Most proteins, particularly those that accomplish complicated tasks, form assemblies with other proteins and molecules that are critical to their function. Established structural biology tools are most effective for highly purified samples that have limited conformational variability, which makes it challenging to apply those methods to capture a systems-wide understanding of the structures, interactions, and dynamics that are present under different cellular conditions. The Bush Lab develops and applies mass spectrometry based techniques that are fast, sensitive, and tolerant of heterogeneity for characterizing the native structures of biological assemblies.

Modelling biomolecular assemblies using constraints from mass spectrometry (MS) and ion mobility (IM) experiments. The masses and identities of individual proteins (subunits) are determined using proteomics (a). The stoichiometry of the intact assembly is determined using MS from a native-like buffer (b). Subassemblies are generated by disrupting the assembly in solution and characterized by MS (c). Collision cross sections (Ω) of the intact assembly and subassemblies provide conformational information (d). The masses of subunits and subassemblies from MS are used to generate 2D interaction maps (e). 3D models are constructed using these maps, Ω values, and any complementary structural information (f). Atomic structures and models can then be docked into these models (g).

Native Mass Spectrometry. Gas-phase ions of assemblies can retain significant memories of their native structures in solution. Many measurements of stoichiometry, connectivity, and shape have shown that these aspects of assembly structure can be strongly correlated in both environments. Gas-phase techniques, including mass spectrometry, ion mobility, and ion chemistry are used to probe the native structures of biological assemblies. Accurate models of protein assemblies, including those that are heterogeneous, dynamic, and/or membrane-bound, are built based on results from both gas-phase experiments and complementary techniques.

  • Lab Contacts: Seoyeon (Cece) HongMeagan Gadzuk-Shea
  • Hexamers of the Type II Secretion ATPase GspE from Vibrio cholerae with Increased ATPase Activity Connie Lu, Stewart Turley, Samuel T. Marionni, Young-Jun Park, Kelly K. Lee, Marcella Patrick, Ripal Shah, Maria Sandkvist, Matthew F. Bush, Wim G.J. Hol. Structure 2013, 21, 1707–1717. (Link|PUBMED)
  • Ion Mobility Mass Spectrometry of a Circadian Clock Protein Complex Reveals a Ligand-Dependent Conformational Switch Samuel T. Marionni, Weiman Xing, Ning Zheng, Matthew F. Bush. 60th American Society for Mass Spectrometry Conference 2012 (Poster). SCFFBXL3 ubiquitin ligase targets cryptochromes at their cofactor pocket Weiman Xing, Luca Busino, Thomas R. Hinds, Samuel T. Marionni, Nabiha H. Saifee, Matthew F. Bush, Michele Pagano, Ning Zheng. Nature 2013496, 64–68. (Link|PUBMED)

CAPTR_toolboxExpanding the toolbox for Native MS: Cation to Anion Proton Transfer Reactions (CAPTR). Charge state assignments in native mass spectrometry are often ambiguous difficult due to narrow charge state distributions and wide mass spectral peaks. This can result in significant error (>3%) in mass assignments of native-like ions. In order to overcome this challenge, we have implemented Cation to Anion Proton Transfer Reactions (CAPTR), which generates a wide range of charge-reduced product ions thus making charge state assignments unambiguous. CAPTR differs from electron transfer disassociation in that a proton is transferred rather than an electron, and CAPTR does not result in peptide fragmentation. Additionally, we can use CAPTR to separate ions that are unresolved by their mass-to-charge ratio, but differ in their charge state.

  • Lab Contacts: Meagan Gadzuk-Shea, Ken Laszlo
  • Analysis of Native-Like Proteins and Protein Complexes Using Cation to Anion Proton Transfer Reactions (CAPTR). Kenneth J. Laszlo; Matthew F. Bush. J. Am. Soc. Mass Spectrom. 2015, 26, 2152-2161. (Link|Link to PUBMED)

RF Confining Drift Cell

A new RF confining drift cell developed for use on our Waters Synapt G2 HDMS. We use this cell to measure absolute collision cross sections for ions of intact protein complexes.

Ion Mobility (IM) Mass Spectrometry (MS) Development. IM is an emerging technology for characterizing the structures of biomolecules that can be used in tandem with MS. IM separates ions based primarily on their charge state and collision cross section, which depends on ion shape and other factors. We are developing new IM cells to enable the measurement of high accuracy collision cross sections and new methods for interpreting those results, in the contexts of both native MS and systems biology.

  • Lab Contact: Sam Allen & Rae Eaton
  • Analysis of Native-Like Ions using Structures for Lossless Ion Manipulations. Samuel J. Allen, Rachel M. Eaton, Matthew F. Bush. Anal. Chem. 2016, 88, 9118–9126. (Link)
  • Radio-Frequency (RF) Confinement in Ion Mobility Spectrometry: Apparent Mobilities and Effective Temperatures. Samuel J. Allen, Matthew F. Bush. J. Am. Soc. Mass Spectrom. 2016, DOI: 10.1007/s13361-016-1479-9. (Link)
  • Ion mobility mass spectrometry of peptide, protein, and protein complex ions using a radio-frequency confining drift cell. Samuel J. Allen, Kevin Giles, Tony Gilbert, Matthew F. Bush. Analyst 2016141, 884-891. (Link)


Effects of Charge on the Structure of Protein Ions.  Despite the effort and progress in using gas-phase measurements to probe the structures of biological molecules in solution, a robust understanding of the relationship between the charge state of the gas-phase ion, the structure of the gas-phase ion, and the original structure in solution remains elusive. An improved understanding of the phenomena underlying these observations is important for maximizing the structural information that can be drawn from IM experiments. Towards that end, we apply a combination of mass spectrometry, ion chemistry, ion mobility, and theory.

  • Lab Contact: Ken Laszlo
  • Folding of Protein Ions in the Gas Phase after Cation-to-Anion Proton-Transfer Reactions (CAPTR). Kenneth J. Laszlo, Eleanor B. Munger, and Matthew F. Bush. J. Am. Chem. Soc. 2016, 138, 9581–9588. (Link|PUBMED)
  • Effects of Polarity on the Structures and Charge States of Native-like Proteins and Protein Complexes in the Gas Phase Samuel J. Allen, Alicia M. Schwartz, Matthew F. Bush. Anal. Chem. 2013, 85, 12055–12061. (Link|PUBMED)


Electrospray Fundamentals. In native mass spectrometry, intact protein complexes are transferred from solution into the gas phase using nanoelectrospray ionization. We use a range of mass spectrometry and ion mobility experiments, coupled with computational chemistry and Monte Carlo simulations, to gain fundamental insights into our implementations of nanoelectrospray ionization. These projects probe many stages of this process, from the initial nanodroplets to the final multiply charged ions. The outcomes of these projects are guiding our long term objectives of increasing the sensitivity of native mass spectrometry experiments and the information content of the resulting native mass spectra.

  • Lab Contacts: Sam Allen & Kim Davidson
  • Nonspecific Aggregation in Native Electrokinetic Nanoelectrospray Ionization. Kimberly L. Davidson; Derek R. Oberreit; Christopher J. Hogan; Matthew F. Bush. Int. J. Mass Spectrom. 2016, in press.
  • Effects of Polarity on the Structures and Charge States of Native-like Proteins and Protein Complexes in the Gas Phase Samuel J. Allen, Alicia M. Schwartz, Matthew F. Bush. Anal. Chem. 2013, 85, 12055–12061. (Link)

Ion Mobility Arrival Time Distribution of (AAHAL+2H)^2+

The ion mobility arrival time distribution of doubly protonated AAHAL is sensitive to subtle details of ion structure. (Full Article)

Structures of Peptide Ions & their Dissociation Products. In collaboration with the Turecek group, we are investigating the structures of peptide ions and their gas-phase dissociation products. Ions are activated using either collision-induced dissociation or electron-transfer dissociation prior to characterization using ion mobility. These studies provide fundamental insights into the structures and reactivities of peptide ions, which can increase the information content of  proteomics experiments.

  • Lab Contact: Ken Laszlo
  • Does Thermal Breathing Affect Collision Cross Sections of Gas Phase Peptide Ions? An Ab Initio Molecular Dynamics Study. Robert Pepin, Alessio Petrone, Kenneth J. Laszlo, Matthew F. Bush, Xiaosong Li, and František Tureček. J. Phys. Chem. Lett. 2016, 7, 2765–2771. (Link)
  • Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization. Robert Pepin, Kenneth J. Laszlo, Aleš Marek, Bo Peng, Matthew F. Bush, Helène Lavanant, Carlos Afonso, František Tureček. J. Am. Soc. Mass Spectrom. 2016, DOI: 10.1007/s13361-016-1437-6. (Link)
  • Comprehensive Analysis of Gly-Leu-Gly-Gly-Lys Peptide Dication Structures and Cation-Radical Dissociations Following Electron Transfer: From Electron Attachment to Backbone Cleavage, Ion-Molecule Complexes, and Fragment Separation Robert Pepin, Kenneth J. Laszlo, Bo Peng, Aleš Marek, Matthew F. Bush, František Tureček. J. Phys. Chem. A 2014118, 308–324. (Link|PUBMED)

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