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Determination of gas phase protein ion densities via ion mobility analysis with charge reduction

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We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase measurements to be correlated to biomolecular structures in solution, low charge state ions should be analyzed. Further, to determine if different solution conditions give rise to ions of different structure, ions of similar charge state should be compared. Non-denatured protein ion densities are found to be in excellent agreement with non-denatured protein ion densities inferred from prior DMA and drift tube measurements made without charge reduction (all ions with densities in the 0.85-1.10 g cm(-3) range), showing that these ions are not strongly influenced by Coulombic stretching nor by analysis method.
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Cite this:
Phys. Chem. Chem. Phys
., 2011, 13, 21630–21641
Determination of gas phase protein ion densities via ion mobility analysis
with charge reduction
Anne Maißer,
b
Vinay Premnath,
a
Abhimanyu Ghosh,
a
Tuan Anh Nguyen,
c
Michel Attoui
c
and Christopher J. Hogan Jr.*
a
Received 29th June 2011, Accepted 19th October 2011
DOI: 10.1039/c1cp22127b
We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a
differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a
condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C,
lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM
aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge
reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal
concentrations of positive and negative ions. Ions produced by the source collide with and reduce the
charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading
to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to
effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions.
Density inference is made via determination of the mobility diameter, and correspondingly the spherical
equivalent protein volume. Through this approach it is found that for both non-denaturing and
denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of
0.97gcm
3
and 0.86 g cm
3
, respectively. Ions from non-denaturing ESI are found to be slightly more
compact than predicted from the protein crystal structures, suggesting that low charge state protein ions
in the gas phase are slightly denser than their solution conformations. While a slight difference is
detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are
found to be much more dense than those examined previously by drift tube mobility analysis, in which
charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to
non-compact ions in the gas-phase, and suggests that for gas phase measurements to be correlated to
biomolecular structures in solution, low charge state ions should be analyzed. Further, to determine if
different solution conditions give rise to ions of different structure, ions of similar charge state should be
compared. Non-denatured protein ion densities are found to be in excellent agreement with non-
denatured protein ion densities inferred from prior DMA and drift tube measurements made without
charge reduction (all ions with densities in the 0.85–1.10 g cm
3
range), showing that these ions are not
strongly influenced by Coulombic stretching nor by analysis method.
Introduction
Gas phase ion mobility spectrometry (IMS) of large protein
ions produced by electrospray ionization (ESI) is a potential
route for structural characterization of proteins and protein
complexes,
1
and a number of recent studies
2–6
display the
ability of IMS to distinguish large (450 kDa) ions of identical
mass but different gas-phase structure from one another. Most
often, however, it is not the gas-phase structure which is of interest,
but rather the structure in solution. To correlate gas-phase
measurements with protein structure in solution, it is essential
that during the transition from solution to gas phase (the electro-
spray process), the structure of proteins is minimally perturbed.
Early studies
7–9
of biomolecular ion generation via non-denaturing
ESI (ESI in near-neutral pH solution of sufficiently high ionic
strength) clearly demonstrate that non-covalent bonds in proteins
are preserved during the electrospray process, permitting analysis
of intact protein complexes. Similarly, many mobility measure-
ments of protein ions
10–11
introduced into the gas-phase with both
denaturing and non-denaturing ESI suggest that the gas-phase
structures and solution phase structures are not dissimilar.
That non-covalent bonds typically remain intact in proteins
during ESI-based introduction into the gas-phase is encouraging
for the potential of IMS characterization of protein and protein
a
Department of Mechanical Engineering, University of Minnesota,
Minneapolis, MN, USA. E-mail: hogan@me.umn.edu;
Fax: 1-612-625-6069; Tel: 1-612-626-8312
b
Faculty of Physics, University of Vienna, Vienna, Austria
c
Faculty of Physics, University Paris-Cre
´teil Val de Marne East,
Paris, France
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complex ions. Nonetheless, there are a number of possible
reasons that protein structure could be altered during
ESI which are hitherto unexamined. First, as suggested by
Breuker and McLafferty,
12
the removal of solvent (water) may
promote collapse of protein structures in the gas-phase.
Second, the ESI process, even under non-denaturing condi-
tions, leads to a significant level of excess charge (close to the
Rayleigh limit of a similar sized water drop) on the protein
surface.
13–16
Such high charge levels may lead to Coulombic
stretching,
17–19
in which case the gas phase structure need not
resemble the structure in solution. Finally, many IMS schemes
function at reduced pressure, and prior to analysis, they
require that ions pass through several high voltage gates and
high pressure drop orifices.
20
These types of ion introduction
schemes, while well-suited for mass spectrometry, may give
rise to structural modification in protein ions. At present, the
influence of solvent loss, Coulombic effects, and IMS inlet
effects have not been decoupled from one another, and
without detailed studies of these effects, the comparison of
gas-phase ion structures to solution phase ions will remain
tenuous.
To better utilize IMS measurements in the analysis of
proteins, improved understanding of the influence of the ESI
process on protein structure is clearly necessary. Along these
lines, the purpose of this study is two-fold. First, we examine
charge reduced protein ions
21–24
(1–3 net charges per protein
ion) produced via ESI under both non-denaturing and
denaturing conditions with an atmospheric pressure, low
electric field strength IMS instrument (a differential mobility
analyzer, DMA
25
). The use of charge reduction and
atmospheric pressure measurements mitigates the possible
influences that Coulombic stresses and high energy gas
molecule collisions may have on the structure of gas phase
protein ions, leaving only the influence of the solution to
gas-phase transition, i.e. the influence of the aerosolization
process on protein structure. Second, while charge reduction
ESI-DMA measurements are reported numerously in the
literature,
26–32
the collision cross sections/mobility diameters
of protein ions examined in prior work has recently been
brought into question.
33
This study, using a distinctly different
DMA from prior work but a similar charge reduction ioniza-
tion source, allows for a more critical examination of these
prior measurements. From mobility measurements, we infer
the gas-phase protein density (based on the mobility
diameter
34
of the protein ions, described subsequently), a
metric which can be directly compared to protein densities in
solution. We compare densities inferred from our measure-
ments to those made using drift tube IMS
35–36
and DMAs,
and in doing so we show most prior density calculations, based
on the collision cross section of the protein ion, lead to a
drastic underestimation of the density of gas phase protein
ions. We further show that earlier charge reduced protein ion
DMA measurements
26,28
give rise to anomalously low protein
ion densities relative to measurements made here as well as
most other reported mobility measurements. Finally, we
compare the mobilities and inferred densities of protein ions
electrosprayed under denaturing conditions to those electro-
sprayed under non-denaturing conditions of the same reduced
charge state.
Materials and methods
Protein solutions
The following chemicals were purchased from Sigma Aldrich
(St. Louis, MO, USA) and used for all experiments conducted:
bovine heart cytochrome c (C2037), equine heart myoglobin
(M1882), chicken egg white lysozyme (L6876), bovine serum
albumin (A9647), chicken egg white albumin (L6876), water
(HPLC grade), ammonium acetate, methanol (HPLC grade)
and formic acid. 20 mM aqueous ammonium acetate buffer
was prepared for use as a non-denaturing protein solvent.
28
Each protein was dissolved in this buffer and purified in an
Eppendorf (5418) centrifuge using Nanosep 3 K Omega
centrifuge vials (Pall Co.). On purification, individual protein
stock solutions of 10–50 mg mL
1
of the buffer were prepared.
Formic acid buffer containing 1% formic acid by volume in a
mixture of 49.5% methanol and 49.5% water by volume was
made for use as a denaturing solvent. From the stock solutions,
further dilutions were carried out using ammonium acetate and
formic acid buffers to produce non-denatured and denatured
protein solutions respectively. For ion mobility measurements,
the protein solution concentrations were 1–370 mM, and for
mass spectrometry studies, the concentrations were 50–370 mM.
Ion mobility measurements
A schematic of the experimental system used is shown in Fig. 1.
ESI was performed in positive mode using a commercial
electrospray source
37
(model 3480, TSI, St. Paul, MN, USA).
A fused silica capillary (ID 40 mm and OD 150 mm) with a
tapered end was used for electrospraying each protein solution.
A backing pressure of 3.4 psi (pounds per square inch) was
applied to drive the electrospray solution through the capillary
and the electrospray was operated in a stable cone jet mode
38
with an operating voltage between 1.8–2.6 kV. Filtered carbon
dioxide was introduced at a flowrate of 0.4 lpm (liters per
minute) near the capillary tip to prevent corona discharge.
Fig. 1 Schematic of the experimental setup used to measure the
mobility distribution of denatured and non-denatured proteins. Protein
ions were produced using electrospray ionization following which their
charge state was reduced using a Po-210 radioactive source. Electrical
mobility based classification of the charge reduced ions was carried out
using a 1/2 mini DMA. An electrometer and a condensation particle
counter were used to detect aerosol particles exiting the DMA.
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Additionally, dehumidified and filtered air at a flowrate of
12–15 lpm was introduced to improve transmission of ions out
of the electrospray source chamber. Following ESI, the
produced highly charged drops (B130–150 nm in original
diameter with non-denaturing solutions, 90–125 nm in original
diameter with denaturing solutions, measured with electro-
sprayed sucrose solutions in the respective buffer liquid, the
droplet diameter can be derived from the size of the dry
residue particle diameter knowing the concentration of sucrose
in solution) quickly exited the ESI chamber and entered a
second chamber, which contained a Po-210 radioactive source.
The Po-210 source produced a high concentration of positive
and negative ions (in roughly equal concentrations), which are
highly diffusive and decrease the charge levels of drops
through ion-drop collisions. The charge reduction process
depends on the ion concentration and residence time of the
particles exposed to the ion cloud. If both are sufficient the
charging process results in a steady state charge distribution,
39
at which most drops are uncharged (neutral), with few drops
having one excess surface charge and an even smaller
fraction carrying two charges. The electrospray source
was operated with high flow rates of the carrier gas through
the charging chamber which resulted in short residence time
and thus incomplete neutralization. This enabled measure-
ment of protein ions of up to three excess charges. The
removal of charge from drops prior to their complete evapora-
tion has several consequences: (1) Coulombic fissions are
hindered as drops do not reach the Rayleigh limit during the
evaporation process.
24
Therefore, in order to produce isolated
protein ions in the gas-phase, only one protein may be present
in each initial drop.
40
Furthermore, as the number of proteins
per droplet is roughly Poisson distributed,
41
the formation of
some non-specific multiprotein ions
33,41
is unavoidable with
charge neutralization. (2) Upon the evaporation of solvent, the
remaining protein and multi-protein ions are not stretched by
Coulombic stresses; rather, structural modifications are solely
due to the removal of solvent. (3) As low protein concentra-
tions in ESI solutions must be used to observe isolated
gas-phase protein ions and most proteins do not exit as ions
in the gas-phase but rather as neutral aerosol particles, the
gas-phase protein ion concentration is substantially below
what is commonly used in protein ESI-MS measurements.
Following the evaporation of solvent, single and multi-
protein ions remain, which adopt the charge state of their
parent drop and thus have charge states of 3 or less. The low
charge state protein ions were directed into the differential
mobility analyzer, which was a recently commercialized
1/2-mini DMA (NanoEngineering Corp., FL, USA). The
operation of DMAs,
42–44
and specifically their operation for
the measurement of charge-reduced ions such as those examined
here is described in great detail elsewhere.
26,28,45–49
Briefly,
DMAs operate as ion mobility filters, such that under a given
set of operating conditions (DMA geometry, sheath flowrate,
and voltage difference between electrodes), only ions of a
prescribed mobility are transmitted through a DMA. Therefore,
by stepping through a series of operating conditions (i.e. changing
the voltage difference between electrodes) and measuring ion
concentrations at the DMA outlet, ion mobility spectra can be
determined. Here, the 1/2 mini DMA was operated with its long
bullet installed, which enables investigation of ions with
collision cross section/unit charge of B160 nm
2
. The DMA
was operated in closed loop mode with sheath air flowrates of
a few hundred lpm, well below the DMA reaches sonic
conditions (B700 lpm for this DMA). At these moderate flow
conditions, chosen to cover a relatively broad size range, this
DMA device shows moderate resolving powers, R(defined from
the full width at half maximum, FWHM, of a peak, V
FWHM
,and
the peak position V
0
,R=V
0
/V
FWHM
), which was examined
through the use of monodisperse mobility standards.
50
It was
found that the conditions employed here gave a resolving power
of B20, which, while lower than what has been achieved with
different high resolution DMAs,
43
is still considerably higher
than what has been used previously in the examination of charge
reduced protein ions (resolving powers B10).
28
Ion mobility distributions for each electrosprayed protein
solution were recorded by stepping the voltage between elec-
trodes in the DMA from 0 to 5 kV (in steps of 10 V). With this
voltage difference employed, electric fields were sufficiently low
such that the measured mobilities were low field mobilities,
51
and no ion heating occurred (temperature increase o1K
52
)
during DMA measurement. A negative voltage was applied to
the DMA outlet electrode while the upper electrode was
grounded to establish the voltage difference. Although this
ensured safe operation of the DMA, ions exiting the DMA
were at a lower potential, and had to reach ground potential
prior to measurement. This was accomplished via use of a
leaky dielectric tube (ensital SD, a natural copolymer acetal),
which ions were transmitted through upon exiting the DMA
and prior to entering the detector. Two detectors were utilized
in this work: a custom made Faraday cage electrometer
(FCE), which measured the current of ions, and a condensa-
tion particle counter
53
(CPC, TSI 3025), in which the hetero-
geneous condensation of micrometre sized butanol drops onto
ions allowed for single ion detection for ions larger than B2.5 nm
(in diameter). The current measured by the FCE can be related to
number concentration through knowledge of the charge state and
the flow rate through the detection instrument. Both detectors
were operated simultaneously during measurements.
Because of the inherent difficulties of precisely measuring
flowrates of several hundred lpm, DMA calibration was
necessary to establish a mobility scale. Only measurement of
a single mobility standard was necessary, as the DMA is a
linear mobility spectrometer. For calibration, a home-made ESI
source similar to that used by Ude and Fernandez de la Mora
50
was used to electrospray a solution of 5 mM tetra-dodecyl
ammonium bromide (TDDAB) in chromatography grade
methanol. The DMA voltage (V
std.
) at which [Tetra-dodecyl
ammonium]
+
ions were transmitted was recorded and its known
mobility
50
in air (Z
std.
)of0.713cm
2
V
1
s
1
at 20 1C(the
laboratory temperature) was used to determine the mobilities (Z)
of all measured ions (transmitted at V
DMA
)usingeqn(1):
Z¼Zstd:Vstd:
vDMA
ð1Þ
Electrospray ionization-mass spectrometry (ESI-MS):
ESI-MS studies using a commercial quadrupole—time-of-
flight mass spectrometer (API QSTAR Pulsar, Sciex, Toronto,
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CA, USA) were also conducted to examine protein ions
electrosprayed under non-denaturing conditions. For these
measurements, the home-made electrospray source mentioned
in the previous section was also used. The capillary tip was
positioned close to the inlet orifice (B1 mm) of the MS to
maximize transmission of protein ions into MS inlet. Each
protein solution was electrosprayed using capillaries of ID
40 mm (OD 360 mm. Samples were driven through the smaller
diameter capillary (ID 40 mm) with a backing pressure of 3 psi.
A voltage of 1.8–2.2 kV was applied between the electrospray
solution and the MS inlet plate to produce a stable
electrospray cone jet that was visually verified with a digital
microscope, as well as by checking the constancy of the
electrospray back current. Nitrogen curtain gas was used to
prevent entry of non-charged particles into the MS inlet
orifice. The QSTAR Pulsar instrument settings used for all
experiments were as follows: Focusing potential FP 100,
Collision gas (CAD) 3, Ion Release Delay (IRD) 11, Ion
Release Width (IRW) 10, Ion source gas 1 (GS1) 20, Ion
source gas (GS2) 0. The declustering potentials, DP and DP2,
were kept at 0 V for each electrosprayed protein solution to
examine protein ions with minimal high energy gas molecule
collisions in the MS inlet.
Results and discussion
Mobility measurements
Mobility spectra (with the mobility represented as the inverse
mobility) for all measured protein ions produced with non-
denaturing ESI and denaturing ESI are shown in Fig. 2 and 3,
respectively. Spectra measured with the electrometer and the
CPC are overlaid with one another. In each of the electro-
meter-determined mobility spectra, a high intensity peak in the
inverse mobility of 0.5–1.0 V*s cm
2
range is apparent, which
is not detectable by the CPC. This signal derives from positive
ions produced by the Po-210 radioactive source, which have
mobilities ranging from the polarization limit in air (close to
2.2 cm
2
V*s),
51
down to half of the polarization limit. These
ions, which may vary drastically in chemical composition from
one another, do not give rise to heterogeneous condensation of
butanol, and were therefore not detected by the CPC. The
remaining detected signal can be attributed to single and
multiprotein ions which are primarily singly or doubly
charged, and are composed of 1–4 proteins. As in prior IMS
studies
26,28,48
of charge-reduced protein ions, by varying the
protein concentration in ESI suspensions and noting that the
formation of non-specific multiprotein ions (such as those
Fig. 2 Mobility spectra of protein ions produced via electrospray ionization under non-denaturing conditions.
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21634 Phys. Chem. Chem. Phys., 2011, 13, 21630–21641 This journal is cthe Owner Societies 2011
observed here) in charge reduction ESI follows Poisson
statistics,
40,41,54,55
singly charged monomer, dimer, and higher
order multimeric ions can be identified in mobility spectra.
Similarly, by noting that the mobility of a doubly charged ion
composed of a given number of proteins is roughly twice the
mobility of its singly charged counterpart, doubly charged ions
can also be identified. This identification of peaks can be
confirmed additionally by determination of the detection
efficiency of the CPC by comparing the number concentrations
measured by both detection devices. All identifiable ions in
mobility spectra are labeled in Fig. 2 and 3.
Width of the mobility distributions
Striking in Fig. 2 and 3 are the extremely broad FWHM for all
measured protein peaks, ranging from 14–32% for those
electrosprayed under non-denaturing conditions, and from
8–24% for those electrosprayed from a strongly denaturing
solution (determine from spectra gathered with the CPC).
Even with the modest instrument resolving powers employed,
these FWHM are much higher than the capabilities of the 1/2
mini DMA, appear to vary greatly between proteins, and are
dependent on ESI solution conditions. Higher FWHM than
instrument limits, similar to those found in this study, have
also been observed by Kaufman et al.
48
in earlier studies of
charge reduced protein ions with a similar ESI source. It was
speculated that these higher FWHM were either due to
machine imperfections in the DMA employed (thereby
reducing its resolving power), or differential amounts of
residual solute/solvent adducts bound to protein ions during
mobility measurement. This study utilized a DMA under
drastically different operating conditions but an essentially
identical electrospray source, which, similar to the observations
of Kaufman et al.
48
suggests that the charge reduced ionization
process leads to significant and variable levels of adduction on
protein ions or to variability in protein ion conformation
following electrospray ionization (as a different DMA is utilized
here we rule out the possibility of machine imperfections
influencing both studies). Certainly, due to the suppression of
Coulombic effects, additional adduct formation is possible with
charge reduction ESI, as any non-volatile solutes within the
same drop as a protein would eventually non-specifically bind
with the protein in the absence of Coulombic fissions.
56
To
determine if the broadness of the protein peak distributions
arises from the presence of non-volatile adducts, ESI-MS with
non-denaturing solutions was performed without charge
reduction but at the same electrospray flowrate and with the
same solution as used in DMA measurements. These spectra
were measured using minimal declustering potentials in the MS
in an effort to preserve adducts in the mass spectrometer,
Fig. 3 Mobility spectra of protein ions produced via electrospray ionization under strongly denaturing conditions.
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though we note that in the MS inlet the dissociation of adducts
both ionized
44
and as neutrals
57
is in many instances unavoidable.
Mass spectra for cytochrome C, lysozyme, and myoglobin are
shown in Fig. 4. Even with minimal declustering, few adducts are
visible (primarily sodium adducts). This level of adduction and
the sharp mass peaks observed suggest that if the broad FWHM
in mobility spectra are caused by variable levels of protein ion
adduction, then these adducts must be easily removed as ions pass
through the low pressure interface at the mass spectrometer, or
that the level of adduction is significantly reduced by Coulombic
fissions (and hence not visible in the MS spectrum). While,
based on MS measurement, we can rule out the possibility of
non-volatile adduct attachment as the origin of broad protein
ion peaks, we cannot eliminate the possibility of differential
amounts of residual solvent (water) or a volatile solute bound
to proteins during atmospheric pressure IMS measurements,
which evaporates prior to analysis at low pressure. In this case,
the FWHM would be determined not by instrument limita-
tions (nor by the polydispersity of the electrospray drops,
54
which was the same for all examined protein ions), but by each
protein’s ability to retain solvent in the gas phase
58
and the
protein ion to protein ion variation in the amount of residual
water bound. Conversely, as mentioned above, a second
possibility is a range of similar but unresolvable conforma-
tions of the gas-phase protein ion. As with differential residual
water, this would lead to apparent FWHM that vary on a
protein by protein basis. With available analytical techniques,
it is not possible to clearly determine whether peak width is
controlled by differential amounts of adduction, or varying
gas-phase conformations.
18
It is noteworthy, however, that
protein ions electrosprayed from non-denaturing solutions
have broader FWHM than their counterparts from strongly
denaturing solutions, which suggests some structural differ-
ence between the two ion types, discussed subsequently in this
manuscript.
Gas phase protein ion densities
Mobility measurements can be used to infer a relative size of
each protein ion, and typically the goal of protein ion mobility
analysis is size/structure inference. Most often, in IMS studies
of biomolecules (high mass ions) a collision cross section (O)is
evaluated from the mobility using the equation:
51
O¼3ze
8Zpngas ffiffiffiffiffiffiffiffiffiffiffiffiffiffi
p
2kTmg
rð2Þ
where Z
p
is the protein ion mobility, zis the number of excess
charges on the protein ion, eis the unit charge on an electron,
n
gas
is the number density of the background gas molecules,
kT is the thermal energy, and m
g
is the background gas
molecular mass. A common practice in studies with reduced
pressure (drift tube or T-wave) IMS measurements of protein
ions
59
is to infer Ofrom mobility measurements using eqn (2),
and then compare measured Oto predicted Obased on
momentum transfer from gas molecules to a model structure
of the ion, treating the gas molecules as hard spheres (the
EHSS model
60,61
) or considering potential interactions between
the gas molecule and ion
62
(the trajectory method, TM). A
third method, approximating Oas the orientationally averaged
projected area of the ion (the projection approximation, PA), is
Fig. 4 Mass spectra of selected proteins electrosprayed under non-denaturing conditions without charge reduction.
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21636 Phys. Chem. Chem. Phys., 2011, 13, 21630–21641 This journal is cthe Owner Societies 2011
also often employed, but this method ignores the influence
of multiple collisions between a gas molecule and ion (which
is a molecularly rough surface) and thus leads to drastic
underestimations in O.
61
While the EHSS and TM approaches
have been successfully employed to determine Oin helium
background gas,
63
difficulties arise in their application to
diatomic gases (e.g. N
2
and O
2
). Both approaches approximate
the gas molecule as a single sphere which collides specularly
with the protein ion surface. In diatomic gases, impinging gas
molecules will no doubt undergo multiple collisions with an
ion (even if the ion surface were a perfect sphere). Without
utilizing detailed structural models, not only of the protein ion
but also diatomic gas molecules,
64
as well as conservation of
linear and angular momentum during collision, the accuracy
of EHSS and TM calculations for diatomic gases is uncertain.
As a simplified alternative, in the absence of potential inter-
actions, Ocan be decomposed into two components: (1) a size
component, i.e. the orientationally averaged projected area of
the structure formed by the ion and gas molecule during
collision, and (2) a momentum scattering coefficient,
6,33,34,65,66
x, equal to unity for single, specular collisions between gas
molecules and the ion and approaching 1.39 for completely
diffuse gas molecule–ion collisions.
67
With this approximation,
Ocan be calculated as:
O¼p
4xðdpþdgÞ2ð3Þ
where d
p
is the protein ion ‘‘mobility diameter’’, and d
g
is the
effective gas molecule diameter (0.3 nm for air
34,68
). The value
of xhas been measured indirectly
69–73
in a number of gases to
be 1.36, dating back to the work of Millikan.
74
Recently,
numerous measurements by Ku and Fernandez de la Mora
68
as well as Larriba et al.
34
show that x=1.36 holds valid in air
for ions substantially smaller than the protein ions
examined here.
We infer both Oand d
p
from mobility measurements. Before
doing so, we note that eqn (2) applies rigorously only in the
free molecular drag limit. To correct for small non-continuum
effects on protein mobilities measured at atmospheric
pressure, to infer d
p
from Z
p
we apply the Stokes-Millikan
equation, given as:
Zp¼
1þl
2ðdpþdgÞ1:257 þ0:4 exp 0:55ðdpþdgÞ
l

3pmðdpþdgÞð4Þ
where lis the mean free path of the gas (66.5 nm for air at
atmospheric pressure and room temperature) and mis the air
dynamic viscosity (1.82 10
5
Pa*s). In lieu of applying
eqn (2), we apply eqn (4) to infer d
p
from Z
p
and subsequently,
use eqn (3) to determine Ofrom d
p
. Diameter values and
corresponding collision cross sections inferred with this
approach are listed in Table 1 for each protein ion analyzed.
As each protein peak examined is anomalously broad, we also
report upper and lower limits on the protein diameter, taken at
the point of 50% maximum intensity on the sides of each peak.
An advantage to estimating d
p
from mobility measurement
is that unlike O, it is relatively insensitive to the background
gas properties (considering hard sphere potentials and a
constant xin all gases); thus, it permits further calculation
of protein ion properties which can be compared to those
measured for proteins in solution, namely, the gas-phase ion
protein density. It has been shown for a number of materials,
including polymers,
75,76
ionic liquids,
34,68
metals,
77
and organic
salts, that the gas-phase density is in excellent agreement with
the bulk density for ions in the 2–10 nm diameter range, when
calculated from mobility measurement with d
p
inference.
Striking about these results is that these ions, while dense,
are not necessarily spherical, yet the analysis approach yields
unambiguously the bulk density of each ion. We therefore
follow the same analysis approach to determine the gas-phase
density of protein ions. Fig. 5 shows the 1/3rd power of the
mass of the examined protein ions as of a function of their
lower limit, upper limit, and peak diameters for ESI under
non-denaturing conditions, with corresponding data shown in
Fig. 6 for ESI with denaturing solution. For comparison, we
can estimate the volume, and hence the density of the mono-
mers of four of the five proteins examined from their crystal-
lographic structures. This was accomplished here using a
modification to Visual Molecular Dynamics (VMD, http://
www.ks.uiuc.edu/Research/vmd/) developed by Andriy Anishkin
(University of Maryland) in which the cross-sectional area of a
protein is determined as function of position (z-coordinate) for
each protein. By then dividing up the protein into thin sheets of
known area and small length, the volumes of these sheets can be
determinedandsummedtogivethetotalproteinvolume.For
each calculation, a spherical probe of specified radius must be
used, which was taken to be 0.18 nm and approximate a thin
layer of water remaining on proteins after ESI. For calculations,
crystal structures were taken from the following RCSB
protein data bank files: cytochrome C (3CYT.pdb), lysozyme
(2LYZ.pdb), myoglobin (1MBN.pdb), and ovalbumin
(1OVA.pdb). From volume calculations, a volume diameter
(volume = pdiameter
3
/6) is inferred for monomer, dimer, and
trimer protein ions. Ion mass as a function of volume diameter
is also shown in both Fig. 5 and 6 from VMD calculations. All
mass-diameter plots from mobility measurements as well as
VMD calculations display a power law relationship with
a scaling exponent close to 1/3, as expected for compact
structures of similar density. The average densities inferred
for each protein, considering all single and multiprotein ions
as well as all charge states, are shown in Table 2, for ESI under
non-denaturing conditions, denaturing conditions, and from
VMD calculations.
Comparison to prior measurements and nondenaturing to
denaturing comparison
With mobility diameters and protein ion densities inferred
from DMA measurements, we first compare results from our
measurements to those from prior mobility measurements
made similarly with ions generated with non-denaturing
electrosprays. Fig. 7 shows the cube root of masses of measured
protein ions as a function of inferred mobility diameter for each
ion. With the technique described above to infer mobility
diameter, also plotted in Fig. 7 are the mass-mobility diameter
results from Bush et al.
35
(measured in a drift tube in both
He, with an inferred gas molecule diameter of 0.19 nm, and
N
2
, with an inferred gas molecule diameter of 0.3 nm),
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Hogan & Fernandez de la Mora
33
(in air, using a parallel-plate
DMA-MS without charge reduction ESI), Kaufman et al.
48
(in air, using a low resolution DMA and charge reduction
ESI), and lastly the combined data sets of Kaddis et al.
26
and
Bacher et al.
28
(again using a low resolution DMA and charge
reduction ESI). Most measured protein ion mass-diameter
values agree well with one another, and are bound within
the range 0.85 g cm
3
ogas-phase protein ion density o
1.10 g cm
3
. Contrary to the conclusions of prior reports,
5,35
however, the measurements of Kaddis et al.
26
and Bacher
et al.
28
are not in agreement with any other data sets, and the
inferred mobility diameters from these studies are anoma-
lously large, with correspondingly small inferred densities
of B0.70 0.05 g cm
3
. The misinterpreted agreement found
in prior works derives from the manner in which protein ion
density has been previously calculated; in Kaddis et al.
26
and
Bacher et al.,
28
protein ion density is correctly inferred using
the technique also applied here. However, in a number of drift
tube based IMS studies
5,35
where comparison is made to prior
DMA measurements, ion density has been inappropriately
calculated treating all ions as spheres with projected areas
equivalent to the measured collision cross sections, or similarly
mobility diameters have been treated as the square root of
O/p. This leads to a drastic (B36%) underestimation of the
ion density and the conclusions of agreement with prior DMA
based measurements.
The common feature that the works of Kaddis et al. and
Bacher et al. share, which is distinct from other works, is the
use of a commercial nanoDMA (TSI, Inc),
78
and although the
performance of this nanoDMA has been tested rigorously,
79
it
consistently produces anomalous results in the examination of
ESI generated protein ions.
27
In spite of the confusion brought
about by prior size inference techniques from mobility measure-
ments, it is clear from this work that for proteins electrosprayed
under non-denaturing conditions, measurements of charge
reduced proteins with well-behaved DMAs are in very good
Table 1 Summary of protein ion collision cross sections, peak diameters, the upper and lower estimates on protein diameters from mobility
spectra, and the full width half maximum (FWHM) of each peak in mobility spectra. **BSA- Bovine Serum Albumin
Protein name
Number of
multimers
Mobility
(cm
2
V
1
s
1
)Charge
Collision cross
section (nm
2
)
Peak
diameter
(nm)
Lower limit
(nm)
Upper limit
(nm) FWHM (%)
Buffer: 20 mM Ammonium acetate in water
BSA 1 0.0537 + 13.24 5.94 5.70 6.25 17.6%
BSA 1 0.1579 +++ 13.49 6.00 5.81 6.55 26.0%
BSA 1 0.1039 ++ 13.62 6.03 5.76 6.37 17.7%
BSA 2 0.0327 + 21.81 7.71 7.32 8.05 17.9%
BSA 2 0.0654 ++ 21.81 7.71
Cytochrome C 1 0.2835 ++ 4.96 3.52 3.19 3.80 31.7%
Cytochrome C 1 0.1454 + 4.83 3.47 3.24 3.71 24.7%
Cytochrome C 2 0.0935 + 7.54 4.41 4.19 4.75 19.8%
Lyzozyme 1 0.1346 + 5.28 3.64 3.39 3.86 22.4%
Lyzozyme 1 0.2648 ++ 5.41 3.69 3.49 3.99 25.2%
Lyzozyme 2 0.0869 + 8.16 4.60 4.35
Myoglobin 1 0.1206 + 5.83 3.84 3.68 4.05 17.7%
Myoglobin 1 0.2347 ++ 6.00 3.90 3.75 4.18 20.8%
Myoglobin 2 0.0804 + 8.77 4.78 4.59 5.12 20.9%
Myoglobin 2 0.1601 ++ 8.88 4.81
Myoglobin 3 0.1610 + 11.60 5.54 5.23 5.78 18.5%
Myoglobin 4 0.0506 + 14.01 6.12
Ovalbumin 1 0.1350 ++ 10.47 5.25 5.10 5.51 15.6%
Ovalbumin 1 0.0670 + 10.55 5.27 5.09 5.48 14.7%
Ovalbumin 2 0.0930 ++ 15.26 6.40 6.11 6.82 22.1%
Ovalbumin 2 0.0462 + 15.35 6.42 6.16 7.12 30.4%
Buffer: 0.1% Formic acid in 50% water and 50% methanol
BSA 1 0.0460 + 15.45 6.44 6.17 6.89
BSA 1 0.0886 ++ 16.05 6.57 6.24 7.05 22.9%
Cytochrome C 1 0.1474 + 4.78 3.45 3.37 3.54 10.0%
Cytochrome C 1 0.2834 ++ 4.96 3.52 3.40 3.61 10.8%
Cytochrome C 2 0.0955 + 7.38 4.36 4.24 4.56 14.3%
Cytochrome C 3 0.0698 + 10.14 5.16 4.87 5.40 11.6%
Cytochrome C 4 0.0569 + 12.44 5.75 5.41 5.94 8.6%
Lyzozyme 1 0.1307 + 5.39 3.68 3.59 3.76 8.5%
Lyzozyme 1 0.2563 ++ 5.49 3.72 3.64 3.83 10.4%
Lyzozyme 2 0.0845 + 8.36 4.66 4.55 4.76 8.7%
Lyzozyme 2 0.1660 ++ 8.36 4.66 4.59 4.79 8.7%
Lyzozyme 3 0.0663 + 10.66 5.30 5.21 5.45 8.5%
Lyzozyme 4 0.0567 + 12.49 5.76 5.62 6.06
Myoglobin 1 0.1130 + 6.23 3.98 3.88 4.10 11.8%
Myoglobin 1 0.2194 ++ 6.43 4.05 3.97 4.15 9.1%
Myoglobin 1 0.3177 +++ 6.67 4.13 4.01 4.24 13.0%
Myoglobin 2 0.0693 + 10.21 5.18 5.00 5.38 14.9%
Ovalbumin 1 0.0602 + 11.76 5.58 5.41 5.81 13.4%
Ovalbumin 1 0.1152 ++ 12.28 5.71 5.52 5.94 14.2%
Ovalbumin 2 0.0815 ++ 17.43 6.86 6.60 7.33 23.9%
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21638 Phys. Chem. Chem. Phys., 2011, 13, 21630–21641 This journal is cthe Owner Societies 2011
agreement with drift tube measurements in He and N
2
,aswell
as with recent parallel-plate DMA measurements. As the
compared data sets were collected in different background
gases, at different pressures, and with different ion introduction
schemes, we can conclude that the influence of these parameters
is minimal, and any structural modifications to protein ions
produced under non-denaturing conditions brought about
during the IMS process occur during ESI drop formation and
evaporation itself. Further, while not all of these studies utilized
charge reduction, the charge states of examined non-denatured
protein ions in prior work appear sufficiently low that Coulombic
stretching does not occur; otherwise differences between values
inferred from our measurements and those from measurements
without charge reduction would be discernable. We note that
these conclusions regarding the lack of influence of ion intro-
duction scheme as well as Coulombic influences are by no
means universal, and only apply to the protein ions measured.
In the examined mobility diameter range (3–8 nm), however,
almost none of the measured proteins from any study display a
gas-phase density outside of the 0.85–1.10 g cm
3
range,
suggesting that most globular proteins do indeed have similar
gas-phase density.
80
Although VMD inferred densities are bracketed between the
densities inferred from the upper and lower limits on ion
mobility and are below that of bulk peptide material
(B1.3–1.4 g cm
3
),
13,81
with the exception of Cytochrome C,
all densities based on the peak diameter in mobility spectra
(for non-denaturing ESI) are higher than the expected density
for the native state (including Lysozyme, which contains
disulfide bridges). Though a small difference, this clear and
consistent result suggests that rather than broad spectra
originating from water adduction (or in addition to water
adduction), proteins undergo slight and varying degrees of
compaction in the absence of Coulombic stresses. A similar
conclusion was recently reached for GroEL ions produced via
electrospray and subsequently analyzed with an atmospheric
pressure DMA operating at much higher resolution,
6
and the
densities inferred from peak diameters are in outstanding
agreement with those inferred from recent DMA-MS measure-
ments
33
where density calculation was made in the same
Fig. 5 Protein Ion diameter versus the 1/3rd power of the protein mass
with non-denaturing ESI, showing the experimental data points for the
peak diameter and the upper and lower diameter limit taken at 50%
particle concentration of the peak. VMD predicted values are based
upon the density of the Cytochrome C, Lysozyme, Myoglobin, and
Ovalbumin monomer, dimer, and trimer volume equivalent diameters.
Fig. 6 Protein Ion diameter versus the 1/3rd power of the protein mass
with denaturing ESI, showing the experimental data points for the peak
diameter and the upper and lower diameter limit taken at 50% particle
concentration of the peak. VMD predicted values are based upon the
density of the Cytochrome C, Lysozyme, Myoglobin, and Ovalbumin
monomer, dimer, and trimer volume equivalent diameters.
Table 2 Summary of protein ion densities (in g cm
3
), based on the
upper limit of their diameters, the lower limit of their diameters, and
the diameters from peak in mobility spectra. VMD denotes the density
inferred from the volume of the native-state crystal structure. **BSA-
Bovine Serum Albumin
Non-denaturing ESI Denaturing ESI
Protein
Upper
limit
Lower
limit Peak
Upper
limit
Lower
limit Peak VMD
BSA 0.81 1.10 0.96 0.62 0.88 0.77 N/A
Cytochrome C 0.73 1.14 0.91 0.80 1.01 0.89 0.98
Lysozyme 0.75 1.11 0.93 0.83 0.97 0.91 0.86
Myoglobin 0.82 1.12 0.98 0.76 0.90 0.83 0.92
Ovalbumin 0.84 1.14 1.01 0.70 0.91 0.82 0.91
Fig. 7 Comparison of experimental data from this study with prior
studies.
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manner to this work (average density of 0.95 g cm
3
). Evidence
for compaction is also in agreement with the predictions of
Breuker and McLafferty, that evaporation of solvent can lead
to the collapse of protein structure.
12
Along these lines, recent
research efforts have been focused on finding appropriate salt
additives to ESI solutions which can stabilize proteins during
the ESI process, preventing compaction while conversely
readily dissociating from protein ions prior to mobility
measurement.
82,83
Our results, combined with the results of
prior studies, further suggest that slight protein compaction
during ESI is commonplace, and such salt additives are indeed
necessary to prevent ESI-induced compaction.
Virtually all prior reports of mobility measurements of
protein ions from strongly denaturing ESI
63,84
note that the
produced ions are highly charged and that the ions adopt
stretched conformations. Conversely, in this work, charge
reduced ESI of non-denatured protein ions reveals that,
although the inferred densities for protein ions from non-
denaturing solutions are slightly lower than those from non-
denaturing solutions (average density of 0.86 g cm
3
as
compared to 0.97 g cm
3
), relatively compact ions are formed
when high charge levels are mitigated, with densities similar to
those predicted for crystal structures. For direct comparison,
mass-mobility diameter values for protein ions from denatur-
ing solutions are plotted in Fig. 7 (blue circles). Also plotted
are the inferred mass-mobility diameter values (charge states
of +3 or less) for ubiquitin, cytochrome C, lysozyme, and
apomyoglobin, produced by strongly denaturing ESI from the
Clemmer cross section database,
63,84–86
as well as for +1
and +2 charge state MALDI generated ions from Fernandez-
Lima et al.
36
(both measured in He). Although the inferred
density from the MALDI measurements is anomalously high
(above the densities of bulk peptides in some cases), all three sets
of measurements indicate that low-charge state protein ions are
compact, irrespective of whether they are produced from non-
denaturing or denaturing solutions. Further confirmation of this
is found in the post-ESI charge reduction work of Badman and
coworkers,
87,88
wherein highly charged protein ions, originally
stretched, monotonically decrease in collision cross section as
their charge state is decreased. Overall, ion mobility spectrometry
of low-charge state protein ions, performed in this work and in
others, indicates that irrespective of the electrospray solution
utilized, relatively compact (and possibly compressed denser than
the crystal structure) ions will be generated, and that stretched
conformations in the gas-phase are usually formed and/or
stabilized by Coulombic forces. Although this conclusion is by
no means new,
17
it has been frequently disregarded in IMS
studies, and direct comparisons between the structures of low
charge state ions and higher, Coulombic stretched ions have been
used to draw conclusions about solution phase protein
structures.
19,89
Conclusions
Five proteins were electrosprayed from both non-denaturing
and strongly denaturing solutions. Charge reduction was
subsequently used to prevent Coulombic fission of produced
droplets as well as to minimize the produced protein ion
charge states. Protein ion mobility spectra were measured with
a DMA coupled to both a condensation particle counter and
Faraday cage electrometer. From mobility measurements, the
mobility diameters and gas phase densities of proteins were
inferred and compared to the mobility diameters and densities
from prior protein ion mobility measurements. Based on this
work, we conclude the following:
1. Low charge state protein ion mobility spectra generally
display broad peaks where the FWHM is not limited by
instrument resolution. Based on comparison of inferred
protein ion densities to the densities of crystal structures, it
appears likely that upon introduction to the gas-phase and in
the absence of Coulombic stretching, proteins undergo
compaction, with the broad FWHM attributable to variable
degrees of compaction. However, it is not possible at present
to rule out the possibility of adduction further broadening
mobility peaks.
2. The inferred densities from mobility measurements of
protein ions from non-denaturing solutions made via drift
tubes and DMAs are in excellent agreement, with densities
typically falling in the range 0.85 g cm
3
to 1.10 g cm
3
. Prior
work has quoted lower densities due to the omission of the
momentum scattering coefficient in quantifying the size
(mobility diameter) of protein ions. Therefore, most examined
proteins ions from non-denatured ESI solutions are undis-
turbed by Coulombic stretching, and also appear slightly
compacted by introduction into the gas-phase.
3. Also in the absence of Coulombically-induced stretching,
protein ions generated from non-denaturing solutions are also
relatively compact, with densities comparable to densities
determined for crystal structures.
4. While the observed differences in charge-state distribution
between low charge state and high charge state ions strongly
imply that the protein ions do indeed have different structures
in solution,
14–16,90,91
this cannot be directly inferred from
comparison of low and high charge state ions. As the charge
level required to induce Coulombic stretching will vary from
protein to protein, we suggest that when efforts to compare
protein ion structures from different solutions are made,
comparison only be made between ions of identical/similar
charge state. Therefore, to truly distinguish between different
gas-phase conformers, extremely high resolving power IMS is
desirable.
92
Without such high resolving powers, examination
of the FWHM for distributions, shown here to vary between
ions from denaturing and nondenaturing solutions, may aid in
detecting multiple ion conformers.
Acknowledgements
We thank Professor Peter McMurry (University of Minnesota)
for providing the charge reduction electrospray ionization
source and condensation particle counter used in this work,
Professor Wladek Szymanski and the University of Vienna
Faculty of Physics for providing support for A.M.’s visit
to the University of Minnesota, and Dr Andriy Anishkin
(University of Maryland) for advice in the use of Visual
Molecular Dynamics. This work was partially supported by
NSF-CHE-1011810.
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... The thermodynamic properties of both gas phase protein ions and aerosol phase protein particles have been investigated in many studies (e.g. Adler, Unger, & Lee, 2000;Butcher, Miksovska, Ridgeway, Park, & Fernandez-Lima, 2019;Hogan & de la Mora, 2011;Maißer et al., 2011;Meyer, Root, Zenobi, & Vidal-de-Miguel, 2016;Mikhailov, Vlasenko, Niessner, & Pöschl, 2004;Pummer et al., 2015;Vörös, 2004). Undenatured protein ions are relatively compact with a density around 1 g cm −3 (Hogan & de la Mora, 2011;Maißer et al., 2011). ...
... Adler, Unger, & Lee, 2000;Butcher, Miksovska, Ridgeway, Park, & Fernandez-Lima, 2019;Hogan & de la Mora, 2011;Maißer et al., 2011;Meyer, Root, Zenobi, & Vidal-de-Miguel, 2016;Mikhailov, Vlasenko, Niessner, & Pöschl, 2004;Pummer et al., 2015;Vörös, 2004). Undenatured protein ions are relatively compact with a density around 1 g cm −3 (Hogan & de la Mora, 2011;Maißer et al., 2011). However, protein particles generated with spray-drying processes can be aggregated or dense, amorphous spheres depending on the spray-drying conditions. ...
... The relative humidity (RH) of the aerosol sample was gradually reduced in the dryer by radical diffusion of water vapor from the aerosol to the densely packed silica gel surrounded. The reduction of RH, quantified as aerosol drying rate, highly depends on the aerosol flow rate in the diffusion dryer Table 1 Molecular weight (M), bulk density (ρ bulk ) and gas phase density (ρ ions ) of BSA and OVA at 295 ± 1 K (Haynes, 2014;Maißer et al., 2011;Muramatsu & Minton, 1988;Neurath & Bull, 1936;Nisbet, Saundry, Moir, Fothergill, & Fothergill, 1981 (hereinafter referred to as aerosol drying flow rate). A list of the investigated aerosol drying flow rates and corresponding drying rates is shown in Table 2. ...
... where A is the fit parameter from eq 1, MM mono is the molar mass of the monomer, N A is the Avogadro number, d is the (volumetric mass) density reported to a monomer unit, and ξ is the momentum scattering coefficient. 46 Table 2 displays the fitting parameter of the common trend lines of the nine polymers analyzed in this study. By comparing these parameter A values and by using the above-extracted > PEO). ...
Article
Ion mobility-mass spectrometry (IM-MS) experiments are mostly used hand in hand with computational chemistry to correlate mobility measurements to the shape of the ions. Recently, we developed an automatable method to fit IM data obtained with synthetic homopolymers (i.e., collision cross sections; CCS) without resorting to computational chemistry. Here, we further develop the experimental IM data interpretation to explore physicochemical properties of a series of nine polymers and their monomer units by monitoring the relationship between the CCS and the degree of polymerization (DP). Several remarkable points of the CCS evolutions as a function of the DP were found: the first observed DP of each charge state (ΔDPfirst DP), the DPs constituting the structural rearrangements (ΔDPrearr), and the DPs at the half-rearrangement (DPhalf-rearr). Given that these remarkable points do not rely on absolute CCS values, but on their relative evolution, they can be extracted from CCS or raw IM data without accurate IM calibration. Properties such as coordination numbers of the cations, steric hindrance, or side chain flexibility can be compared. This leads to fit parameter predictions based on the nature of the monomer unit. The interpretation of the fit parameters, extracted using solely experimental data, allows a rapid screening of the properties of the polymers.
... To achieve this, different types of DMAs have been developed, namely Caltech radial DMA (RDMA), nanoRDMA, the Grimm nanoDMA and the Karlsruhe-Vienna DMA (Jiang et al. 2011). These high resolution DMAs have been used for the measurement of sub-2 nm particles in a variety of applications, such as atmospheric nucleation of aerosols (Kulmala et al. 2004), determination of gas phase protein densities (Maißer et al. 2011), understanding the early stages of particle formation and growth in aerosol reactors (Wang et al. , 2015, quantifying sub-2 nm filtration efficiency of fibrous filters (Chen et al. 2016;Kim, Kang, and Pui 2016). ...
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While there are several computational studies on differential mobility analyzers, there is none for high flow differential mobility analyzers (DMA) to classify nanoparticles less than 3 nm. A specific design of a high flow differential mobility analyzer, a Half Mini DMA, is investigated to predict its performance through numerical modeling in the incompressible flow regime. The governing equations for flow field, electric field and aerosol transport are solved using COMSOL 5.3. The transfer function of the Half Mini DMA is compared with that of a Nano DMA (TSI 3085). The results show that both the height of the transfer function and resolution (R) of the Half Mini DMA are much better than those of Nano DMA in sub-2 nm particle size range. Finally, the transfer function of Half Mini DMA is evaluated for different values of aerosol flow rate to the sheath flow rate (q/Q). Comparison of the simulated transfer function with existing models from Knutson-Whitby (Knutson and Whitby 1975) and Stolzenburg (Stolzenburg 1988) is also elucidated. It is found that the former model overestimates the resolution; whereas the latter is close to the simulation results for q/Q above 0.067. This work provides a useful method to study the flow regimes and transfer function of a high flow DMA.
... The singly self-charged generated particles were then classified based on their electrical mobility by passing them through a high flow differential mobility analyzer half-mini DMA; (De Juan & Fernández de la Mora, 1998; Fernández de la Mora & Kozlowski, 2013; Fernández de la Mora, de Juan, Eichler, & Rosell, 1998;Kangasluoma et al., 2014;Maißer et al., 2011;Maißer, Barmpounis, Attoui, Biskos, & Schmidt-Ott, 2015;Wang et al., 2014). Mobility classification was achieved by operating the half-mini DMA at a high sheath flow rate in a closed sheath loop configuration. ...
... The utility of Collidoscope for interpreting structures of very large native-like biomolecular ions (with masses approaching ~1 MDa) is also discussed. (1) where g is the initial speed of the collision particle relative to the ion; b is the impact parameter (defined as the initial distance of the approaching particle to the collision axis, see Figure 1); θ, φ, and γ are the angles which define the relative orientation of the ion and collisional particle; k B is the Boltzmann constant; T is the absolute temperature; χ is the scattering angle; and μ is the reduced mass of the system [33,40,41]. (Note that the g 5 term reflects laboratory-frame orientational averaging of the particle and ion velocities, with probabilities given by Maxwell-Boltzmann distributions, as well as momentum transfer.) ...
Article
Ion mobility-mass spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, native-like or denatured proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a "trajectory method" (TM) based CCS calculator, named "Collidoscope," which utilizes parallel processing and optimized trajectory sampling, and implements both He and N2 as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared with those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using X-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions. Graphical Abstract ᅟ.
Article
Measurement of the gas-phase ion mobility of proteins provides a means to quantitatively assess the relative sizes of charged proteins. However, protein ion mobility measurements are typically singular values. Here, we apply tandem mobility analysis to low charge state protein ions (+1 and +2 ions) introduced into the gas phase by nanodroplet nebulization. We first determine protein ion mobilities in dry air and subsequently examine shifts in mobilities brought about by the clustering of vapor molecules. Tandem mobility analysis yields mobility-vapor concentration curves for each protein ion, expanding the information obtained from mobility analysis. This experimental procedure and analysis is extended to bovine serum albumin, transferrin, immunoglobulin G, and apoferritin with water, 1-butanol, and nonane. All protein ions appear to adsorb vapor molecules, with mobility "diameter" shifts of up to 6-7% at conditions just below vapor saturation. We parametrize results using κ-Köhler theory, where the term κ quantifies the extent of uptake beyond Köhler model expectations. For 1-butanol and nonane, κ decreases with increasing protein ion size, while it increases with increasing protein ion size for water. For the systems probed, the extent of mobility shift for the organic vapors is unaffected by the nebulized solution pH, while shifts with water are sensitive to pH.
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In structural biology, collision cross sections (CCSs) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density. Although assigning protein shapes purely on CCS data is not possible, we find that we can distinguish oblate- and prolate-shaped protein complexesby using the CCS, molecular weight, and oligomeric states to mine the Protein Data Bank (PDB) for potentially similar protein structures. Furthermore, comparing the CCS of a ferritin cage to the solution structures in the PDB reveals significant deviations caused by structural collapse on the gas phase. We then apply the strategy to an integral membrane protein by comparing the shapes of a prokaryotic and a eukaryotic sodium/proton antiporter homologue. We conclude that mining the PDB with IM-MS data is a time-effective way to derive low-resolution structural models.
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The initial stages of particle formation are important in several industrial and environmental systems; however, the phenomenon is not completely understood due to the inability to measure cluster size distributions. A high resolution differential mobility analyzer with an electrometer was used to map out the early stages of Si particle formation from pyrolysis of SiH4 in a furnace aerosol reactor. We detected for the first time subnanometer stable clusters from silane pyrolysis, and the diameter was measured to be about 0.7 nm. This diameter is within the range of probable sizes that the reported families of critical silane clusters could have based on their actual molecular structure. The size distributions of negative clusters are also mapped out. In addition, gas chromatography mass spectrometry, and transmission electron microscopy characterizations of the clusters and primary particles are used to assess their mechanistic roles in aerosol dynamics of the initial stages of particle formation.
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Measurement systems for particle sizing starting at 1 nm are used to bridge the gap between mass spectrometer measurements and traditional aerosol sizing methods, and thus to enable measurement of the complete size distribution from molecules and clusters to large particles. Such a measurement can be made using a scanning mobility particle sizer equipped with a diethylene glycol growth engine (e.g. TSI Model 3777 Nano Enhancer) along with a condensation particle counter, and a differential mobility analyzer (DMA) appropriate for such small sizes. Previous researchers have used high-resolution DMA (HRDMA) and also the TSI Nano-DMA (Model 3085) in such an SMPS system. In this study, we evaluate the performance of the recently introduced TSI 1 nm-DMA (Model 3086). The transfer function was characterized using 1–2 nm monomobile molecular ion standards. The same measurements were repeated on a TSI Nano-DMA, with good agreement to previously published values. From the measured transfer function, the resolution of each DMA model was determined as a function of particle size and sheath flow rate. Resolution of the TSI 3086 in the 1–2 nm range was 10–25% higher than the TSI 3085. Measured resolutions of the TSI 3086 were 10–20% lower than theoretically predicted values while those of the Model 3085 were 0–10% lower.
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While flame aerosol reactor (FLAR) synthesis of nanoparticles is widely used to produce a range of nano-materials, incipient particle formation by nucleation and vapor condensation is not well understood. This gap in our knowledge of incipient particle formation is caused by limitations in instruments, where, during measurements , the high diffusivity of sub 3 nm particles significantly affects resolution and transport loss. This work used a high resolution differential mobility analyzer (DMA) and an atmospheric pressure interface-mass spectrometer (APi-TOF) to observe incipient particle formation during flame synthesis. By tandemly applying these two instruments, differential mobility analysis-mass spectrometry (DMA-MS) measured the size and mass of the incipient particles simultaneously, and the effective density of the sub 3 nm particles was estimated. The APi-TOF further provided the chemical compositions of the detected particles based on highly accurate masses and isotope distributions. This study investigated the incipient particle formation in flames with and without the addition of synthesis precursors. Results from FLAR using two types of precursors including tetraethyl orthosilicate (TEOS) and titanium isopropoxide (TTIP) are presented. The effect of the precursor feed rates on incipient particle growth was also investigated.
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Full-text available
The slip correction factor has been investigated at reduced pressures and high Knudsen number using polystyrene latex (PSL) particles. Nano-differential mobility analyzers (NDMA) were used in determining the slip correction factor by measuring the electrical mobility of 100.7 nm, 269 nm, and 19.90 nm particles as a function of pressure. The aerosol was generated via electrospray to avoid multiplets for the 19.90 nm particles and to reduce the contaminant residue on the particle surface. System pressure was varied down to 8.27 kPa, enabling slip correction measurements for Knudsen numbers as large as 83. A condensation particle counter was modified for low pressure application. The slip correction factor obtained for the three particle sizes is fitted well by the equation: C = 1 + Kn (α + β exp(-γ/Kn)), with α = 1.165, β = 0.483, and γ= 0.997. The first quantitative uncertainty analysis for slip correction measurements was carried out. The expanded relative uncertainty (95 % confidence interval) in measuring slip correction factor was about 2 % for the 100.7 nm SRM particles, about 3 % for the 19.90 nm PSL particles, and about 2.5 % for the 269 nm SRM particles. The major sources of uncertainty are the diameter of particles, the geometric constant associated with NDMA, and the voltage.
Article
Full-text available
The validity of the Stokes-Millikan equation is examined in light of mass and mobility measurements of clusters of the ionic liquid 1-ethyl-3-methyl-imidazolium tetrafluoroborate (EMI-BF4) in ambient air. The mobility diameter dZ based on the measured mobility and the Stokes-Millikan law is compared with the volume diameter dv, which generalizes the mass diameter for binary substances such as salts. dv is based on the sum of anion and cation volumes in the cluster corrected for the void fraction of the bulk ionic liquid. For dv > 1.5 nm, dZ is within 1.4% of dv + 0.3 nm. For smaller clusters 3.84 and 14.3% deviations are observed at dv = 1.21 nm and 0.68 nm, respectively. These differences are smaller than expected due to a cancellation of competing effects. The increasing difference seen for dv < 1.5 nm is due primarily to the interaction between the cluster and the dipole it induces in the gas molecules. Other potential sources of disagreement are non-globular cluster geometries, and departures of the cluster void fraction from the bulk value. These two effects are examined via molecular dynamics simulations, which confirm that the volume diameter concept is accurate for EMI-BF4 nanodrops with dv as small as 1.6 nm.
Article
The charge state of ions produced in electrospray ionization (ESI) was reduced in a controlled manner to yield predominantly singly charged species by exposure of the aerosol to a bipolar ionizing gas. Analysis of the resulting ions on an orthogonal time-of-flight mass spectrometer yielded mass spectra greatly simplified compared with conventional ESI spectra. The decreased spectral complexity afforded by the charge reduction facilitates the analysis of mixtures by ESI mass spectrometry.
Article
Coefficient of viscosity for carbon dioxide, determined by the oil drop method, was found to be 1.478×10-4 for 23° C and 76 cm pressure. This agrees well with Van Dyke's value 1.472×10-4 obtained by the constant deflection method. The constant A in the modified form of Stokes' law came out 0.815. Motion of oil droplets in carbon dioxide, oxygen, and helium, at low pressures.-Measurements in these gases were extended to pressures of 1.9, 2.0, and 10 mm respectively, and to values of la, ratio of mean free path to oil drop radius, of 76, 130, and 71, respectively. The empirical correction factor to Stokes' law suggested by Knudsen and by Millikan, viz. f(la)=1+(la)(A+Be-Cal) was found to hold for all. Moreover the value of (A+B) comes out the same for all within the experimental error, and equal to 1.175. The gases were carefully purified. A new atomizer for use at these low pressures was developed; otherwise the apparatus and methods were the same as those used by Ishida. Comparison with motion of large ions. The formulas of Lenard and Langevin for large ions are both the same as the empirical law for droplets, but the Wellisch formula cannot be extended to droplets whose mass is large with respect to the mass of a molecule.
Article
A slip correction factor is used to correct Stokes' law for the fact that the no-slip boundary condition is violated for small aerosol particles moving with respect to the gaseous medium. The Knudsen-Weber form of the slip correction is given by C(Kn)= 1 + Kn[α + β exp(—γ/Kn)]. The parameters α, β, and γ are customarily those based upon the experiments reported in 1917 and 1923 by R. A. Millikan for aerosol droplets of oil. Because of differences in molecular interactions with the surfaces of solid particles and oil drops, different parameters should be appropriate for solid particles.In this study an improved version of the Millikan apparatus was designed, built, and used to measure the slip correction factors for 90 solid, spherical particles in air. Measurements were made on 11 polystyrene latex-divinylbenzene particles, 25 polyvinyltoluene particles, and 54 polystyrene latex particles, spanning a Knudsen number range from 0.03 to 7.2. The fitted nonlinear least squares values of α, β, and γ are 1.142 (±0.0024 SE), 0.558 (± 0.0024 SE), and 0.999 (±0.0212 SE), respectively, for the assumed mean free path of 0.0673 μm for air at sea level and 23°C with viscosity of 183.245 micropoise. This value of α agrees very closely with Millikan's result (adjusted to the same mean free path). The sum of α + β = 1.700 (applicable in the free molecular regime) was 4.45% higher than Millikan's value for oil droplets; this observation is consistent with the expectation that a higher percentage of molecules undergo specular reflections from the surface of a solid particle than from the surface of an oil droplet.
Article
The demand for analysis of nanosized particles and assemblies of biologic and inorganic origin has increased in the recent decade together with the growing development of biotechnology and nanotechnology. Recent developments of electrostatic differential mobility analysis (DMA) provide an excellent characterization tool in the nanometer size range. With an increasing number of available nano-DMA (nDMA) systems, the question of data comparability and implementation of possible calibration procedures arise. Here we present analysis of proteins in a range between 3 nm (5.7 kDa) and 15 nm (660 kDa) with five different nDMA systems. Results show differences in the obtained sizes up to 15% between different nDMA systems, which consequently leads to the conclusion that a calibration procedure for each nDMA is necessary when applying such systems for the analysis of nanoparticles with respect to size and molecular mass.
Article
A new technique for studying the time dependence of conformational changes of gas-phase protein ions is described. In this approach, a short pulse of electro-sprayed protein ions is introduced into an ion trap and stored. After a defined time period, the distribution of ions is ejected from the trap into an ion mobility/time-of-flight mass spectrometer. Combined measurements of mobilities and flight times in the mass spectrometer provide information about the abundances of different conformer types and charge-state distributions. By varying the storage time in the trap, it is possible to monitor changes in ion conformation that occur over extended time periods (∼10-200 ms). The method is demonstrated by examining changes in cytochrome c ion conformations for the +7 to +10 charge states.
Article
Recent progress in adding a mobility dimension to preexisting API–MS systems without modifying the MS itself is discussed, based on inserting a differential mobility analyzer (DMA) as part of the MS's atmospheric pressure ion source. Design criteria leading to high DMA resolving power R and transmission efficiency η are discussed. Various DMA prototypes have been interfaced to several triple quadrupoles, a single quadrupole and a quadrupole-TOF, all demonstrating R>50 and η>50%. We obtain two-dimensional DMA–MS spectra of the multiply charged clusters formed in electrosprays of concentrated solutions of tetrahexylammonium bromide (A+Br−). These reveal systematic loss of (ABr)A+ fragments from unstable multiply charged clusters, and provide mobility measurements in air on mass resolved (ABr)n(A+)z clusters with n>100 and z up to 10. Well-defined bands of ions not individually resolved are clearly visible at considerably larger n and z values.
Article
The dual goals of retaining native solution structure in the gas phase and facilitating accurate mass measurement by mass spectrometry often require conflicting experimental parameters. Here, we use ion mobility–mass spectrometry to investigate the effects of aqueous buffer removal on the structure of an archetypal ring complex, GroEL, an 800kDa chaperone protein complex from Escherichia coli. Our data show that subjecting the protein complex ions to energetic collisions in the gas phase removes aqueous buffer from the assembly in a manner indicative of at least two populations of adducts bound to the complex. Adding further energy to the system disrupts the quaternary structure of the assembly, causes monomer unfolding, and eventual dissociation at higher collision energies. Including additional salts of lower volatility in a typical ammonium acetate buffer produces gas-phase protein complex ions that are seemingly stabilised relative to changes in gas-phase structure. These data are combined to offer a general picture of the desolvation and structural transitions undergone by large gas-phase protein complexes.
Article
Aerosol particle detection and sizing techniques have recently been extended downwards to a size range commensurate with the size of many important biomolecules. Although such large molecules do not naturally occur individually as aerosol particles, electrospray- drying makes it feasible to generate an aerosol of isolated single macromolecules from macromolecule solutions. The combination of this electrospray generation with the improved aerosol techniques makes possible a new method of size analysis for biomolecules. We present characteristics of this new system and review some recent results.