I On Mobility Instruct B

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Ion Mobility in Structural Biology

Timothy M. Allison*,1, Michael Landreh†
*Biomolecular Interaction Centre and School of Physical and Chemical Sciences, University of Canterbury,
Christchurch, New Zealand
†
Science for Life Laboratory, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet,
Solna, Sweden
1
Corresponding author: e-mail address: timothy.allison@canterbury.ac.nz
Contents
1. Introduction 1
2. Structural Aspects of Proteins in the Gas Phase 4
3. Instrumentation 7
3.1 Established IM Types and Platforms 8
3.2 Emerging Technologies 11
4. IM for CCS Determination 11
4.1 Common Approaches to Measuring CCS 13
4.2 Considerations of Accuracy in CCS Determination by TWIMS 15
5. Using IM–MS to Study Protein Folding in the Gas Phase 16
6. Complementary and Supporting Computational Resources 18
6.1 Theoretical CCS Calculations 18
6.2 Software for Analysis of IM–Mass Spectra 21
7. IM for Separation 22
8. Combining IM–MS With Other Structure Analysis Methods 24
9. Conclusions 27
References 27
1. INTRODUCTION
Understanding how the structure of proteins relates to their function is a
fundamental tenet of structural biology. Advances in the field of structural
biology have been dependent on a variety of different technologies for pro-
viding the structural information required for making this connection.
A notable example is protein crystallography, which has been a workhorse
technique for many years, resulting in more than 140,000 structures of pro-
teins deposited in the Protein Data Bank [1]. More recently, there has been a
rapid rise in the use of cryo-electron microscopy (cryo-EM) [2], driven by
various technological advances, which has resulted in numerous new
Comprehensive Analytical Chemistry # 2019 Elsevier B.V. 1

ISSN 0166-526X All rights reserved.
https://doi.org/10.1016/bs.coac.2018.10.001
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2 Timothy M. Allison and Michael Landreh
structures of protein complexes. While both of these techniques focus at the

atomic level on the three-dimensional shape of proteins, there are many other
aspects to protein structure. For example, this may include lower resolution
structural parameters such as the gross size of a protein, or the composition and
quaternary structure of a protein complex, and its local movements, or
dynamics. Information on these other structural attributes of proteins beyond
their tertiary structures can be obtained in various ways by other techniques,
such as small-angle X-ray scattering (SAXS) [3], nuclear magnetic resonance
[4], and mass spectrometry [5], to name just a few. All of these technologies
have, in their often complementary ways, helped to reveal the structural
secrets of proteins—the crucial molecules that drive life.
One of these important technologies is mass spectrometry (MS) (Fig. 1)
[5]. MS is often associated with the study of small molecules in the various
Fig. 1 The structural biologist’s toolbox is packed full of different and often comple-
mentary tools for unravelling the structure–function relationships of proteins, some
of which are annotated. Mass spectrometry is one of these tools, and we classify it into
different types. These types depend on the size of the analyte and whether the
approach retains noncovalent interactions. Due to the preservation of structure and
interactions in native MS, this approach is of particular interest to structural biologists.
It is in this context where IM is coupled to MS, where structural biologists use IM for both
separation and determination of CCS.
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Ion Mobility in Structural Biology 3
fields of omics, such as lipidomics, glycomics, metabolomics, and proteo-

mics. In this latter field of proteomics, the MS approach applied to proteins
is very often peptide centric, with the proteins first digested in solution into
peptides, before analysis and ultimately identification [6]. However, MS in
principle does not have a mass limit, and it is adept at studying much larger
molecules, and as is relevant to structural biology: whole and intact proteins.
In some cases, this is termed denaturing MS, where the protein molecules
are first denatured (but in this case not cleaved) in solution [6]. Such a dena-
turation step destroys noncovalent interactions, such as those within all levels
of protein structure, including protein–protein interactions (pertinent for
quaternary structure), as well as those between proteins and other molecules,
such as ligands. This limits the breadth of structural information that can be
obtained to the identification of individual protein components. This is use-
ful for identifying by mass the presence of a particular protein, and through
top-down proteomics analyses (where protein sequences and modifications
are analysed using peptide fragments generated in the gas phase) denatur-
ation in solution eases the gas-phase sequencing and identification of
proteoforms.
The field of native MS is concerned with analysing proteins from solu-
tion states in which the native forms of the proteins are preserved [7,8]. This
means that protein quaternary structure and other noncovalent interactions
that occur in solution are retained in transfer to the gas phase of the mass
spectrometer. This allows the composition, topology, and binding charac-
teristics of different protein systems, from small to large, to be directly
detected and studied [8]. Owing to the way in which native MS preserves
these important biological interactions and the direct detection afforded
based on mass, this technique is very clearly a useful tool in the structural
biologist’s arsenal.
Where then does IM come in? It is through a tight integration with
native MS where IM plays its biggest role in structural biology, exploiting
that for both techniques the analyte must be ionised and the breadth of pro-
tein structural information retained under the native regime and thus avail-
able to study. This is a long-standing integration and consequently the
conjoined techniques, known as IM–MS, have developed side by side
[9]. A distinct advantage of this coupling of technologies is that it provides
partially orthogonal separation. While MS separates molecules based only on
their mass and charge, the addition of IM adds a dimension of separation
based on IM; this is dependent on the size and shape of the ions, in addition
to their charge [10]. However, the utility of IM is not limited to separation:
common in its application in structural biology is using it to measure the
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CCS of ions [11]—this gives valuable information on the size and shape of
the ions, and therefore the protein structure. This is particularly useful when
information about the structural architecture of a protein is unknown and
offers access to structural information on par with the resolution afforded
by SAXS [12].
In this chapter, we focus on the use of IM with native MS. In the context
of structural biology, we will introduce the common IM–MS instrumenta-
tion and describe how IM is utilised for both separation and measurement.
We will also introduce some crucial concepts in MS related to ion chemistry
and the structural stability of proteins in the gas phase that is important to
consider when using IM for structural biology purposes. Lastly, we will
exemplify the ways in which IM is applied to the study of proteins and their
structure.
2. STRUCTURAL ASPECTS OF PROTEINS IN THE

GAS PHASE
The key to using IM for the study of biological macromolecules is
the integration with MS, as IM spectrometry (IMS) is a gas-phase tech-
nique. Hence, structural biology applications of IM are restricted to anal-
ysis of gaseous protein ions. Thus, we must consider the pertinent
implications of making these measurements in the gas phase of the mass
spectrometer.
The notion that MS may be suitable for the direct analysis of protein
structure and interactions was first put forward following reports that specific
protein–ligand complexes could be detected in mass spectra following
electrospray ionisation (ESI) from buffered solutions [13–15]. These obser-
vations provided the first indications that proteins are able to retain features
of their solution structure after transfer to the gas phase. To understand
which structural information can be obtained from a gaseous protein ion
and how it relates to the solution structure, we have to consider how the
ionisation process and subsequent analysis in the gas phase affects the protein
structure.
In ESI, the most commonly used method for ionisation of intact pro-
teins, the analyte solution is sprayed from a fine-tipped emitter (typically
2–20 μm) [16] to which a voltage is applied to produce charge-carrying
microdroplets that contain the protein molecules of interest. Evaporation
of solvent molecules from the droplets leads to an increasing charge density
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and eventually fission due to repulsive forces. This process is repeated

multiple times, until the size of the droplet approximates that of the
contained protein (disordered proteins tend to be ejected during the evap-
oration process, and ionise by a different mechanism [17]). With the evap-
oration of the last solvent molecules, the droplet charges are deposited on the
protein surface, generating protein ions that can be analysed in the mass
spectrometer [18].
Recently, all-atom molecular dynamics simulations have been used to
investigate the transition of proteins from an aqueous environment to the
gas phase. In general, the removal of the last layer of solvent molecules
and the attachment of charge carriers to the protein have a very modest effect
on the overall fold, the protein retaining the native tertiary structure at the
completion of the ESI process [19,20]. The key to retaining structure
appears to be the gradual solvent evaporation at minimal thermal activation.
A striking example of gentle solvent removal is the observation that three
specific water molecules can be retained at the interface between a tyrosine
kinase and its phosphopeptide ligand in the gaseous protein–ligand complex
ion [21]. A useful strategy for gentle desolvation and transfer into the mass
spectrometer is the creation of an intermediate pressure regime in the ion
source region. This process, termed ‘collisional cooling’, dampens the accel-
eration of the ions as they transition from atmospheric pressure to high vac-
uum, and thus minimises high-energy collisions with residual gas molecules
that would otherwise increase the thermal energy of the ions and promote
unfolding [22–24].
While the effect of solvent removal can be minimised via careful instru-
mental tuning, the structure of the ionised protein does not remain unaf-
fected by the subsequent exposure to the temperature regimes in the ion
source and vacuum inside the mass spectrometer. By studying the fragmen-
tation pattern of intact ionised proteins as a function of trapping time or
acceleration voltage, it has been shown that different parts of the protein
backbone become available to cleavage by electron capture dissociation,
depending on how much time the protein has spent under vacuum condi-
tions, or the degree of thermal activation it has been exposed to [25,26].
Multiple molecular dynamics studies paint a detailed picture of the evolution
of protein structures when transferred to the gas phase, as outlined by
Breuker and McLafferty [27]. To summarise (Fig. 2), three steps occur: first
within picoseconds after desolvation, the charged side chains collapse due to
the loss of counter ions, and form new salt bridges or hydrogen bonds in
their immediate environment, as well as with the protein backbone. These
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Fig. 2 Proteins will adopt a number of structures and conformations after transfer into
the gas phase. These will inherently arise through time spent in vacuum, but are also a
function of thermal activation. This scheme represents the structural events that occur
as a function of time spent in the gas phase, and the resulting possible outcomes in
terms of collision cross section of the ion.
interactions or ‘cross-links’ may help to preserve an overall native-like

fold of the protein, which can remain largely unchanged for milliseconds.
At the second stage, a gradual loss of intramolecular contacts is believed
to occur, as electrostatic interactions, which no longer face competition
from surrounding solvent molecules, appear strengthened and hydro-
phobic interactions are weakened [28]. However, other studies have shown
this not to be universally true, as even purely hydrophobic interactions
can be preserved in the gas phase [29,30] and might in some cases be more
stable than under condensed-phase conditions [31]. In the third step, the
gradual loss of native structure allows for the formation of new noncovalent
bonds between distant parts of the protein, which enable the protein to
adopt conformation(s) that are energetically favourable in the gas phase
[32,33].
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The timescale for a native MS experiment using a standard quadrupole-

time of flight mass spectrometer is in the low millisecond range [27]. There-
fore, the conformational changes that have to be considered when analysing
native MS data relate to the first and second step of the gas-phase structural
evolution. Multiple molecular dynamics studies have established the equil-
ibration process of protein structures under MS conditions within this
timeframe [34–37]. Common to these studies is the observation that, while
the overall structure of the desolvated protein is retained, unsupported loops,
and exposed hydrophilic sites tend to collapse onto the protein core, leading
to some compaction relative to the crystal structure [38]. On the other hand,
buried binding sites, protein–protein contact surfaces, and other structural
motifs supported by inter- or intramolecular interactions, remain largely
unaffected in these models.
Taken together, the experimental and computational evidence strongly
suggests that the overall structure of globular proteins, and interactions such
as protein–protein interactions, ligand binding, and folding states, can all be
preserved under the experimental conditions used for IM–MS. As a conse-
quence, IM–MS is a versatile tool for structural studies, where the size and
shape of proteins and their complexes under a variety of conditions can be
determined. However, finer structural features, such as loops and side chain
orientations, as well as ensemble equilibria of, e.g., intrinsically disordered
proteins (IDPs), are likely more sensitive to gas-phase conditions, and
IM–MS does not necessarily provide a direct picture of their condensed-
phase behaviour [39]. In these cases, the use of complementary computa-
tional approaches is often required to allow detailed interpretation of
IM–MS data.
3. INSTRUMENTATION
How is the combination of IM and MS practically achieved? Various
mass spectrometer architectures integrate IM, with different IM devices, and
range from in-house-built instruments to those that are commercially avail-
able. The choice of IM technology is influenced by whether it is being used
primarily as a separation tool (where resolution may be more important), or
for calculating the CCS of a protein (where as we will explain later, direct
measurements are sometimes favoured). The coupling of IM with MS means
that the mass spectrometer is responsible for sample ionisation, ion transfer,
and ion detection. In addition to the separation afforded by IM, the coupling
of technologies also provides for partially orthogonal separation of the ions
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based on m/z, and in conjunction, the possibility to employ the full gamut of
other possible ion manipulations using the rest of the mass spectrometer.
The instrument architecture, that is, where the IM device is located rel-
ative to the other components of the mass spectrometer, can be an important
parameter in determining the feasibility of experiments. For membrane pro-
teins, which are usually ionised while encapsulated in detergent micelles, to
interrogate the ‘naked’ protein ion requires removal of the micelle and
detergent molecules before IM separation [40–42]. This requires that the
IM device is located after the part of the instrument in which collisional
activation for detergent removal takes place. Often in-source activation is
not sufficient for this purpose [43,44], and therefore the IM device would
need to be after a dedicated collision cell. Likewise, for experiments requir-
ing tandem MS, where a particular m/z window needs to be chosen prior to
IM, then the IM cell must be located after the quadrupole mass analyser [43].
Instruments in which the IM device is located immediately after the ion
source are therefore limited in their general capabilities for structural biology
investigations.
3.1 Established IM Types and Platforms

There are three types of IMS that have found use in structural biology: dif-
ferential [45], drift tube (DTIMS) [11,46], and travelling wave (TWIMS)
[11,46]. Important criteria in many cases in the choice of technology are
ion transmission and resolving power, which differ for each IM type. The
ability to directly measure CCS and commercial availability means this
choice is frequently DTIMS and TWIMS, respectively, and are therefore
the types on which we will focus here.
It would be remiss not to recognise the enormous contribution of cus-
tom instruments and associated investigations into the gas-phase behaviour
of proteins have played, enabling the application of IM–MS in structural
biology. While these studies are too numerous to individually cite, they have
been critical to establish the relevance and manipulations of protein gas-
phase structure and have also lead to the development of new methodologies
for exploring the structure–function paradigm of proteins using IM–MS.
3.1.1 Drift Tube IMS

Drift tube IMS separates ions based on their mobility under a constant
potential gradient (Fig. 3). Here, the ion beam is radially confined by
applying a radio frequency, while the potential gradient and the coun-
teracting drag force separate the ions traversing the tube [10]. A distinct
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Fig. 3 There are two types of IM commonly used for structural biology applications: drift
tube and travelling wave. DTIMS has the advantage of direct CCS measurements,
whereas although TWIMS can offer higher resolution IM, CCS determination requires
the formation and use of a calibration to convert measured drift times into CCS. The
illustrative graphs underneath each IM device cartoon represent the static and dynamic
electromagnetic fields of DTIMS and TWIMS, respectively.
advantage of this type of IM device is that the CCS of ions can be directly
calculated from the measured arrival times. However, these devices can also
suffer from poorer sensitivity, and IM resolution can sometimes be limiting
[46]. As the resolving power of DTIMS devices is proportional to the square
root of the applied drift field and the length, the only practical way to
increase resolution is longer drift tubes, but this is normally associated with
a loss in sensitivity.
The resolution of the IM of a protein ion is also affected by other factors.
One compounding factor is the size of the ions themselves, and the associ-
ated ensemble of conformations a protein can reside in. This inherent
ensemble of states, on the assumption it is at least in part retained in transfer
to the gas phase, and the ion packet width both increase the observed peak
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widths in the mobility domain. This is not exclusive to DTIMS of course

and is a general limitation because of the difficulty in discerning the instru-
mental and sample contributions to the observed peak width. Nevertheless,
instruments with these devices have been crucial in the use of IM in
structural biology and are consistently used for absolute CCS measurements
in validation and calibration frameworks [47–52].
3.1.2 Travelling-Wave IMS

To overcome the limitations of DTIMS, most commercially available
instruments feature travelling-wave IM (TWIMS) developed by Waters,
which uses pulses of direct currents that traverse the drift tube to separate
ions (Fig. 3) [53]. Instead of setting a linear field strength, the transport of
the ion packets is controlled by the velocity and height (i.e. direct current
potential amplitude) of the travelling wave. This means that separation can
be tuned to the molecules of interest, meaning a shorter drift tube can be
used, leading ultimately to better resolution than can be obtained for a linear
electric field DTIMS device of the same length. In TWIMS ion guides, the
direct current pulses that comprise the travelling wave are superimposed on
the radio frequency that focuses the ion beam, thus minimising diffusion of
ion packets and improving resolution.
Despite these advantages, TWIMS poses a challenge when used in
structural biology: as a result of travelling-wave separation, the relationship
between ion CCS and drift time becomes nonlinear, as the transport efficiency
now originates from a complex interplay where wave height and velocity act
differently on ions with different charge and size. Unlike DTIMS, a theoret-
ical conversion of ion drift times to mobility constants, which is required for
accurate ion size determination, is not feasible as a routine method [54].
Instead, TWIMS drift times have to be converted to absolute ion CCSs using
multipoint calibrations fitted with an exponential function [47], as described
vide infra. Recently, however, a method to relate TWIMS drift times to CCS
has been developed that can be used in many instances to directly calculate
CCS with similar error to calibration-based approaches [55].
With regard to commercial availability, the vast majority of IM–MS
instruments today utilises TWIMS technology, such as the Synapt instru-
ment family from Waters. Furthermore, the commercial IM–MS set-ups
share a common architecture where the IM cell is placed in line with a
quadrupole mass filter and a collision cell to allow selection and activation
of the ions prior to IM separation [56].
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3.2 Emerging Technologies

In the quest for high-resolution IM of proteins, two new technologies have
recently emerged. The first, travelling-wave enabled multipass cyclic IMS,
uses a cyclic drift tube in which ions can be continually cycled, thus dramat-
ically increasing the effective length of the drift tube and obtainable resolu-
tion [57]. The second, structures for lossless ion manipulation (SLIM)
[58,59], promises very long drift tube lengths without loss of sensitivity,
and a means to directly measure CCS. Interestingly, the lower than expected
resolving power of the SLIM approach when applied to protein ions suggests
that an ensemble of structures are present in the gas phase, rather than a single
conformer [60]. This is consistent with results from using a new variable
temperature high-resolution drift tube, where the broad arrival time distri-
butions observed were hypothesised to correspond to a high number of
different but stable structures rather than interconverting conformers [61].
Time-dependent experiments have also utilised the SLIM architecture to
explore the lifetime and evolution of protein ion structure in the gas phase.
Deviations in the CCS that occurred on the timescale of other IM separa-
tions were in line with the associated reported uncertainties, suggesting that
the initial gas-phase structures of protein ions can have relatively long
lifetimes [62].
4. IM FOR CCS DETERMINATION

During IM ions are separated based on their size and shape (which is a
measure of protein structure at low resolution) and charge [10]. The size and
shape of the ion affect the probability of collisions to occur during IM, which
in turn through momentum transfer in these collisions affects the time it
takes an ion to transit through a drift cell. This size and shape are known
as the CCS, which are a rotationally averaged measurement representing
the structure of, in this case, a protein. The mobility of an ion, and therefore
the relationship between its charge, CCS, and drift time is known through
the Mason–Schamp equation for separations performed by DTIMS [63].
Importantly, this means that the time it takes an ion to transit through an
IM drift tube is directly proportional to its CCS. Thus, by measuring the
drift time, the CCS can be calculated directly.
In the absence of other structural information about a protein, a crude
structural metric of the volume it occupies is useful. Simplistically, CCS,
which is correlated with protein mass, is another measure of the size of a
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protein, akin to its volume or radius of gyration as measured in SAXS exper-

iments. This coarse information is useful for integrative modelling [64–67],
especially of protein complexes, and for determining the conformational
state(s) of proteins. These states could include a folded state, or any other
numerous more extended conformations, which may arise because of struc-
tural changes in the protein, related to unfolding, or other rearrangement
events (Fig. 4).
The CCS is a single number representation of the structure of a protein
and is therefore not unique to a particular protein or protein complex,
or even a particular protein conformation. For example, it is possible for
the CCS of the same protein in two different conformational states to be
the same. Many parameters affect the CCS of a protein ion, including the
protein conformation at the time of measurement [68], and the method
Fig. 4 Using IM the CCS of a protein ion can be measured. On the basis of the ability of
IM to separate on the basis of size, different proteins of different size but same m/z will
have different CCS. The CCS for a single protein however could correspond to one of
many different possible conformational states. Determining which of these states
corresponds to the protein can be ambiguous, for example, a collapsed and unfolded
form of a protein may possess a CCS coincident with that for a folded form, or two dif-
ferent conformations of a protein may share the same CCS.
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of measuring the CCS, such as the choice of neutral gas molecule used for
the IM separation [69]. Notably, the CCS of an ion depends on the identity
of the gas—a molecular species will have a different CCS in each gas used for
measurement. The single number nature of CCS is to some degree a
limitation, but this is compensated for by being able to measure the width
of the IM drift time distribution, which represents the diversity of confor-
mations present in the sample. Therefore, in some cases not only is it possible
to measure the CCS of an ion but also the distribution of different structural
forms that are present [70–73].
4.1 Common Approaches to Measuring CCS

As outlined earlier, the CCS of a protein ion can be directly calculated from
the measured drift time on drift tube instruments (Fig. 5). In these cases, the
measured arrival time is composed of both the drift time itself, and what is
known as a t-zero time, which is the time the ion spends in transfer post-IM
separation before detection. The t-zero time is calculated by measuring the
arrival time at different voltages applied over the drift cell, and extrapolating
a line fitted to the inverse drift voltage plotted vs arrival time to the arrival
time intercept. This can then be subtracted from the measured arrival time to
determine the drift time of the ion. Assuming that the t-zero component of
the measured arrival time is constant, which under the same instrument
conditions and using the same m/z ion should hold true, higher throughput
approaches are then enabled [41], as the requirement for multiple measure-
ments at different applied drift voltages to determine the CCS of an ion is
negated. In support of this approach is that the variation in t-zero time
for different ions is usually very small relative to the drift time itself (on
the order of 1%), leading to only small potential errors in the drift time used
for calculation of the CCS.
For TWIMS, although the drift time is related to the CCS of an ion,
because of the complexity of the wave-based separation, it has been too
difficult to directly relate CCS to drift time. In this case the Mason–Schamp
equation is modified by the incorporation of empirical factors, which
approximate, over a small range, the exponential relationship between
CCS and drift time in these devices. This relationship is established through
the construction of a calibration using ions with similar mobility to an
analyte and known CCS, forming a calibrated relationship between drift
time and CCS (Fig. 5) [47–52]. This calibration can then be used for the
calculation of CCS for unknown protein ions. The ions used in a calibration
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Fig. 5 The CCS of a protein can be determined directly if using DTIMS or indirectly via
calibration by TWIMS. The DTIMS approach requires measuring the arrival time over a
series of different applied drift fields to determine the time taken for the ion in transfer
post-IM to detection (T-zero). The TWIMS approach requires measuring the arrival times
of other proteins with DTIMS-determined CCS in order to form a calibration, which over
a small range and for molecules with similar mobility correlates arrival time to CCS.
Example CCS determined by each method written in the proposed nomenclature
for CCS.
each must have CCS measurements determined (in most cases) by DTIMS,
on an instrument with similar architecture. This carries the assumption that
the CCS of an ion measured on a DTIMS instrument will be the same on a
TWIMS instrument. This should hold true, so long as the level of collisional
activation applied in both instances is the same, for example that no
collision-induced unfolding (CIU) of a protein ion has taken place.
Databases of experimentally measured CCS of protein ions obtained
used DTIMS are available [47–50]. Some of these databases list CCS for
denatured as well as native samples, and so it is important to use ion infor-
mation that matches the state of the unknown protein. These databases also
often list CCS measurements made in different gases, for example helium
and nitrogen, and for different charge states of a protein. When determining
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a CCS through calibration, the literature CCSs measured in a different gas

type can be used, in which case the calibrated CCS value will become an
‘effective’ value for that gas.
In some cases, the CCS can be directly calculated from the TWIMS drift
times. Mortensen et al. [55] developed a method that models the TWIMS
potential wave, and equations to describe the velocity of ions in the travel-
ling electric field. Under low wave velocity conditions, this method can be
used to obtain CCS on TWIMS devices without calibration. The CCS have
similar values to DTIMS measurements and errors similar to calibration-
based TWIMS approaches.
Within the native MS community, there are moves afoot to standardise
the reporting of CCS information [74]. The EU COST action on native MS
has discussed reporting standards to specify nomenclature for referring to a
CCS to include the gas and measurement type [75]. In addition, there has
been discussion reinforcing the importance of including important method-
ological details for these measurements. In the case of TWIMS, this would
include details necessary to reproduce exactly the calibration, that is, the
proteins and charge states used as standards in the calibration. Given the
variation in CCS, it is important for cementing the reputation of the mea-
surement that a consistency in reporting is established.
4.2 Considerations of Accuracy in CCS Determination

by TWIMS
There are several sources of error in calculating a CCS using the calibrated
TWIMS approach. These include the natural variation between replicates,
from the calibration curve, from the protein standards that comprise the
calibration curve, and from changes in gas between the protein standard
measurements and the analyte [51]. Underlying this calculation of error is
an assumption that an appropriate choice of proteins used to form the
calibration has been made. The key criterion for IM calibration is choosing
ions with similar mobility [47,54]. This means the ions should be of similar
mass, charge, and CCS. Ergo, because on this basis different molecular ion
types have different mobility, ions of the same molecular type as the analyte
should be used in construction of a calibration. For example, salt ions should
most probably not be used to calibrate protein ions. Likewise, because of
differences in CCS and density, unfolded, or denatured protein ions should
not necessarily be used for the calibration of a native-like folded protein and
vice versa. The consequence of this requirement is it frustrates the develop-
ment and use of a single reference standard (in much the same way that CsI
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clusters of different sizes can be used to calibrate a mass spectrum) to calibrate

TWIMS-based CCS determinations. Conceivably, PEG ions, which span a
range of sizes may make good candidate calibrant, but due to differences in
mobility to protein ions, would not be suitable.
Related to the requirement of mobility matching between analyte and
ions used for calibration is the challenge of calibrating for charge-reduced
membrane proteins [76]. While membrane proteins themselves are not
naturally charge-reduced under native IM–MS conditions, in their analysis
they are often subject to charge reduction due to the use of volatile deter-
gents [40–42,77,78]. This charge reduction is especially useful for preserving
the structure of membrane proteins in the gas phase, as enhanced activation
(relative to soluble proteins) is needed to remove bound detergent [42]. This
effect pushes these ions into a different mobility space than the soluble
proteins that are used in the creation of calibrations. Significantly, it has been
shown that forming the calibration with proteins of higher mass than the
protein of interest can generate an accurate calibration, however it is impos-
sible to know the accuracy a priori [76].
5. USING IM–MS TO STUDY PROTEIN FOLDING IN THE

GAS PHASE
Considering that IM–MS can be used to monitor the CCS, and thus
the overall conformation of a protein ion under a variety of conditions, it is
no surprise that it has become a method of choice for protein folding studies.
Although protein structure analysis by IM–MS has yielded numerous
important insights into biological processes, it is crucial to remember that
IM–MS data reflect gas-phase conformations of proteins, and therefore,
the impact of desolvation has to be considered when extrapolating to
solution-phase behaviour.
In line with this consideration, the first protein folding studies by
IM–MS focused on gas-phase conformations of proteins. In one of the ear-
liest native MS studies using IM, Clemmer and coworkers revealed a close
correlation between ion charge and CCS. They showed that ubiquitin ions
with a low number of charges preferentially retained compact conformations
with a CCS close to that of the crystal structure, while more highly charged
ions possessed structures better represented by elongated conformations
[79]. Similarly, they were able to demonstrate that extensive trapping of
gaseous proteins prior to IM resulted in progressive loss of compact
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conformers and unfolding, the rate of which again turned out to be charge-
state dependent [80].
Analogous to unfolding through extended trapping times, protein ions
can be thermally activated in an ion trap or collision cell prior to IM by
increasing the collision voltage. Here, raising the collision or trap voltages
increases ion acceleration and leads to inelastic collisions with residual
buffer gas. The collisions increase the internal energy of the protein ion
and dissociate its internal noncovalent interactions, thus breaking up the
native tertiary structure and facilitating rearrangement of the polypeptide
chain into new conformations. These processes are accompanied by a
change in CCS of the ion, which was demonstrated by Jarrold and
coworkers by plotting the CCS as a function of collision voltage for individ-
ual charge states [81,82]. Interestingly, the resulting CIU plots contain
multiple unfolding intermediates, which have been suggested to stem from
unfolding events of individual (sub)domains [83]. The complexity of
unfolding plots for larger proteins has been used to ‘fingerprint’ isobaric
species such as antibodies, where differences in the disulphide bond pattern
amount to notable differences in their CIU plots [84,85].
Moving beyond the application of IM–MS to investigate the physical
properties of individual protein ions, the approach has yielded valuable
insights into the structures of protein complexes. In an early study, Loo
and coworkers used CCS measurements to observe a change in the 20S
proteasome structure upon addition of an unfolded protein substrate [86].
The group of Carol Robinson developed an IM–MS-based strategy to study
supramolecular assemblies of the trp RNA-binding protein. By comparing
the CCSs of the ring-shaped trap protein complexes calculated from crys-
tallographic data and MD simulations to the drift times for each charge state
of the complex in IM–MS, they were able to demonstrate that the size of
the lower charge states corresponds to the intact ring, while higher charges
had collapsed structures [87]. Through collisional activation of the intact
complexes prior to IM analysis, it is possible to unravel the relationship
between protein folding and complex stability. For example, studies on
the transthyretin tetramer revealed that individual subunits unfold prior to
dissociation from the complex [88].
In recent years, the combination of CIU and native MS has been
extended to protein–ligand complexes. Since ligand binding is often accom-
panied by the formation of additional charge interactions or hydrogen
bonds, this also results in changes in the CIU profile of the protein–ligand
complex. Interestingly, similar effects have been observed for specific [89]
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and nonspecific [90] ligand binding, as well as for cation and anion adducts
[91–93]. Hence, the mere degree of gas-phase stabilisation imparted by a
ligand cannot be used as a proxy for interaction specificity. It can, however,
inform about structural incorporation of a ligand into a protein or protein
complex. This concept has been exploited to study binding of phospholipids
to membrane proteins in the gas phase. Laganowsky and coworkers have
shown that detergent-solubilised membrane protein complexes exhibit a
surprising selectivity towards lipids that give rise to significant stabilisation
against CIU [41,94,95]. These lipids are easily incorporated into the deter-
gent micelles surrounding solubilised membrane proteins, where they com-
pete with detergent molecules for lipid-binding sites to yield protein–lipid
complexes that can be monitored by MS [96]. IM–MS and MD simulations
of closely related membrane transporters revealed that the most broadly
stabilising lipids are preferentially incorporated into grooves and clefts in
the protein structure where they strengthen intersubunit contacts and
reduce gas-phase unfolding [97].
6. COMPLEMENTARY AND SUPPORTING

COMPUTATIONAL RESOURCES
Computational tools play a crucial role in many techniques used in
structural biology. These can be classified into tools that are used for data
processing, for example the processing of images in X-ray crystallography
to cryo-EM, and those that are used for analysis, such as the various visual-
isation tools for protein structure. Another important class of computational
tools are those specialising in theoretical calculations, for example those
used in molecular modelling and molecular dynamics simulations. These
classifications represent a common approach to solving structural biology
problems, where a complete structural model is often achieved through
combining structural information attained both experimentally and theoret-
ically. For the use of IM in structural biology this approach is no different,
with various software tools providing theoretical calculation capabilities, and
others which are used for the processing and analysis of IM–mass spectra.
The use and development of IM for structural biology have been critically
supported by the coemergence of these software tools.
6.1 Theoretical CCS Calculations

In the simplest sense, a common desire is to compare an experimentally
determined CCS for a protein ion with that expected from a particular struc-
tural form of a protein. This has many uses, for example determining if the
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protein form observed in an IM–MS experiment is representative of a

native-like structure of the protein. Alternatively, this could be used in
the context of comparing models derived in silico of a protein complex
to an experimentally determined CCS to ascertain whether they may be
accurate. To do this comparison requires a method to theoretically calculate
CCS from structural models of proteins.
Fortunately, theoretical calculations of CCS from structural models are
possible, and there is a variety of different algorithms for performing this
calculation [98–101]. These include the trajectory method, elastic hard
sphere scattering, projection approximation, and projected superposition
approximation. Each method has its own requirements, computational cost,
and practical relevance to the molecule being analysed. This is particularly
the case for proteins, where because of the size of these molecules approx-
imations can be made (such as ignoring gas scattering effects and long-range
interactions) that ultimately only modestly affect the accuracy of the
resulting CCS. This is useful because this lowers the computational cost;
the methods incorporating these parameters do not scale well to molecules
the size of proteins [10,102]. As such, for protein calculations, the favoured
methodology has frequently been the projection approximation.
In the case of molecular modelling of protein complexes (Fig. 6), many
thousands of models can be generated in the process. While the computa-
tionally cheaper algorithms are therefore useful when there is a need to
Fig. 6 Theoretical CCS calculations can be used to match putative models of protein
complexes to experimentally derived CCS. In these instances, the experimental CCS
acts as a filter to select plausible models that fit the observed CCS of the protein com-
plex. In many cases, thousands of different models may be generated, so efficient
methods to calculate theoretical CCSs from models are advantageous to reduce the
computational cost.
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calculate many thousands of CCSs from different models, the speed of the
implementation, even of the relatively fast projection approximation, begins
to play an important role. For example, recently >100,000 different models
of lipid binding to membrane proteins were generated and for every protein
and lipid combination, the CCS was computed [41]. At this scale, not only
must the method be fast but the implementation also. Uses of CCS like this,
and the requirement to compute so many CCS calculations have helped to
spur the development of new and faster implementations of the algorithms
for computing theoretical CCS [102,103]. These tools (Table 1) will now
make it easier to use IM-based restraints in computational modelling
projects.
It is important to note that some implementations of these algorithms
incorporate statistical sampling from distributions of parameters. Therefore,
the CCS calculated using such approaches is reproducible only within
statistical limits and has an associated standard deviation. It is important when
using theoretical CCS values to perform the calculations multiple times, and
to be aware of the magnitude of the error in the value relative to the pre-
cision required.
The structural rearrangements that proteins undergo in transfer to the gas
phase are also relevant for interpretation of CCS calculated theoretically and
obtained experimentally. Assuming that the level of activation necessary to
promote active unfolding of a protein in the gas phase is not present [112], an
inevitable consequence of this transfer to vacuum is that the protein structure
will collapse, to some varying degree, in the gas phase. As has been already
described, this undoubtedly will include the side chains on the surface of a
protein, but also any regions of proteins which have large holes (such as those
in pore-like proteins), and proteins with extended regions also risk adopting
energetically more favourable compact conformations (so-called collapsed
states). Of note, the CCS of a collapsed protein form that has been activated
to then undergo CIU may be coincident with the CCS of that expected for a
native-like form [113], so care must always be taken to try to rule out this
possibility. Some of the more general features of the structural
rearrangements that occur to proteins are taken into account by an empirical
correction factor that converts theoretical CCSs obtained from crystal struc-
tures, computational models [97,114], or EM data [114] into those that
match experimentally measured CCS [115]. This correction factor is
thought to be compensating for the effects of side chain collapse on the
surfaces of proteins.
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Table 1 Community-Developed Software Tools Are an Important Contribution to the

Use of Ion Mobility in Structural Biology
Software Category Features
PULSAR [94] IM IM viewing; mass spectrum deconvolution; unfolding
fingerprints; arrival time distribution extraction; CCS
calibration; stability analysis
Amphitrite [104] IM IM viewing; mass spectrum deconvolution; arrival
time distribution extraction; CCS calibration
Benthesikyme IM Automatic fingerprint generation
[105]
CIUSuite [106] IM IM viewing; unfolding fingerprint generation,
analysis, and feature detection; stability analysis
UniDec 2D IM IM viewing; 2D mass spectrum deconvolution
[107]
Collidoscope CCS TM
[103]
Impact [102] CCS PA, calibrated TM
Mobcal [98] CCS TM, EHSS, PA
ImOS [108] CCS TM, EHSS, PA
EHSSrot [109] CCS EHSS
Sigma [110] CCS PA
CCSCalc [111] CCS PA
These software include those for processing and analysis of experimental IM spectra, and for the calcu-
lation of theoretical CCSs from structural models.
EHSS, exact hard-sphere scattering; PA, projection approximation; TM, trajectory model.
6.2 Software for Analysis of IM–Mass Spectra

The coupling of IM to MS, and the additional dimension of separation it
adds, requires advanced software tools to visualise, extract, and analyse
the detected signals. In particular, software has been useful in implementing
methods to extract arrival times associated with a articular ion, fitting of the
peaks observed in the arrival time, and the tools to calculate and calibrate
CCS from the measurements (Table 1) [94,104–107]. These software tools
in some instances have been developed for specific studies, but have since
through their general utility become to exist in their own right. Owing
to the volume of data that IM–MS experiments can generate, these tools
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have enabled through workflow improvements analyses that were not pre-
viously practical. The visualisations they provide also help to illuminate
aspects of protein ion behaviour in the gas phase, which has important fun-
damental implications for practitioners to understand, and in the application
of IM for structural biology.
IM–mass spectra viewing and some analysis are supported by vendor
software and community-developed software. Vendor software typically
provides the tools for viewing mobiligrams (graphs of arrival time vs m/z;
such as DriftScope from Waters) but is now also providing tools for CCS
calculation or calibration (for example, Agilent MassHunter IMMS Browser
and Tofwerk Tofware). Within the native MS community, individual
research groups have developed their own tools that perform these, and
many other more specialised tasks. This has been for two reasons: first,
because the vendor tools at the time did not exist, and second because
the implemented tools were critical for the development of new analytical
techniques, impractical, and too specialised for vendors to support.
An area where custom researcher-developed software for IM analysis has
focused is in the area of ‘activated’ MS and the analysis of CIU data. These
software typically implement tools to enable the generation of differences
between CIU plots or fingerprints, and also ways in which to analyse for
differences in transition points between different structural forms of proteins
as they are observed to unfold in the gas phase. These tools automate many
aspects of the analysis processes and are beginning to address the foreseeable
demands for reproducible quantification in these new types of IM-based
assays. The cumulative effect is that these new software tools are opening
new avenues to discovery.
7. IM FOR SEPARATION
In this chapter, so far we have talked about how IM is used in struc-
tural biology to measure the CCS of protein ions but this is not the only use
of IM for structural biology investigations in the gas phase. While MS sep-
arates based on mass and charge IM also separates, but in this case based on
mobility, which in turn depends on CCS and charge. This separating ability
is complementary to that provided by MS, and it has been exploited in sev-
eral different ways in MS experiments.
Often mass spectra can be difficult to assign, especially when charge-state
series deriving from many different species are present. Assignment can be
particularly challenging when the charge-state envelopes for different
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species have overlapping signals at the same mass-to-charge values. In these

cases, the power of orthogonal mobility separation can dramatically assist
identifying charge-state series, and assigning a mass spectrum. In these cases,
the approach is to extract a two-dimensional area from the mobility space (an
area bounded in both m/z and arrival time), and to then calculate the
corresponding mass spectrum by summing across the arrival time dimension,
as illustrated in Fig. 7. In surface-induced dissociation (SID) experiments,
symmetric dissociation is common and can predominate, which leads to
charge-state series with signals that overlap at the same m/z values [116].
Mobility separation here can also provide a means to help assign spectra.
Fig. 7 Orthogonal mobility separation can be used to ‘clean-up’ a mass spectrum.

Exploiting the different mobilities of species observed in a mass spectrum, individual
regions can be isolated in mobility-space and the corresponding mass spectrum
regenerated. In this example peaks corresponding to a protein charge-state series
can be isolated from the overlapping presence of detergent clusters in the spectrum,
revealing a true intensity baseline.
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Wysocki and others have used this to help assign mass spectra of designed
protein cages, where SID was used to interrogate and confirm the interfacial
design of the complexes [117].
In the case of membrane proteins, mobility separation can also help to
clean-up spectra. Membrane proteins are most commonly analysed by
MS encapsulated in detergent micelles, which means empty detergent
micelles are present in the sample being analysed and often show in the mass
spectrum (Fig. 7). When the proteomicelle is disrupted and the detergent
molecules removed from the protein in the gas phase, signals corresponding
to these detergent molecules appear at low m/z. These signals do not overlap
with the signals from the protein of interest. However, also present are
empty detergent micelles, and often these are not dissociated to low mass
species, and will appear as a smear in mobility space, and a large background
signal in the corresponding mass spectrum. This can decrease the relative
intensity of obscure peaks corresponding to the protein of interest. Mobility
selection for just the region of the IM–mass spectrum in which the protein
peaks reside substantially improves spectral quality, and can reveal protein
signals of greater intensity, devoid of background signal. This is an especially
useful strategy to apply in cases where quantification of protein signal
intensity is performed.
Mobility separation can also be used in combination with collision-
induced dissociation (CID) to deconvolute and thereby measure the stoichi-
ometries and CCSs of polydisperse proteins [118]. In the m/z domain, the
signals for these polydisperse proteins overlap meaning that without disso-
ciation the underlying stoichiometries are hidden. However, performing
CID before IM would result in an inability to record the arrival time infor-
mation and therefore calculate the CCS of the intact assemblies. Taking
advantage of instrument architecture where CID can be invoked after IM
separation allows the arrival time of the precursor ion to be determined
directly from the CID products: the dissociation products will have the same
arrival time as the parents from which they were dissociated. This is a
useful and innovative use of the orthogonal means of separation that IM pro-
vides to MS.
8. COMBINING IM–MS WITH OTHER STRUCTURE

ANALYSIS METHODS
As outlined in Section 1, the biological function of a protein is a com-
plex picture that spans from the molecular to the cellular level. Its basic
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building blocks are the sequence and fold of the individual protein compo-
nents, which govern local conformational dynamics and quaternary interac-
tions that in turn control the actions in a biological context. The use of
IM–MS as described here is a powerful means of probing the relationship
between protein folding and interactions, but as with all structural biology
methods, its use as a stand-alone method does not usually provide a full
account of a protein’s molecular function. Therefore, IM–MS is often used
as a complementary approach with other non-MS methods to create
‘hybrid’ structural biology strategies [5]. From the wealth of important
studies that include IM–MS, we have selected a few examples from small
to large protein systems to highlight different contexts in which its specific
qualities can be harnessed.
The structural determinants of amyloid formation are the focus of intense
research and may hold clues to the treatment of diseases such as Alzheimer’s
and diabetes [119]. However, aggregation intermediates formed by
amyloidogenic peptides are short-lived and heterogeneous. Pagel and
coworkers have used the resolving power of IM–MS to facilitate structural
analysis of amyloid oligomers. By measuring the CCS of each oligomer
population, they were able to identify anisotropic species that represent early
aggregation intermediates, and isolate these for secondary structure analysis
by gas-phase infrared spectroscopy [120,121]. Their findings demonstrate
that the formation of transient β-turn motifs across a range of oligomer
stoichiometries is one of the earliest steps in amyloid formation.
IDPs pose similar problems to structural biologists. Although implicated
in a number of important cellular processes, their extreme conformational
flexibility makes them poorly suited for most structure determination
approaches. The group of Perdita Barran has used IM–MS extensively to
understand the conformational space sampled by IDPs. A direct comparison
of the two IDPs, α-synuclein and apolipoprotein CII, using IM–MS and
hydrogen/deuterium exchange (HDX)–MS revealed that despite a lack of
discernible secondary structures in both cases, the latter protein preferen-
tially adopts more compact conformations in the gas phase [122]. Based
on these and similar findings, Borysik and coworkers combined IM–MS
with SAXS to reveal that the unique electrostatic landscapes of different
IDPs give rise to distinct collapsed states in the gas phase that are not
observed in solution [123]. Although the gas-phase conformations of IDPs
do therefore not necessarily represent exclusively physiological relevant
solution structures, IM–MS reveals that IDP sequences encode subtle
conformational preferences.
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Beyond its ability to capture heterogeneous and short-lived folding

states, IM–MS can also be employed to analyse dynamic changes in the
architecture of protein complexes. One such example is the allosteric inhi-
bition of phosphoribosyltransferase (PRT) by histidine. Pacholarz et al. used
native MS to show that the oligomeric state of the 189 kDa PRT hexamer is
regulated by pH, while IM–MS and analytical ultracentrifugation revealed
that binding of one histidine molecule per subunit induces global compac-
tion of the hexamer. HDX–MS was then used to identify how histidine
binding translates to tightening of the active site and enzymatic inhibition
by steric hindrance [124].
Perhaps the most well-known application of IM–MS is its ability to gen-
erate constraints for structural modelling of protein complexes. Here, olig-
omeric protein assemblies are subjected to IM–MS to generate CCS values
for all observable stoichiometries. Using crystallographic data for individual
subunits, multiple hypothetical architectures are generated, filtered, and
refined using the experimentally determined CCS values as constraints to
yield low-resolution models (reviewed in [125]). This approach has been
successfully applied to homooligomers such as small heat-shock chaperones
[114], as well as heterogeneous assemblies with multiple components
[126,127].
IM–MS has also been employed to facilitate structural analysis of large
protein complexes by electron microscopy (EM). A simple example is the
structure of the Toll-like receptor in complex with a dimeric ligand, where
the combination of native MS and IM separation was employed to reveal an
unexpected 2:2 stoichiometry that enabled interpretation of the low-
resolution maps obtained by EM [128]. More recently, cryo-EM analysis
of the CRISPR interference complex CSM revealed an elongated mul-
tiprotein structure of unknown subunit stoichiometry and connectivity.
Having established that the complex contained several directly connected
subunits, Rouillon et al. used IM–MS to show that these subunits indeed
assemble into rod-like structures that could be fitted into the EM densities
[129]. Given the demonstrated ability of IM–MS to generate such con-
straints for fitting EM structures, Degiacomi and Benesch developed the
EM\IM software package, which allows for the determination of theoret-
ical CCS values from density maps [130]. Moving even further towards the
macromolecular architectures of whole organisms, Albert Heck and
coworkers used IM–MS to elucidate the assembly of viral capsid proteins.
By comparing different oligomers assembled from individual crystal struc-
tures to CCS values obtained by IM–MS, they were able to define a set
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of sheet-like assembly intermediates that matched the connectivity pattern

observed in the EM structures of complete capsids [131]. Lastly, going
wholly towards in vivo analysis, the group of Michal Sharon has developed
a simple strategy for IM–MS analysis of intact protein complexes from
whole-cell lysates, revealing an overall close match of their CCS in crude
lysates and as purified proteins [132]. Although currently limited to bacterial
overexpression systems and buffer changes, this approach may be combined
with new ionisation strategies for nonvolatile buffers [133] and holds prom-
ise for IM measurements of native complexes in the presence of their native
solvent and interaction partners.
9. CONCLUSIONS
In this chapter we have given an overview of how IM is used in struc-
tural biology, spanning from technical descriptions of the approach to exam-
ples of the applications of the technique from small to large protein systems.
Like all advanced techniques, practitioners should have a thorough under-
standing of ‘how it works’. As IM–MS further develops, meeting this need is
the important work of many towards understanding the behaviour of pro-
teins in the gas phase. This fundamental knowledge is critically important for
guiding and placing appropriate limits on the interpretation of data, and as
further evidence towards the robustness of the technique.
Many advancements on the horizon in IM–MS for structural biology are
particularly exciting. New IM devices we have mentioned that are capable
of yielding substantial increases in resolution will enable new and very
detailed insight into the structure and dynamics of protein complexes. These
instrumental improvements will no doubt be matched by further develop-
ment of the various software that are used to analyse IM data, leading to new
methodologies. The new insights that will be generated will be important
and useful for integrative modelling approaches, and it seems likely that
the complementary role of IM to other structural biology methods will only
strengthen.
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Ion Mobility in Structural Biology

Comprehensive Analytical Chemistry # 2019 Elsevier B.V. 1

2 Timothy M. Allison and Michael Landreh

structures of protein complexes. While both of these techniques focus at the

Ion Mobility in Structural Biology 3

fields of omics, such as lipidomics, glycomics, metabolomics, and proteo-

4 Timothy M. Allison and Michael Landreh

2. STRUCTURAL ASPECTS OF PROTEINS IN THE

Ion Mobility in Structural Biology 5

and eventually fission due to repulsive forces. This process is repeated

6 Timothy M. Allison and Michael Landreh

interactions or ‘cross-links’ may help to preserve an overall native-like

Ion Mobility in Structural Biology 7

The timescale for a native MS experiment using a standard quadrupole-

8 Timothy M. Allison and Michael Landreh

3.1 Established IM Types and Platforms

3.1.1 Drift Tube IMS

Ion Mobility in Structural Biology 9

10 Timothy M. Allison and Michael Landreh

widths in the mobility domain. This is not exclusive to DTIMS of course

3.1.2 Travelling-Wave IMS

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3.2 Emerging Technologies

4. IM FOR CCS DETERMINATION

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protein, akin to its volume or radius of gyration as measured in SAXS exper-

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4.1 Common Approaches to Measuring CCS

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a CCS through calibration, the literature CCSs measured in a different gas

4.2 Considerations of Accuracy in CCS Determination

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clusters of different sizes can be used to calibrate a mass spectrum) to calibrate

5. USING IM–MS TO STUDY PROTEIN FOLDING IN THE

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18 Timothy M. Allison and Michael Landreh

6. COMPLEMENTARY AND SUPPORTING

6.1 Theoretical CCS Calculations

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protein form observed in an IM–MS experiment is representative of a

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Table 1 Community-Developed Software Tools Are an Important Contribution to the

6.2 Software for Analysis of IM–Mass Spectra

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species have overlapping signals at the same mass-to-charge values. In these

Fig. 7 Orthogonal mobility separation can be used to ‘clean-up’ a mass spectrum.

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8. COMBINING IM–MS WITH OTHER STRUCTURE

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26 Timothy M. Allison and Michael Landreh

Beyond its ability to capture heterogeneous and short-lived folding

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of sheet-like assembly intermediates that matched the connectivity pattern

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[3] D.A. Jacques, J. Trewhella, Small-angle scattering for structural biology—expanding

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