Structural studies of metal ligand complexes by ion mobility-mass spectrometry

Collision cross sections (CCS) have been measured for three salen ligands, and their complexes with copper and zinc using travelling-wave ion mobility-mass spectrometry (TWIMS) and drift tube ion mobility-mass spectrometry (DTIMS), allowing a comparative size evaluation of the ligands and complexes. CCS measurements using TWIMS were determined using peptide and TAAH calibration standards. TWIMS measurements gave significantly larger CCS than DTIMS in helium, by 9 % for TAAH standards and 3 % for peptide standards, indicating that the choice of calibration standards is important in ensuring the accuracy of TWIMS-derived CCS measurements. Repeatability data for TWIMS was obtained for inter- and intra-day studies with mean RSDs of 1. 1 % and 0. 7 %, respectively. The CCS data obtained from IM-MS measurements are compared to CCS values obtained via the projection approximation, the exact hard spheres method and the trajectory method from X-ray coordinates and modelled structures using density functional theory (DFT) based methods. © 2013 Springer-Verlag Berlin Heidelberg

Effects of Drift Gas on Collision Cross Sections of a Protein Standard in Linear Drift Tube and Traveling Wave Ion Mobility Mass Spectrometry

There has been a significant increase in the use of ion mobility mass spectrometry (IM-MS) to investigate conformations of proteins and protein complexes following electrospray ionization. Investigations which employ traveling wave ion mobility mass spectrometry (TW IM-MS) instrumentation rely on the use of calibrants to convert the arrival times of ions to collision cross sections (CCS) providing “hard numbers” of use to structural biology. It is common to use nitrogen as the buffer gas in TW IM-MS instruments and to calibrate by extrapolating from CCS measured in helium via drift tube (DT) IM-MS. In this work, both DT and TW IM-MS instruments are used to investigate the effects of different drift gases (helium, neon, nitrogen, and argon) on the transport of multiply charged ions of the protein myoglobin, frequently used as a standard in TW IM-MS studies. Irrespective of the drift gas used, recorded mass spectra are found to be highly similar. In contrast, the recorded arrival time distributions and the derived CCS differ greatly. At low charge states (7 ≤ <i>z</i> ≤ 11) where the protein is compact, the CCS scale with the polarizability of the gas; this is also the case for higher charge states (12 ≤ <i>z</i> ≤ 22) where the protein is more unfolded for the heavy gases (neon, argon, and nitrogen) but not the case for helium. This is here interpreted as a different conformational landscape being sampled by the lighter gas and potentially attributable to increased field heating by helium. Under nanoelectrospray ionization (nESI) conditions, where myoglobin is sprayed from an aqueous solution buffered to pH 6.8 with 20 mM ammonium acetate, in the DT IM-MS instrument, each buffer gas can yield a different arrival time distribution (ATD) for any given charge state

Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome <i>c</i>, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry

Thermally induced conformational transitions of three proteins of increasing intrinsic disordercytochrome <i>c</i>, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome <i>c</i>, dimers of significant intensity are also observed. Cytochrome <i>c</i> [M + 5H]⁵⁺ ions present in one conformer of CCS ∼1200 Ų, undergoing compaction in line with the reported <i>T</i>_melt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H]⁷⁺ cytochrome <i>c</i> monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H]¹¹⁺ dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome <i>c</i> but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Ų at 365 K, in line with the low solution <i>T</i>_melt = 315–317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for <i>z</i> > 9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Ų at 300 K. This undergoes conformational tightening in line with the solution <i>T</i>_melt = 348 K before unfolding at the highest temperatures. The more extended charge states present in two or more conformers at room temperature, undergoing thermally induced unfolding before significant structural collapse or annealing at high temperatures. Variable-temperature IM-MS is here shown to be an exciting approach to discern protein unfolding pathways for conformationally diverse proteins