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

The field of ion mobility mass spectrometry (IM ‐ MS) has developed rapidly in recent decades, with new fundamental advances underpinning innovative applications. This has been particularly noticeable in the field of biomacromolecular structure determination and structural biology, with pioneering studies revealing new structural insight for complex protein assemblies which control biological function. This perspective offers a review of recent developments in IM ‐ MS which have enabled expanding applications in protein structural biology, principally focusing on the quantitative measurement of collision cross sections and their interpretation to describe higher order protein structures.


| INTRODUCTION
There is no doubt that mass spectrometry (MS) has had an enormous impact in biological research. In particular, the emergence of proteomics has led to major technological advances and workflow designs that allow for identification and quantification of ever larger sets of proteins, which in turn has revolutionized our understanding of the proteome and its diverse roles in biological function. Such advances have not only benefitted high-throughput proteomics, but have also

| BASIC PRINCIPLES OF CCS MEASUREMENT
In general terms, IM separates ions based on the rate at which they migrate through a buffer gas under the influence of an external electric field. Importantly, the mobility of an ion is related to structural features such as charge and CCS, the latter of which can be used to infer information about three-dimensional conformations. Modern IM-MS instrumentation utilizes this basic separation principle in a variety of ways, and in combination with a range of MS platforms. For biomacromolecules, the IM separation technologies available for quantitative size determination are essentially of four types; drift tube, travelling wave, trapped ion and differential mobility analyzers. Aspects of these IM-MS techniques and their applications have been described in detail in several recent notable reviews. [2][3][4][5][6][7] Consequently, only a brief summary of these IM separation approaches is provided here.

| Drift tube ion mobility
Time-dispersion forms the basis of separation in the oldest and conceptually simplest form of IM spectrometry, namely drift tube ion mobility spectrometry (DTIMS). Here, ion packets are gated into a drift tube (with lengths of single linear drift tubes typically ranging from a few centimeters up to two meters), where they are propelled by a static uniform low electric field. Usually a neutral, inert gas such as helium, argon or nitrogen at pressures of 1-15 mbar is used to fill the drift tube, which restricts ion motion. The velocity of ions is related to their mobility in the buffer gas, which, in the 'low field limit' where the ratio between electric field strength and gas density is small (≤2 × 10 −17 V cm 2 ), 8 is proportional to the ion CCS (Ω) according to the Mason-Schamp equation: 9 where K 0 is the reduced mobility (measured at standard temperature and pressure), z is the charge state of the ion, e is the elementary charge, N is the number density of the drift gas, μ is the reduced mass of the ion-neutral drift gas pair, k B is the Boltzmann constant and T is the gas temperature.
The CCS parameter represents the rotationally averaged surface area of the ion which is available for interaction with the buffer gas; a smaller CCS results in fewer collisions with the buffer gas which otherwise impede ion motion, and therefore a shorter drift time through the drift tube. Consequently, measuring the drift time or arrival time distribution of a population of ions provides gas-phase structural information ( Figure 1A). Although DTIMS offers comparatively high IM resolving power (often defined in terms of the centroid arrival time Characteristics of different ion mobility separation methods for the measurement of collision cross section. A, Drift tube ion mobility: Packets of ions travel along a potential gradient opposing a constant gas flow. Ions with larger CCS experience more collisions with the gas, and hence traverse the cell with reduced velocity (greater drift time). B, Travelling wave ion mobility: Alternating phases of RF voltage are applied to a stacked ring ion guide (top), to which a sequence of symmetric potential waves is superimposed (bottom). The ions propagate through a reverse flow of buffer gas along the potential wave front; ions with low mobilities experience the most wave roll over events as a result of increased collisions with the gas, and exit the cell last. C, Trapped ion mobility: Ions are trapped in the TIMS tunnel at the point where the force of a gas flow matches the opposing force of an electrical field in the tunnel (top). Ions are portrayed aligned along the electrical field gradient (bottom), and are selectively released in order of increasing mobility as the electrical field is ramped down. D, Differential mobility analyzer: In this geometry, ions are introduced through a slit on the upper plate, and dispersed into a fan with trajectories defined by combined action of orthogonal gas flow and applied electric field. Ions of a selected mobility are drawn through an exit aperture to the mass spectrometer of the IM peak, normalized to peak width, i.e. t/Δt) it suffers from poor ion transmission efficiency, which, until more recent developments, limited applications in structural biology. Newer generation DTIMS-MS instruments have encompassed noteworthy innovations to the basic drift tube design, including the use of electrostatic and electrodynamic fields to accumulate ions before the drift cell 10 and focus radially diffuse ions. [11][12][13] Tandem DTIMS has also been used to interrogate mobility-selected ions in a manner analogous to MS/MS experiments, and assess gas-phase stability and microconformational states. 14,15

| Travelling wave ion mobility
Travelling wave ion mobility spectrometry (TWIMS), first reported by the buffer gas present in the TWIMS sector gives rise to a drag force, meaning those ions with greater CCS (low mobility) slip behind the wave front more frequently (also known as 'roll over') and therefore take longer to traverse the mobility cell ( Figure 1B).

| Trapped ion mobility
Trapped ion mobility spectrometry (TIMS) coupled with MS is a relatively recent addition to the field. Rather than moving ions through a stationary gas, as in DTIMS, ion separation in TIMS is achieved by holding ions stationary in a moving column of gas, and selectively ejecting these ions based on differences in mobility. A TIMS device is a modified ion funnel, through which ions are propelled by a gas flow in the presence of an axially variable, opposing electric field ( Figure 1C). An ion with a mobility of K is trapped at the point where the electric field strength is such that the ion drift velocity equals the carrier gas flow velocity. Following the trapping event, ions are sequentially eluted with ascending mobilities by gradually reducing the electric field strength. A key advantage of this approach is that the physical dimensions of the IM cell can be significantly reduced (approximately 5 cm) while achieving high resolving power (R ∼ 300), duty cycle (100%), and efficiency (∼80%). 6 Importantly, the fundamental physics of TIMS is the same as that in DTIMS and the relationship between mobility and CCS still applies; hence, it is possible to measure ion CCS using first principles for structural characterization. The performance of DMAs with nanometer-size objects is severely limited by Brownian motion; however, resolving powers in excess of 50 have recently been described using high-transmission parallel-plate DMAs, 25 which can be coupled as a front-end device to almost any mass spectrometer with an atmospheric pressure ion source.

| Differential mobility analyzers
Consequently, unlike most other IM separations, the DMA can be used to measure the mobility of electrosprayed ions without the need for energetic confinement or declustering, or even vacuum interfaces.  A correct interpretation of IM data on any protein must therefore reconcile these effects, particularly when considering comparison across platforms or between experimental and theoretical values. In this work, coarse-grained and homology modelling was used to 'fill-in' missing details from high-resolution experimental structures at both subunit and oligomer levels. Topological models were then constructed by determining the packing arrangements via known archetypal shapes and/or crystal symmetries that provide the best fit to the experimental CCS values ( Figure 3A). The method therefore refines each building block using experimental data before larger oligomeric complexes are sequentially constructed.

| IM-MS for the masses
The success of this approach was illustrated by application to multimeric protein complexes within the Escherichia coli replisome. replisome, and is likely to become more commonplace as the accuracy and precision of CCS measurements continue to improve.

| Membrane proteins
Structure determination of membrane protein assemblies in particular continues to define a substantial challenge in structural biology.
Membrane proteins constitute approximately 30% of a typical genome, and more than half of all membrane proteins are predicted to be pharmaceutical targets. 59   and cyclic traveling wave 69 devices currently in development, it is expected that quantitative IM methods will be more routinely coupled with high-resolution MS measurement using, for example, Fourier transform ion cyclotron resonance (FTICR) and orbitrap mass analyzers.
IM devices for coupling with orbitrap mass analyzers already exist, including the recent release of FAIMS-Orbitrap instrumentation.
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 Model of early aggregation states of β 2 microglobulin. Monomeric β 2 microglobulin co-populates distinct native-like, partially folded, and unfolded states at acidic pH, while the dimer is a highly dynamic species. Initial association of the monomer to the dimer results in a dynamic trimer that undergoes structural change to a more stable species. The tetramer evolves in a similar manner, first forming with a dynamic structure that subsequently becomes stabilized. Further association proceeds by stacking elongated monomeric subunits. Reproduced with permission from Smith et al 65 achievable, 70  In general, broad agreement has been observed between IM CCS values recorded for proteins and protein complexes with corresponding published X-ray structures. 42 However, while this holds true for the currently studied protein analytes, it is yet to be seen whether existing approaches are suitable to describe the multitude of diverse structures predicted across the proteome. Furthermore, a significant improvement is required in the computational prediction of protein structures or coarse-grained approximations in the absence of X-ray or NMR datasets in order to increase the scope and throughput of structure determination by IM-MS.
In summary, the measurement of protein CCS values by IM-MS must be carefully considered and evaluated. However, it can provide a valuable experimental constraint to define protein structures, often for systems which elude traditional structure biology approaches.
With continued technological advancement, and motivation to stretch this technology to ever more complex systems, it is expected that the applications of quantitative IM-MS in structural biology will greatly increase in coming years.