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Cite This: Anal. Chem. 2019, 91, 7554−7561 pubs.acs.org/ac

Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility

Spectrometry Traveling Wave Device
Charles Eldrid,† Jakub Ujma,‡ Symeon Kalfas,† Nick Tomczyk,‡ Kevin Giles,‡ Mike Morris,‡
and Konstantinos Thalassinos*,†,§
†
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, WC1E 6BT, United
Kingdom
‡
Waters Corporation, Wilmslow, SK9 4AX, United Kingdom
§
Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, WC1E 7HX, United Kingdom
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

*
S Supporting Information

ABSTRACT: Ion mobility mass spectrometry (IM-MS)

allows separation of native protein ions into “conformational
Downloaded via 109.136.230.168 on January 2, 2021 at 17:08:46 (UTC).

families”. Increasing the IM resolving power should allow finer

structural information to be obtained and can be achieved by
increasing the length of the IM separator. This, however,
increases the time that protein ions spend in the gas phase and
previous experiments have shown that the initial conforma-
tions of small proteins can be lost within tens of milliseconds.
Here, we report on investigations of protein ion stability using
a multipass traveling wave (TW) cyclic IM (cIM) device.
Using this device, minimal structural changes were observed
for Cytochrome C after hundreds of milliseconds, while no
changes were observed for a larger multimeric complex
(Concanavalin A). The geometry of the instrument (Q-cIM-
ToF) also enables complex tandem IM experiments to be performed, which were used to obtain more detailed collision-induced
unfolding pathways for Cytochrome C. The instrument geometry provides unique capabilities with the potential to expand the
field of protein analysis via IM-MS.

T he invention of soft-ionization techniques1−3 has allowed

the transfer of intact biomolecules and proteins into the
gas phase.4−6 Early electrospray mass spectrometry experi-
dynamics of disordered proteins.15,16 Deliberate activation of
ions through increasing the internal energy via collisional
heating can cause unfolding,17 which can provide important
ments (ESI-MS) showed pronounced differences in charge information on the structural stability of proteins under
state distribution depending on the solution conditions, different conditions or with different states, the effect of
suggesting that solution phase protein structure can be probed
modifications or ligands on protein structure and dynam-
using ESI-MS.7 In their landmark work, Clemmer and Jarrold
constructed an ESI−ion mobility−mass spectrometer (ESI- ics,16,18,19 and the subunit organization of oligomers.19,20 This
IM-MS), which revealed that a single charge state of a protein can also be applied to protein complexes to investigate
can be present in a range of conformations.8 This sparked a oligomerization pathways and subunit organization of com-
significant interest in studies of proteins in the gas phase. IM- plexes.12,21−24
MS has the advantage of being able to detect multiple and IM-MS functions by introducing analyte ions into a drift-cell
lowly populated conformational ensembles from small sample containing an inert buffer gas such as helium or nitrogen. Ions
volumes, which are comparable to those seen in solution when drift through the cell under the influence of an electric field
the ions have a low internal energy (Eint).9 Being able to detect and collide with buffer gas molecules. Drift velocity of ions is
these conformational states provides information on protein governed by their mobility which is inversely proportional to
folding dynamics and requires far lower sample concentrations
their collision cross-section (CCS). Species that are extended
when compared to other structural techniques such as X-ray
crystallography and NMR. Introduction of the first mainstream (larger CCS) will undergo a greater number of collisions with
commercial IM-MS instrumentation (Synapt HDMS, Waters the buffer gas and have a longer drift time compared to more
Corporation, 2006)10 accelerated implementation of the
technique in protein studies. IM-MS has been used to show Received: December 7, 2018
the basic dynamic behavior of proteins,11−13 protein domain Accepted: May 22, 2019
organization,14 and to identify and investigate the structural Published: May 22, 2019

© 2019 American Chemical Society 7554 DOI: 10.1021/acs.analchem.8b05641

Anal. Chem. 2019, 91, 7554−7561
Analytical Chemistry Article

compact species (smaller CCS). Mass to charge ratios (m/z) that solution specific conformations transform to new gas
are then measured for each ion by a mass spectrometer. phase structures in the absence of solvent after a period of 30
Increasing the resolution of the IM device should, in ms. More recently, Allen et al. have utilized a SLIM-based
principle, enable separation of overlapping features, allowing tandem IM instrument45 to study the Cytochrome C ions. It
greater understanding of protein structures in the gas phase. was shown that structural changes occurred an order of
The resolution of the IM device depends on the temperature, magnitude later than reported previously. This was attributed
ion charge, electric field, and the path length.25 There are primarily to pressure-related differences in effective ion
several examples in the literature where attempts were made to temperatures46 in the QIT and tandem IM systems and also
increase the resolution of the IM apparatus by decreasing the solution-dependent effects, further confirmed by collision-
temperature,26,27 increasing the electric field,28 and increasing induced unfolding (CIU) experiments.45 Collectively, the
the path length.29,30 Conventionally, the latter is a physical previous work suggests that IM separation time scales
distance that ions travel, and thus, a number of several meters appropriate for native protein ions is likely to be on the
long drift tube (DT) type instruments have been realized.31−33 order of tens to hundreds of milliseconds, especially for larger
There are practical limits in this approach related to the ions.
physical size of the apparatus and high voltages required. Importantly, Badman et al. utilized a relatively short, linear
Alternatively, an experimental setup with inverted “frame of field DT device, where transit times are on the order of tens of
reference” can be envisaged, where the ions are trapped in a milliseconds, comparable to trapping times in the QIT.
stream of moving gas by an opposing electric field, as in the Moreover, due to a relatively low velocity of ions in such
case of trapped ion mobility spectrometry technique devices, their effective temperatures are essentially equal to
(TIMS).34 Here, the “effective path length” can be influenced that of the buffer gas. The latter is not necessarily the case in
by a separation time scale and gas velocity. Another way of the T-Wave based IM separation. It was reported previously
achieving a long separation path is to utilize multipass devices. that some ion heating during the T-Wave based IM separation
The cyclotron mobility spectrometer described by Glaskin et can cause structure perturbation, the effect being especially
al. uses a drift cell made of four curved segments that are pronounced for low molecular weight, high mobility
joined by ion funnels,35 which refocus ions.29 An electric field species.47,48 The native protein ions typically have relatively
applied to subsequent segments/funnels is switched; only the low charge and high mass, thus, representing a contrasting
ions with mobilities resonant with the field switching frequency case, in principle. Nevertheless, it is of interest and of
can proceed to the following segments, while others are lost. importance to evaluate the extent of native structure
An IM spectrum is obtained by scanning the field switching perturbation upon prolonged exposure to T-Waves, partic-
frequency. Several developments of high resolution IM ularly in relation to the possibility of high-resolution IM
instrumentation have been facilitated by traveling wave (T- separations in the future.
Wave, TW) technology,36 which relies on a series of voltage Here, we utilize a prototype cIM-MS instrument enabling
pulses that propel ions across the device. T-Wave technology custom experiments designed to further explore the stability of
eliminates problems related to high voltages, required for the monomeric (Cytochrome C, β-Lactoglobulin) and multimeric
traditional, linear field DT-IM apparatus. A T-Wave based, (Concanavalin A) proteins, in a T-Wave based separator. Ions
multipass cyclic IM (cIM) separator was first introduced by were subjected to trapping in a region of the cIM device, and
Giles et al.,37 and separation at a path length over 50 m was native conformations were found to be stable for hundreds of
demonstrated.38 Further advances in a T-Wave technology milliseconds. Additional experiments were also performed to
include structures for lossless ion manipulation (SLIM), show that extended time within the cIM device under typical
notably by Smith et al.39 A multipass SLIM device has allowed separation conditions does not significantly impact protein
IM separations over an extremely long path length (∼1 km).40 structure in the gas phase. IMS-CIU-IMS experiments revealed
Other recent developments in IM technology include tandem detailed unfolding pathways for Cytochrome C. Our results
methods. A two-stage IM technique was first presented by agree with previous reports suggesting that the native-like
Koeniger et al.,32,41 where an instrument featuring two drift conformation or proteins is maintained in the gas phase under
tubes allowed separation in the first IM stage, mobility extended time scales and, for the first time, show that this is
selection, activation, and separation of product ions in the also true within a cIM device. Collectively, our data show that
second stage. This was further expanded to a three-stage IM the cIM instrument can be used for studying protein dynamics,
method by Merenbloom et al.31 More recently, the multistage stability, and unfolding in the gas phase.
IM technology was combined with a multipass cyclic IM
separator by Giles et al.,38 allowing for IMSn-type workflows.
Increasing the resolution of IM separation via path length
■ METHODS
Sample Preparation. The proteins (equine Cytochrome
typically increases experiment time scales. Understandably, C (Merck Millipore, U.K.), β-lactoglobulin (Sigma, U.K.), and
changes in the nature of analyte ions occurring on the time Concanavalin A (Sigma, U.K.)) were buffer exchanged into
scale of separation are undesirable. Previous work by Badman 200 mM ammonium acetate solution using 3, 10, or 30 kDa
et al. showed that Cytochrome C ions underwent structural Amicon Ultra 0.5 mL centrifugal spin filters (Merck Millipore,
changes with time.42,43 Utilizing a quadrupole ion trap (QIT)- U.K.). The samples were spun a total of three times at 12000
IM-MS instrument, ions were stored for varying amounts of rpm for 15 min at room temperature. The protein was then
time in the QIT, prior the IM-MS measurement. The diluted to 8−10 μM after concentration calculation using a
structural changes started occurring at approximately 30 ms Qubit protein assay (ThermoFisher Scientific, U.K.).
and had stabilized after 60 ms, showing that the initial Mass Spectrometry. The samples were introduced into
population of +7 to +10 ions contains precursors or the instrument using a nano-ESI source (Waters Corp.,
conformational intermediates that unfold in the gas phase.42 Wilmslow, U.K.). The emitters (manufactured using a P97
Similar results were reported for ubiquitin.44 It was suggested Flaming/Brown micropipette puller, coated in gold using a
7555 DOI: 10.1021/acs.analchem.8b05641
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Analytical Chemistry Article

Figure 1. (A) Instrument schematic showing the Q-cIM-ToF geometry; (B) Cartoon showing the orthogonal arrangement of the cyclic IMS and
neighboring optics; (C) Multifunction region.

Quorum Q150R S sputter coater) were held at 1.2 kV. The high-pressure cIM device. First, in the “trapping” mode ions
cIM instrument design is discussed in detail elsewhere49,50 and are stored in the array for a prolonged period of time, before
so will only be briefly covered here. The instrument schematic IM separation takes place (Figure S2). This mode enables the
is presented in Figure 1. Ions are transferred from the source time-resolved assessment of the protein conformation stability
through the first vacuum stages using ion guide arrangements in the absence of any (intentional) activation. Second, the
(StepWave), which propel ions toward the quadrupole mass “spinning” mode was designed to verify the effect of ion
filter. The subsequent trap cell is used for accumulating ions heating during the T-Wave-based IM separation (Figure S3).
prior to IM separation. The resulting ion packets are then Normally, the number of passes around the cIM (and, hence,
transported through an ion guide (IG) and injected into a separation time) is limited by so-called wrap-around, a
helium cell. In this work we utilize the injection energy into the phenomenon where the fastest ions ultimately catch up with
He cell to generate some of the collision induced unfolding the slowest ones.50 To extend the exposure to T-Waves, ions
(CIU) data. The subsequent ion guide (prestore) transports are passed around the cIM device for varying amounts of time
the ions into a multifunction array (Figure 1C) of electrodes (allowing wrap-around) before being recollected in the
forming part of an orthogonal closed loop, the cIM separator prestore and subjected to another IM separation before
(98 cm path length, single pass resolving power (RP) of ∼65 detection. It should be noted that while the ions are being
(CCS/ΔCCS) measured using the inverse-sequence peptide manipulated in the cIM device, the next set of ions are
pair (SDGRG and GRGDS).50 The cIM chamber is filled continually being accumulated in the trap region (∼10−2 mbar
directly with nitrogen to a pressure of ∼2.2 mbar (including of N2) of the instrument. Consequently, at the start of the cIM
some contribution from helium gas leaking in from the He manipulation, the ions will have an average gas-phase lifetime
cell) and the T-wave height was 40 V. The T-Wave direction in of around half of the cIM experiment time. To minimize
the array can be altered to either match those in the cIM activation of ions entering the cIM and retain low-energy
device (i.e., separate) or to inject/eject ions from it. The conformations, generally the injection voltage into the He cell
control software GUI enables creation of custom sequences of is kept as low as possible while still maintaining reasonable
functions to facilitate selective ejection of ions from the cyclic transmission. Experiments to help elucidate the effect of ion
IM device and/or activation followed by further separation of storage in the trap region alone were carried out (see Figures
product ions.50 The typical sequence of events employed in S4 and S5) and indicated no significant activation with time.
single/multipass experiments is presented in Figure S1. Post Lastly, the IMS-CIU-IMS mode of operation is used (Figure
cIM, ions are transported through an ion guide (Post-Store) S6). Here, a mobility-selected ion population can be ejected
and on to the ToF via a segmented quadrupole (XS) transfer and trapped in the prestore, while the remaining ions are
cell. The transfer cell allows activation of mobility separated removed from the cIM. The selected ions are then reinjected
ions. The orthogonal acceleration time-of-flight (oa-ToF) into the array, but with a higher voltage between the two
features an offset V geometry allowing m/z measurements at regions to induce activation. This way, we can probe unfolding
resolutions in excess of 60000 fwhm.38 transitions of selected regions of arrival time space, increasing
In addition to a typical single/multipass operation (Figure the specificity of CIU experiments.18,51 In this work we focus
S1), three custom modes of operation were designed to on the dual stage method; however, multistage experiments
explore protein stability over extended experiment times in the (IMSn) can be performed in an analogous way.
7556 DOI: 10.1021/acs.analchem.8b05641
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Data Analysis. Data were analyzed using Masslynx v4.1

(Waters Corporation) and Driftscope v2.1 (Waters Corpo-
ration). To compare the data, the gross arrival time had the
injection time (10 ms) subtracted to give drift-time. In the case
of trapping experiments, the time spent in the array (0−360
ms) was also subtracted. For spinning, the extended cyclic
motion (0−360 ms) and the reinjection time (45 ms) were
subtracted. CIU fingerprint plots were created using
Benthesikyme.52 Population fitting was performed using in-
house software written in Python 2.7, peak maxima were
identified using the second derivative and manually adjusted to
ensure the same conformational populations were tracked
across the different experiments. During trapping and spinning
experiments the data were aligned according to the most
intense peak to allow the same centroid value for each
Gaussian population for tracking. In the case where a second
conformational population became the maximum peak, the
two most intense peaks were used for alignment. Each
conformational family was approximated by a Gaussian
distribution. The sum of all distributions was optimized to
produce the best fit to the experimental data or trace. No
restraint to the full width half-maximum value of each Gaussian
was imposed like in previous work.52 The code is freely
available at https://github.com/ThalassinosLab/CIVU.

■ RESULTS
Cytochrome C. CytC was analyzed, and a very narrow
charge state distribution was observed, with high abundance of
the +7 charge state (Figure 2A), which was quadrupole
selected for experimentation (1765 m/z). Other charge states
ranging from +8 to +5 could be seen at low abundance.
Minimal activation upon both trapping and spinning experi-
ments was observed for the +7 (Figure 3A−C). Comparing
these structural transitions to the CytC +7 CIU plots (Figure
3C), they are equivalent to less than 5 V of activation. The
time scales of conformational change are similar to previous
studies.45 Figure 2. Mass spectra for the proteins (A) Cytochrome C (CytC),
β-Lactoglobulin. β-Lactoglobulin (βLac) is a 16 kDa (B) β-Lactoglobulin (βLac), and (C) Concanavalin A (ConA). Blue
protein, with several disulfide bridges, that exists as a monomer circle = monomer, green square = dimer, red triangle = tetramer.
and dimer in solution; with the ratio being dependent on
protein concentration and the ionic strength of the solution.53 Increased Resolution. Multipass cIM separation offers
βLac was detected mainly as monomer (Figure 2B), and the increased resolution as a function of the square root of the
charge states +7 to +9 were quadrupole isolated for further number of passes, n (√(nz), where z is the ion charge state).37
analysis (2663, 2294, and 2940 m/z, respectively). The +7 The above experiments have shown that extended time in both
charge state did not display structural changes upon trapping the prestore and, when under T-Wave motion in the cIM, can
or spinning (Figures S8A−C and S11); however, the +8 and cause small conformational changes for some protein ions, but
+9 charges displayed very minimal structural changes over the not others, and this appears to be strongly related to mass and
course of 240 ms (Figures 3D−F, S12, and S13) comparable to charge state. The +7 charge state ions of CytC generated from
10−20 V of intentional activation (Figure S8I). ammonium acetate were subjected to 1−3 passes around the
Concanavalin A. To investigate the effect of prolonged gas cyclic ion guide (Figure 4).
phase exposure on the stability of multimeric complexes we Subjecting the +7 ions to higher resolution separation
analyzed Concanavalin A (ConA), a protein which exists as a revealed some evidence of new features in the broadened ATD
51 kDa dimer or 102 kDa tetramer. The mass spectrum of (Figure 4A−C), however, distinct peaks were not seen. This
ConA contained monomeric, dimeric, and tetrameric species, suggests that the initial width of the protein ATD is due to the
with the most intense peaks belonging to the dimeric states extremely large variety of highly similar conformational
(Figure 2C). The tetrameric +21 charge state was quadrupole families, unresolvable by the cIM separator operating at a
isolated for further analysis (4894 m/z). Here, no structural resolving power of ∼300 (CCS/ΔCCS derived for a +7 ion) at
perturbation upon trapping or spinning (Figure 3H,I, S14) was 3 passes (the peaks of singly charged reserpine, expected to be
observed. In addition, no complex dissociation was observed, diffusion limited, are also shown in Figure 4, for comparison).
apart from under deliberate activation conditions (Figure S9). This, however, is not always the case and is most likely protein,
The CIU experiments revealed that minimal conformational charge state, and solution condition specific. For example, early
changes were observed at up to 50 V activation (Figure 3I). experiments with bovine insulin ions (generated from
7557 DOI: 10.1021/acs.analchem.8b05641
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Figure 3. Trapping (A, D, G), spinning (B, E, H), and CIU (C, F, I) experiments shown for CytC +7 (A−C, purple), βLac +8 (D−F, blue), ConA
+ 21 (G−I, red). Each figure is composed of ATD slices, arranged in increasing experimental increment, i.e., trapping time (ms), from dark to light
shading. Plotted is the drift time against normalized intensity.

denaturing solution) showed that increased number of passes present in initial ATD), appear after the extension of
allows greater resolution of previously unresolvable features conformation δ, between 60−80 V of activation.

■
(Figure S15).
As an alternative approach to probe the presence of DISCUSSION
structural subpopulations in more detail, we used an IMS-
CIU-IMS approach. Our data show that proteins can to a large extent retain their
native and multimeric states over the time scales compatible
IMS-CIU-IMS. An experiment which can be performed on
with high resolution IM separations. Importantly, the effect of
this instrument is multistage IMS (IMSn), where a subset ion
prolonged exposure to T-waves appears, in most cases, similar
population can be selected after IM separation, activated, and
to trapping alone, indicating little structural perturbation
subjected to IM separation again. Due to the geometry of the induced by T-wave based separation itself. In this study we
instrument this can theoretically be done a limitless number of explored time scales up to 360 ms, which would typically
times. Here we will focus our attention on phenomena that can exceed the realistic time scales for separation of protein ions.
be probed in more detail compared to a traditional single stage These time scales (and number of passes around the device)
collision induced unfolding (CIU) analysis. As an example, we are limited by wrap-around in the present setup. The
use the quadrupole isolated, +7 ion of CytC was activated on aforementioned CytC work by Allen et al.,45 explored time
injection to the trap (20 V). The initial ATD is presented in scales up to 33 s, which at the moment is beyond the scope of
Figure 5A. We then use the IMS-CIU-IMS methodology, our work. The magnitude of change observed over several
where the CIU occurs on reinjection to the array from the hundred milliseconds by Allen et al. agrees well with data
prestore, to obtain unfolding profiles of 4 subsets of this initial presented here. This is an order of magnitude longer than
population (B−E). Conformation α, upon activation, directly reported previously by Badman et al.39 It would seem plausible
converts into conformations β, γ, and δ. As longer drift time that this is due to the fact that the quadrupole ion trap (QIT)
conformations are selected, populations β to δ are directly used to retain the protein ions for increments of time operated
accessed. This shows the sequence of unfolding events, at a much lower pressure, approximately 0.0133 mbar42,43
however it cannot be confirmed that this is nonreversible. compared to ∼2.2 mbar in the cIM device and the ∼5 mbar
Two conformations, ε (not directly selected) and ζ (not used in the SLIM based tandem IM. However, the trapping
7558 DOI: 10.1021/acs.analchem.8b05641
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Analytical Chemistry Article

showed that while it does not undergo backbone changes, the

surface residues are rearranged55−57 and it retains fewer salt
bridges when in the gas-phase rather than in solution.58 Gas-
phase salt bridges are thought to play a very important role in
the retention of native or native-like states, due to the much
lower electrostatic permittivity of the vacuum compared to that
of aqueous solution.59−61 Interestingly, subjecting native
protein ions to an increased number of passes around the
cIM device did not resolve overlapping features, suggesting
that the ATDs of native ions consist of a very large number or
even a continuum of conformers.41 This is perhaps not so
surprising if we imagine, for example, a variety of ways in which
solvent exposed residues of the protein can be rearranged
during desolvation. This observation is somewhat parallel with
the previous findings from a study utilizing variable temper-
ature IM instrumentation,26 where only a minimal increase in
resolution was observed for native protein ions at cryogenic
temperatures. Collectively, this suggests that the native protein
ATDs are naturally broad, not due to diffusion or
interconversion, but inherent conformational heterogeneity,
which is consistent with previous findings in the field.41,62 The
above phenomena will be investigated in the future work
utilizing the high resolution capabilities of the instrument
combined with the IMS-CIU-IMS methodology presented
here.

Figure 4. CytC (AmAc) +7 charge state arrival time distribution after

■
*
ASSOCIATED CONTENT
S Supporting Information
multiple passes of the cyclic drift-region 1, 2, and 3 passes in the cIM. The Supporting Information is available free of charge on the
The measured ATD of singly charged reserpine (m/z 609) is shown ACS Publications website at DOI: 10.1021/acs.anal-
to indicate the expected diffusion-limited peak width along with the chem.8b05641.
derived peak for a +7 charge state species.
Detailed instrument information; Stability plots for +7
and +9 βLac; Population tracking for all species; and
region of this instrument operates at similar pressures to the
Mass spectrum for ConA during stability experiments
QIT and our data shows that increasing the time spent in the
(PDF)
trapping cell does not significantly affect ion conformation
(Figure S5). We will attempt to explore this in future work.
The loss of native conformation is less apparent for larger
proteins. For βLac, the lowest charge state observed (+7) did
■ AUTHOR INFORMATION
Corresponding Author
not undergo any detectable change. Higher charge states (+8, *E-mail: [email protected].
+9) were minimally perturbed, and only after 240 ms. This is ORCID
in agreement with previous reports showing that low charge
states are more reflective of the solution conformation of
Charles Eldrid: 0000-0001-5306-3644
proteins.54 No loss of initial conformations was observed for Kevin Giles: 0000-0001-5693-1064
ConA. This suggests that the gas phase longevity of the native Konstantinos Thalassinos: 0000-0001-5072-8428
structures is proportional to the ion mass and, most likely, Author Contributions
inversely proportional to its charge. CytC may indeed be The cyclic ion mobility device was designed and built by K.G.
particularly sensitive to gas phase studies as previous work and J.U. M.M. was involved in scientific discussions around

Figure 5. Slice CIU for an activated +7 CytC ion: (A) arrival time distribution, with the slices which have been removed for further mobility
selection bounded by dotted lines; (B−E) CIU fingerprints for slices 16−17, 19−20, 23−24, 26−27 ms. Populations labeled as α, β, γ, δ, ε, and ζ.

7559 DOI: 10.1021/acs.analchem.8b05641

Anal. Chem. 2019, 91, 7554−7561
Analytical Chemistry Article

this study. N.T. was involved in some of the preliminary (24) Eschweiler, J. D.; Rabuck-Gibbons, J. N.; Tian, Y.; Ruotolo, B.
investigations using the cyclic ion mobility device for this T. Anal. Chem. 2015, 87, 11516−11522.
study. Experiments were carried out by C.E. and J.U. S.K. (25) St. Louis, R. H.; Hill, H. H.; Eiceman, G. A. Crit. Rev. Anal.
wrote the population fitting algorithm and performed data Chem. 1990, 21, 321−355.
analysis. K.T. designed and supervised experiments. The (26) Ujma, J.; Giles, K.; Morris, M.; Barran, P. E. Anal. Chem. 2016,
88, 9469−9478.
manuscript was written through contributions of all authors.
(27) May, J. C.; Russell, D. H. J. Am. Soc. Mass Spectrom. 2011, 22,
Notes 1134−1145.
The authors declare the following competing financial (28) Dugourd, P.; Hudgins, R. R.; Clemmer, D. E.; Jarrold, M. F.
interest(s): Jakub Ujma, Nick Tomczyk, Kevin Giles and Rev. Sci. Instrum. 1997, 68, 1122−1129.
Mike Morris are all employees of Waters Corporation. (29) Glaskin, R. S.; Ewing, M. A.; Clemmer, D. E. Anal. Chem. 2013,

■ ACKNOWLEDGMENTS
C.E. is funded by a BBSRC iCASE Award with Waters BB/
85, 7003−7008.
(30) Merenbloom, S. I.; Glaskin, R. S.; Henson, Z. B.; Clemmer, D.
E. Anal. Chem. 2009, 81, 1482−1487.
(31) Merenbloom, S. I.; Koeniger, S. L.; Valentine, S. J.; Plasencia,
L015382/1. J.U., N.T., K.G., and M.M. are all employees of M. D.; Clemmer, D. E. Anal. Chem. 2006, 78, 2802−2809.
Waters Corporation, which manufactures and sells T-wave IM- (32) Koeniger, S. L.; Merenbloom, S. I.; Valentine, S. J.; Jarrold, M.
MS instruments.

■
F.; Udseth, H. R.; Smith, R. D.; Clemmer, D. E. Anal. Chem. 2006, 78,
4161−4174.
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