Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

Abstract

Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (⍀) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra-and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding. E lectrospray ionization-mass spectrometry (ESI-MS) is a technique able to preserve the non-covalent interactions of protein-ligand complexes in the gas phase [1][2][3]. Since its original discovery, the application of ESI-MS in this area has accelerated rapidly [4,5]. The ESI-MS approach can provide a sensitive and efficient means of obtaining valuable information relevant to binding events allowing, for example, the stoichiometries of noncovalent complexes to be easily obtained [6,7]. The practical information available from ESI-MS measurement is already well documented [8]. In addition to stoichiometry, the affinities of protein-ligand interactions can be quantified using MS [9 -12]. In many cases, binding affinities determined using ESI-MS show good agreement with values obtained using other means, potentially validating MS methods for use in early-stage screening in the drug discovery process [13,14]. ESI-MS is not only suitable for the analysis of protein-ligand interactions, but has widespread utility in studying large protein-protein complexes [15,16], and has been applied to the preservation and detection of very large biomolecular assemblies, including the ribosome [17,18] and the tobacco mosaic virus (Ͼ40 MDa) [19].A key underlying issue in ESI-MS studies of ligand binding is to what extent these findings can be related to solution behavior. Given the great importance of water to the protein fold [20], it seems clear that desolvation should result in catastrophic loss of native structure, with accompanying consequences for ligand binding. Not withstanding the role of solvation spheres around the protein, a recent study has provided evidence that an interior water molecule in FK-binding protein makes significant contribution to the structural integrity of the fo...

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

“…These results are based on the BharshÛ (Figure 2a). The measured Ω values agree with experimental literature data to within 2% or better [3,18,65,70]. Most importantly, Figure 4 demonstrates that virtually identical data are obtained when employing regular ESI or nanoESI.…”

“…For Cyt and hMb, these calculations agree with the experimental TWIMS cross sections quite well (Figure 4b, c). In particular, the Ω EHSS values of these two proteins are virtually identical with the experimental cross sections [70]. Taking into account the ongoing discussion regarding the uncertainties of theoretical Ω calculations [9,28], the agreement between calculated and experimental Ω values in Figure 4b, c is quite remarkable.…”

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

“…Accordingly with N ‐acetyl‐glucosamine (NAG) substrate added to the desorption spray and directed at lysozyme deposited on the stage we observed additional peaks assigned to binding of intact NAG‐5 to lysozyme (Figure 1 c). The rapid turnover of this substrate precludes its observation in solution‐based ES16 but since substrate binding takes place during rapid desorption, analogous to reactive DESI experiments reported for small molecules,17 the transient bound state can be captured using this native DESI approach. …”

Evidence for the Preservation of Native Inter- and Intra-Molecular Hydrogen Bonds in the Desolvated FK-Binding Protein·FK506 Complex Produced by Electrospray Ionization

“…Due to the poor solubility of FK506, both protein and ligand samples were prepared in 10 % propanol. From previous studies with this protein in the presence of other organic modifiers, such as 10 %MeOH [12], the addition of 10 % propanol was not expected to significantly alter the protein structure or the FKBP·FK506 interaction. Each injection mixed 14.03 μL of the ligand solution (200 μM) into the sample cell containing the protein (20 μM) every 3 min.…”

“…Significant effort has been directed towards this question [8]. Ion mobility spectrometry (IMS), a gas-phase electrophoretic technique, is now often used in conjunction with MS analyses and allows low resolution structural information of analytes to be directly accessed in the desolvated MS environment [8][9][10][11][12][13][14]. These studies have provided insights into gas-phase protein structure, and it is generally accepted that on the timescales of IMS-MS experiments, major structural perturbations can be avoided for low charge state ions [15,16].…”