel β β α α (β α)6 Topology. J. Biol. Chem. 1989, 264, 3685–3693.
Stec, B.; Lebioda, L. Refined Structure of Yeast Apo-Enolase at 2.25 Å Resolution. J. Mol. Biol. 1990, 211, 235–248.
Kornblatt, M. J.; Lange, R.; Balny, C. Use of Hydrostatic Pressure to Produce “Native” Monomers of Yeast Enolase. Eur. J. Biochem. 2004, 271, 3897–3904.
Loo, J. A. Electrospray Ionization Mass Spectrometry: A Technology for Studying Noncovalent Macromolecular Complexes. Int. J. Mass Spectrom. 2000, 200, 175–186.
Xie, Y.; Zhang, J.; Yin, S.; Loo, J. A. Top-Down ESI-ECD-FT-ICR Mass Spectrometry Localizes Noncovalent Protein-Ligand Binding Sites. J. Am. Chem. Soc. 2006, 128, 14432–14433.
Granier, T.; Gallois, B.; Dautant, A.; Langlois d’Estaintot, B.; Precigoux, G. Comparison of the Structures of the Cubic and Tetragonal Forms of Horse-Spleen Apoferritin. Acta Cryst. 1997, D53, 580–587.
Johnson, E.; Cascio, D.; Sawaya, M. R.; Gingery, M.; Schroder, I. Crystal Structures of a Tetrahedral Open Pore Ferritin from the Hyperthermophilic Archaeon. Archaeoglobus fulgidus. Structure 2005, 13, 637–648.
Rhee, Y. M.; Sorin, E. J.; Jayachandran, G.; Lindahl, E.; Pande, V. S. Simulations of the Role of Water in the Protein-Folding Mechanism. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6456–6461.
Counterman, A. E.; Clemmer, D. E. Volumes of Individual Amino Acid Residues in Gas-Phase Peptide Ions. J. Am. Chem. Soc. 1999, 121, 4031–4039.
Koeniger, S. L.; Merenbloom, S. I.; Sevugarajan, S.; Clemmer, D. E. Transfer of Structural Elements from Compact to Extended States in Unsolvated Ubiquitin. J. Am. Chem. Soc. 2006, 128, 11713–11719.
Petsev, D. N.; Thomas, B. R.; Yau, S.; Vekilov, P. G. Interactions and Aggregation of Apoferritin Molecules in Solution: Effects of Added Electrolytes. Biophys. J. 2000, 78, 2060–2069.
Bancroft, J. B.; Hiebert, E.; Rees, M. W.; Markham, R. Properties of Cowpea Chlorotic Mottle Virus, Its Protein, and Nucleic Acid. Virology 1968, 34, 224–239.
Speir, J. A.; Munshi, S.; Wang, G.; Baker, T. S.; Johnson, J. E. Structures of the Native and Swollen Forms of Cowpea Chlorotic Mottle Virus Determined by X-Ray Crystallography and Cryo-Electron Microscopy. Structure 1995, 3, 63–78.
Dasgupta, R.; Kaesberg, P. Complete Nucleotide Sequences of the Coat Protein Messenger RNAs of Brome Mosaic Virus and Cowpea Chlorotic Mottle Virus. Nucleic Acids Res. 1982, 10, 703–713.
Durchschlag, H. Specific Volumes of Biological Macromolecules and Some Other Molecules of Biological Interest. In Thermodynamic Data for Biochemistry and Biotechnology Hinz H.-J., Ed.; Springer Verlag: Berlin, 1986; pp 45–128.
Voss, N. R.; Gerstein, M. Calculation of Standard Atomic Volumes for RNA and Comparison with Proteins: RNA is Packed More Tightly. J. Mol. Biol. 2005, 346, 477–492.
Stephen, A. G.; Raval-Fernandes, S.; Huynh, T.; Torres, M.; Kickhoefer, V. A.; Rome, L. H. Assembly of Vault-Like Particles in Insect Cells Expressing Only the Major Vault Protein. J. Biol. Chem. 2001, 276, 23217–23220.
Mikyas, Y.; Makabi, M.; Raval-Fernandes, S.; Harrington, L.; Kickhoefer, V. A.; Rome, L. H.; Stewart, P. L. Cryoelectron Microscopy Imaging of Recombinant and Tissue Derived Vaults: Localization of the MVP N-Termini and VPARP. J. Mol. Biol. 2004, 344, 91–105.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online April 16, 2007
This paper is submitted to honor Professor David E. Clemmer for his well-deserved receipt of the 2006 ASMS Biemann Medal.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Kaddis, C.S., Lomeli, S.H., Yin, S. et al. Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility. J Am Soc Mass Spectrom 18, 1206–1216 (2007). https://doi.org/10.1016/j.jasms.2007.02.015
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2007.02.015