Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X‐ray crystallography and nuclear magnetic resonance spectroscopy measurements

Abstract

The three-dimensional conformation of a protein is central to its biological function. The characterisation of aspects of three-dimensional protein structure by mass spectrometry is an area of much interest as the gas-phase conformation, in many instances, can be related to that of the solution phase. Travelling wave ion mobility mass spectrometry (TWIMS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIMS under denaturing and near-physiological solvent conditions and cross-sections estimated for the charge states observed. Estimates of collision cross-sections were obtained with reference to known standards with published cross-sections. Estimated cross-sections were compared with values from published X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy structures. The cross-section measured by ion mobility mass spectrometry varies with charge state, allowing the unfolding transition of proteins in the gas phase to be studied. Cross-sections estimated experimentally for proteins studied, for charge states most indicative of native structure, are in good agreement with measurements calculated from published X-ray and NMR structures. The relative stability of gas-phase structures has been investigated, for the proteins studied, based on their change in cross-section with increase in charge. These results illustrate that the TWIMS approach can provide data on three-dimensional protein structures of biological relevance.

Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry

“…There is now substantial evidence to support the view that the gas-phase protein structure can reflect, under controlled conditions, the native solution-phase structure [1,6,12]. Multiple studies have shown good agreement between rotationally averaged cross-sectional measurements obtained from X-ray and NMR structures and those obtained by ion mobility experiments [7,[13][14][15][16][17].…”

“…There is now substantial evidence to support the view that the gas-phase protein structure can reflect, under controlled conditions, the native solution-phase structure [1,6,12]. Multiple studies have shown good agreement between rotationally averaged cross-sectional measurements obtained from X-ray and NMR structures and those obtained by ion mobility experiments [7,[13][14][15][16][17].In this study traveling-wave ion mobility mass spectrometry (TWIMS) was used to probe the gasphase conformations of hemoglobin tetramers and their constituents.Address reprint requests to Dr. …”

“…This implies that holo-␣-apo-␣ ␤ is not an essential intermediate within the The typical electrospray ionization (ESI) mass spectrum of a protein consists of an envelope of peaks attributed to a series of multiply charged gas-phase ions that can indicate the stability and compactness of its structure in the gas phase [4,5]. Multiply charged ions are produced by proton attachment, predominantly to exposed basic sites on the protein [6], and those of lowest charge are thought to be most representative of native structure [7]. A highly compact protein would have fewer exposed basic sites than those of an unfolded conformation of the same protein and thus would accept fewer charges [4,8].…”

“…Multiply charged ions are produced by proton attachment, predominantly to exposed basic sites on the protein [6], and those of lowest charge are thought to be most representative of native structure [7]. A highly compact protein would have fewer exposed basic sites than those of an unfolded conformation of the same protein and thus would accept fewer charges [4,8].…”

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

“…Recent years have witnessed the rapid growth of traveling wave (TW) IMS, where ions Bsurf^on DC waves within a stacked-ring rf ion guide [31,[49][50][51]. It has been found empirically [52] that t d in TWIMS is related to Ω according to…”