The Bush Lab is a new research group in the Department of Chemistry and the Biological Physics, Structure & Design Program at the University of Washington. Our research focuses on the development and application of mass spectrometry and ion mobility spectrometry techniques to elucidate the structures and assembly of protein complexes and subcellular machines.
The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry and calibration with compounds of known Ω. Obtaining accurate T-wave Ω-values via calibration, especially for native-like protein ions, remains challenging. Using human insulin oligomer ions we show how to select appropriate calibration standards, find the optimal instrument settings, and validate the resulting T-wave Ω-values. We also probe subtle conformational differences between human insulin and insulin aspart, a fast-acting insulin analog.
These results are also useful for calibrating other ion mobility experiments, particularly for ions of smaller, native-like proteins and protein complexes. Those values have been added to our collision cross section database.
Salbo, R.; Bush, M. F.; Naver, H.; Campuzano, I.; Robinson, C. V.; Pettersson, I.; Jørgensen, T. J. D.; Haselmann, K. M. Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers Rapid Commun. Mass Spectrom. 2012, 26, 1181–1193.
The Bush Lab and collaborators are presenting the following posters at the American Society for Mass Spectrometry annual meeting in Vancouver. Abstracts may be found by searching at for “Bush” using the conference planner.
Matt Bush and Brandon Ruotolo will also co-chair the Ion Mobility MS Workshop on Applying Ion Mobility-Mass Spectrometry to Challenges in Proteomics and Systems Biology (Wednesday, 5:45-7:00, Room 220-222).
We look forward to seeing everyone in Vancouver!
Prof. Bush has been invited to speak at the University of Washington Proteomics Symposium on May 18th in the Brotman Auditorium on the UW South Lake Union campus.
Infrared multiphoton dissociation spectroscopy and ion mobility measurements were used to investigate the structure of gas-phase peptide (AAHAL + 2H)2+ ions produced by electrospray ionization. The experimental data were consistent with properties calculated for the lowest-energy peptide ion conformer obtained by extensive molecular dynamics searches and electronic structure calculations. Traveling-wave ion mobility measurements were employed to obtain the collision cross sections (Ω) of the charge-reduced peptide cation-radicals, (AAHAL + 2H)+●, and c and z fragment ions from electron-transfer dissociation (ETD) of (AAHAL + 2H)2+. The experimental Ω for the ETD charge-reduced and fragment ions were consistent with values calculated for ions that retained specific hydrogen bonding motifs from the precursor ion. These results show that the combination of multilevel theoretical calculations and ion mobility experiments is a powerful tool for assigning the structures of precursor ions and electron transfer intermediates and fragments.
Moss, C. L.; Chamot-Rook, J.; Nicole, E.; Brown, J.; Campuzano, I.; Richardson, K.; Williams, J. P.; Bush, M. F.; Bythell, B.; Paiza, B.; Turecek, F. Assigning Structures to Gas-Phase Peptide Cations and Cation-Radicals. An Infrared Multiphoton Dissociation, Ion Mobility, Electron Transfer, and Computational Study of a Histidine Peptide Ion J. Phys. Chem B 2012, 116, 3445–3456.
Collapse to compact gas-phase structures, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes. Here, we combined experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during activation in the gas phase. Using ion mobility mass spectrometry, we find that all four protein complexes retain their native-like topologies at low collision energies, but that two of the four complexes adopt more compact structures at intermediate collision energies. The extent of collapse was found to depend on charge state, with the surprising observation that the lowest charge states experience the greatest degree of compaction. We compared these experimental results with in vacuo molecular dynamics (MD) simulations, during which the temperature was monotonically increased. During these simulations, low charge state ions of serum amyloid P collapsed prior to dissociation, whereas intermediate and high charge state ions maintained their ring-like topology prior to dissociation. This strong correlation between theory and experiment has implications for understanding the gas-phase dissociation of protein complexes and associated applications to gas-phase structural biology.
Hall, Z.; Politis, A.; Bush, M. F.; Smith, L. J.; Robinson, C. V. Charge-State Dependent Compaction and Dissociation of Protein Complexes – Insights from Ion Mobility and Molecular Dynamics J. Am. Chem. Soc. 2012, 134, 3429–3438.
Collision cross sections for ions of many small molecules, peptides, proteins, and protein complexes, in both helium and nitrogen gases, are now summarized in our database. These results are particularly useful for calibrating results from traveling-wave ion mobility experiments.
Collision cross sections (CCS) for a set of drug-like molecules were measured using RF-confining drift tube ion mobility experiments in both helium and nitrogen gases. These results enabled accurate calibrated CCS using traveling-wave ion mobility experiments and improved accuracy CCS calculations for ions in nitrogen gas. These experiments demonstrate that ion mobility is sensitive to very subtle differences in molecular structure, including differentiation of the diastereomers betamethasone (left) and dexamethasone (right).
Campuzano, I. D. G.; Bush, M. F.; Robinson, C. V.; Beaumont, C.; Richardson, K.; Kim, H.; Kim, H. I. Structural Characterization of Drug-like Compounds by Ion Mobility Mass Spectrometry: Comparison of Theoretical and Experimentally Derived Nitrogen Collision Cross-sections Anal. Chem. 2012, 84, 1026-1033.
One challenge in interpreting results from mass spectrometry experiments is that the structures of protein complexes in the gas phase may differ from those in solution. Here, we investigate the stabilization properties of trisH+, a non-volatile electrospray buffer component, by experimentally characterizing the unfolding and dissociation of three gas-phase tetrameric protein complexes. We find that trisH+ preferentially stabilizes the compact native-like state of these protein complexes.
Freeke, J. F.; Bush, M. F.; Robinson, C. V.; Ruotolo, B. T. Gas-Phase Protein Assemblies: Unfolding Landscapes and Preserving Native-Like Structures Using Noncovalent Adducts Chem. Phys. Lett. 2012, 524, 1-9.
Sam Marionni will present a poster at the PacMass Members’ Night, which will be held at 5:30 PM on November 8th at the Fred Hutchinson Cancer Research Center.
