Determination of Collision Cross-Sections of Protein Ions in an Orbitrap Mass Analyzer

Abstract

We demonstrate a method for determining the collision cross-sections (CCSs) of protein ions based on the decay rate of the time-domain transient signal from an Orbitrap mass analyzer. Multiply charged ions of ubiquitin, cytochrome c, and myoglobin were generated by electrospray ionization of both denaturing solutions and ones with high salt content to preserve native-like structures. A linear relationship between the pressure in the Orbitrap analyzer and the transient decay rate was established and used to demonstrate that the signal decay is primarily due to ion-neutral collisions for protein ions across the entire working pressure range of the instrument. The CCSs measured in this study were compared with previously published CCS values measured by ion mobility mass spectrometry (IMS), and results from the two methods were found to differ by less than 7% for all charge states known to adopt single gas-phase conformations.

Recommendations for reporting ion mobility Mass Spectrometry measurements

“…For these reasons, we devoted special attention to define K 0 and IM‐derived CCS values, and to clarify what influences these quantities. There are other methods than ion mobility spectrometry to determine experimental collision cross sections in a more direct way, for example, by monitoring the loss of ion signal due to collisions (Covey & Douglas, ; Chen, Collings, & Douglas, ; Anupriya, Jones, & Dearden, ; Anupriya et al, ; Dziekonski et al, ; Elliott et al, ; Sanders et al, ). These methods to determine collision cross sections are however not based on ion mobility measurements, and will not be covered here.…”

Importance of collision cross section measurements by ion mobility mass spectrometry in structural biology

“…While the task of coupling DTIMS is not trivial due to the slow MS acquisition rates of these mass analyzers in comparison with IM separation times, recent reports suggest this may be soon achievable, and would enable the ability to measure subtle variations in CCS for heterogeneous systems of similar mass and size, such as post‐translationally modified proteins. A very recent report has even demonstrated the possibility of measuring protein CCSs without the need for an IM cell, by measuring the decay rate of a particular m/z signal in an orbitrap analyzer across the operating pressure range of the instrument . While this approach does not currently match the analytical considerations of dedicated IM‐MS methods, it may contribute to an ever greater integration of CCS measurements in structural biology.…”

“…A very recent report has even demonstrated the possibility of measuring protein CCSs without the need for an IM cell, by measuring the decay rate of a particular m/z signal in an orbitrap analyzer across the operating pressure range of the instrument. 71 While this approach does not currently match the analytical considerations of dedicated IM-MS methods, it may contribute to an ever greater integration of CCS measurements in structural biology.…”

Surface-Induced Dissociation of Noncovalent Protein Complexes in an Extended Mass Range Orbitrap Mass Spectrometer

“…45 In fact, this phenomenon is so apparent that Sanders et al have leveraged the transient decay rate of protein ions in the Orbitrap to make collision cross section measurements. 46 For this reason, we use low resolution (8,750-35,000 as defined at 200 m/z) for acquisition of protein complexes, and only use higher resolution for specific questions (i.e. small ligand binding, PTMs, etc.)…”