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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.

Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers Rune Salbo, Matthew F. Bush, Helle Naver, Iain Campuzano, Carol V. Robinson, Ingrid Pettersson, Thomas J. D. Jørgensen, Kim F. Haselmann. Rapid Commun. Mass Spectrom. 2012, 26, 1181–1193.

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.

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 Christopher L. Moss, Julia Chamot-Rooke, Edith Nicol, Jeffery Brown, Iain Campuzano, Keith Richardson, Jonathan P. Williams, Matthew F. Bush, Benjamin Bythell, Bela Paizs, Frantisek Turecek. 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.

Charge-State Dependent Compaction and Dissociation of Protein Complexes – Insights from Ion Mobility and Molecular Dynamics Zoe Hall, Argyris Politis, Matthew F. Bush, Lorna J. Smith, and Carol V. Robinson. J. Am. Chem. Soc. 2012, 134, 3429–3438.