Deciphering Drift Time Measurements from Travelling Wave Ion Mobility Spectrometry-Mass Spectrometry Studies

TLDR

Understanding protein structure and dynamics is essential for biology and disease, and ion mobility spectrometry–mass spectrometry provides a rapid, single‑experiment method to separate biomolecular components and measure their mass and collision cross‑section, offering 3D‑architectural insight. The study reports a calibration method for a commercial ESI‑TWIMS‑MS using known cross‑sectional areas from conventional IMS‑MS. The authors calibrate the ESI‑TWIMS‑MS by applying known cross‑sectional areas from conventional IMS‑MS, then systematically vary TWIMS drift‑time parameters to assess their impact on calibration accuracy and resolution. The calibrated ESI‑TWIMS‑MS produced cross‑sections that closely matched PDB‑derived values, confirming the method’s accuracy, and when applied to β2‑microglobulin it distinguished folded, partially unfolded, and unfolded conformations under denaturing conditions.

Abstract

Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of individual components in a single, rapid (≤ 2 min) experiment, providing 3D-architectural information directly. Here we report a method of calibrating a commercially available electrospray ionisation (ESI)-travelling wave ion mobility spectrometry (TWIMS)–mass spectrometer using known cross-sectional areas determined for a range of biomolecules by conventional IMS-MS. Using this method of calibration, we have analysed a range of proteins of differing mass and 3D architecture in their native conformations by ESI-TWIMS-MS and found that the cross-sectional areas measured in this way compare extremely favourably with cross-sectional areas calculated using an in-house computing method based on Protein Data Bank NMR-derived co-ordinates. This not only provides a high degree of confidence in the calibration method, but also suggests that the gas phase ESI-TWIMS-MS measurements relate well to solution-based measurements derived from other biophysical techniques. In order to determine which instrumental parameters affect the ESI-TWIMS-MS cross-sectional area calibration, a systematic study of the parameters used to optimise TWIMS drift time separations has been carried out, observing the effect each parameter has on drift times and IMS resolution. Finally, the ESI-TWIMS-MS cross-sectional area calibration has been applied to the analysis of the amyloidogenic protein β 2 -microglobulin and measurements for three co-populated conformational families, present under denaturing conditions, have been made: the folded, partially unfolded and unfolded states.