Effects of Drift Gas on Collision Cross Sections of a Protein Standard

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Effects of Drift Gas on Collision Cross Sections of a Protein Standard in Linear Drift Tube and Traveling Wave Ion Mobility Mass Spectrometry Ewa Jurneczko,† Jason Kalapothakis,† Iain D. G. Campuzano,‡,§ Michael Morris,‡ and Perdita E Barran*,† †

The EastChem School of Chemistry, The University of Edinburgh, Edinburgh, United Kingdom Waters Corporation, Manchester, United Kingdom § Amgen, Inc., California, United States ‡

S Supporting Information *

ABSTRACT: There has been a significant increase in the use of ion mobility mass spectrometry (IM-MS) to investigate conformations of proteins and protein complexes following electrospray ionization. Investigations which employ traveling wave ion mobility mass spectrometry (TW IM-MS) instrumentation rely on the use of calibrants to convert the arrival times of ions to collision cross sections (CCS) providing “hard numbers” of use to structural biology. It is common to use nitrogen as the buffer gas in TW IM-MS instruments and to calibrate by extrapolating from CCS measured in helium via drift tube (DT) IM-MS. In this work, both DT and TW IM-MS instruments are used to investigate the effects of different drift gases (helium, neon, nitrogen, and argon) on the transport of multiply charged ions of the protein myoglobin, frequently used as a standard in TW IM-MS studies. Irrespective of the drift gas used, recorded mass spectra are found to be highly similar. In contrast, the recorded arrival time distributions and the derived CCS differ greatly. At low charge states (7 ≤ z ≤ 11) where the protein is compact, the CCS scale with the polarizability of the gas; this is also the case for higher charge states (12 ≤ z ≤ 22) where the protein is more unfolded for the heavy gases (neon, argon, and nitrogen) but not the case for helium. This is here interpreted as a different conformational landscape being sampled by the lighter gas and potentially attributable to increased field heating by helium. Under nanoelectrospray ionization (nESI) conditions, where myoglobin is sprayed from an aqueous solution buffered to pH 6.8 with 20 mM ammonium acetate, in the DT IM-MS instrument, each buffer gas can yield a different arrival time distribution (ATD) for any given charge state.

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estimate the effective helium CCS of mass-selected ions. There is a drawback to this approach as generally utilized: most measurements with DT IM-MS devices are made in helium, owing to the ability of this small buffer gas to effectively “pick out” molecular detail due to its low scattering cross-section, leading to a higher transmission efficiency and small polarizability volume (0.205 × 10−24 cm3). However, nitrogen is typically used as a buffer gas with commercial TW IM-MS instrumentation,7 and the arrival time of ions in TW IM-MS instruments is usually correlated to a CCS via a calibration procedure which utilizes CCS taken on DT IM-MS devices, effectively converting data taken in one buffer gas to an extrapolated CCS in another.8,9 This has allowed comparison between data on the TW IM-MS devices with the body of work from the DT IM-MS community. For more polarizable gases, such as argon or nitrogen, substantial charge-induced dipoles

n recent years, ion mobility mass spectrometry (IM-MS) has become an informative tool for structural biology.1−6 IM-MS determines the rotationally averaged collision cross-section of mass-selected ions by measuring their mobility through a given buffer gas. In a typical IM-MS experiment, a pulse of ions is injected into a drift cell filled with a buffer gas; ions drift through this under the influence of an electric field and are retarded by collisions with the buffer gas. The velocity of the ions is determined by: the strength of the electric field, the charge of the ion, the number of collisions, and the interaction potential between the neutral buffer gas particle and the ion. This last term is summarized by the collision cross-section (CCS) and relates to the geometry of the ion. There are three principal types of ion mobility instrumentation that have been coupled with mass spectrometry: linear drift tube (DT IM-MS), traveling wave ion mobility mass spectrometry (TW IM-MS), and differential mobility (such as field asymmetric waveform FAIMS). The first two are more commonly applied to the study of absolute conformations. TW IM-MS relies on measurements taken in DT IM-MS devices to © 2012 American Chemical Society

Received: May 9, 2012 Accepted: September 13, 2012 Published: September 13, 2012 8524

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and buffer gas density under the low field conditions is given by kinetic theory:

will influence every collision event, and this effect must be accounted for in structural assignment on the basis of observed momentum-transfer CCS. The existence of empirical parameters which are used to provide a helium CCS from atomic coordinates10−12 has encouraged the conversion of TW IM-MS nitrogen CCS to helium CCS. This situation may soon change since recent work has started to parametrize the momentumtransfer algorithm for nitrogen under ambient temperatures (301K) and low IM (∼2−3 Torr; both TW IM-MS and DT) pressure conditions.13 The established use of helium as the drift gas of choice can be defended. Helium is the lightest and least polarizable of the inert gases; as such, at room temperature and at higher temperatures, it is the atomic gas most amenable to modeling by an idealizing approximation as shown by the Trajectory Method.11 All other gases are more polarizable, and their interactions with isolated ions can no longer be considered as insignificant. Ion induced-dipole interactions will influence each individual ion-neutral scattering event, and a hard-sphere model may no longer be appropriate for representing collisions for polarizable drift gases when the long-range attractive potential has a greater effect.14 Early motivation for ion mobility investigations was to examine the effects of different buffer gases on the mobility of any given ion.15,16 This is beautifully exemplified by the work of Gatland et al.17 who showed that ion mobility measurements can be used to determine ion-neutral interaction potentials. Since it is well-known that a buffer gas will affect the CCS of ions, we were motivated to examine the effect of different buffer gases on the arrival time distributions of a well-studied protein (myoglobin), often used as a standard for conversion of TW IM-MS arrival times to CCS. The effect of buffer gas on CCS has recently been examined by Campuzano et al.13 for small molecules and also for globular proteins18 and has been the subject of a number of studies by Hill and co-workers19−23 as well as several other groups.24,25 Steiner et al.20 used an atmospheric pressure IM-MS to explore the behavior of several classes of amines in five different gases. The results showed an inverse relationship between the observed mobility and the mass and polarizability of the neutral drift gas. Asbury and Hill21 performed a study on a series of low molecular weight aniline compounds and peptides using ambient pressure IMMS. They found that the percentage change of mobility for each ion was different for different gases. Beegle et al.23 demonstrated a linear correlation between the polarizability of the buffer gas and the molecular ion radii, although this was dependent on the size of the ion, an observation we will return to in discussion of our data below. In this work, low-field DT IM-MS and TW IM-MS instruments are utilized. In a typical DT IM-MS experiment, the drift speed of ions, vd, is directly proportional to the electric field strength intensity, E, scaled by the mobility, K, as shown by eq 1. vd = KE

K=

1/2 3ze ⎛ 2π ⎞ 1 ⎜ ⎟ 16N ⎝ μkBT ⎠ Ω

(2)

where z is the charge state of an ion, e is the charge of an electron, μ is the reduced mass of the drift gas-ion pair, kB is the Boltzmann constant, T is the temperature of the drift gas, and Ω is the rotationally averaged CCS. The collision cross-section describes the probability of ion-neutral scattering events and is expected to depend both on the ion and buffer gas size and also on their interaction potential. The latter of these is strongly influenced by the polarizability of the gas. TW IM-MS makes use of a stacked ring ion guide (SRIG) as a mobility separator.26,27 In order to propel the ions through the SRIG, a traveling wave DC voltage is superimposed on top of a high-frequency radially confining RF voltage. This arrangement provides a periodic traveling wave which transports ions along the direction of wave propagation (thereby converting the SRIG to a traveling wave ion guide (TWIG)). At elevated gas pressure, ions of high mobility will be transported more efficiently than ions of lower mobility; their transit time through the device is shorter, permitting for TWIGs to serve as mobility separation devices. The transport phenomenon taking place in TWIG IMS instruments is analogous to Stokes drift; however, the exact dependence of drift velocity and effective diffusivity on field strength, pressure, and other parameters is so far undetermined. We explore here the effect of different drift gases on the recorded arrival time distribution for the protein standard myoglobin using DT IM-MS and TW IM-MS.

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EXPERIMENTAL SECTION Myoglobin (Mb) from equine heart was purchased from Sigma Aldrich (product number M1882; ≥90% essentially salt-free; mr 17 567.3 Da holo-myoglobin) as lyophilized solid and stored at −20 °C before use. Stock solutions of 1 mg·mL−1 (57 μM) were prepared in deionized water and dialyzed overnight (Slide-A-Lyzer Dialysis Cassettes; Thermo Scientific). Buffered myoglobin samples were prepared in 10 mM ammonium acetate at a concentration of 40 μM. Denatured samples were prepared in 1/1 (v/v) water/methanol with 0.2% formic acid added at a concentration of 20 μM. IM-MS experiments were performed on a home-built DT-IM-MS instrument and on a Synapt HDMS (Waters Corporation), described below. All Gases were obtained from BOC Ltd. Drift Tube IM-MS Instrument. The DT IM-MS experiments were performed on a quadrupole time-of-flight mass spectrometer that has been modified in-house to include a 5.1 cm copper drift cell.28 Ions were produced by positive nanoelectrospray ionization (nESI) using a Z-spray source, within a spray voltage range of 1.2−1.8 kV and a source temperature of 80 °C. The drift cell was filled with an appropriate buffer gas (helium, neon, nitrogen, argon), and the pressure was measured using a baratron (MKS Instruments). The average pressures of the drift gases and the average injections energies used during the experiments are listed in the Supporting Information (ST2). The temperature of the drift cell was monitored and recorded. The electric potential across the cell was varied from 12 to 2 V·cm−1. Ion arrival time distributions were recorded by synchronization of the release of ions into the drift cell with mass spectral acquisition. Using the theory described above, the mobility of each ion of interest was

(1) 15

This relationship holds only at the low-field limit, where the ratio of the electric field strength (E) to buffer gas density value (N) is small (less than 6 × 10−17 V·cm2 for singly charged ions drifting in helium at 300K). Under these conditions, the drift velocity is linearly dependent on the electric field strength. At higher E/N, the ions may align in the field and their mobilities become field dependent. The dependencies of ionic mobility on ion charge, temperature, ion and neutral masses, 8525

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Figure 1. nESI mass spectra of myoglobin sprayed from near-native conditions (10 mM ammonium acetate, pH 6.8) obtained on DT IM-MS instrument in (A) helium (combined over 200 scans), (C) nitrogen (combined over 200 scans), and (E) argon (combined over 400 scans). Drift time distributions (DTD) acquired for the [M + 8H] 8+ holo-myoglobin peak (m/z = 2197.0) taken at a drift voltage of 30 V at 300K (measured to 3 decimal places) in (B) helium (zE/N = 43.3), (D) nitrogen (zE/N = 101.3), and (F) argon (zE/N = 211.8).

and from denaturing (1/1 MeOH/H2O) solutions (Supporting Information Figure S1). The drift cell was filled with the selected buffer gas, and the pressure was optimized for signal transmission. Four different drift gases were utilized (helium, neon, argon, and nitrogen) covering a range of polarizability volumes and molecular weights (Supporting Information Table ST1). Irrespective of the drift gas used, the resulting mass spectra were very similar (Figures 1A,C,E, and S1, Supporting Information), and there was no significant change in peak width which could be attributed to differing desolvation effects. From the buffered solution, charge state distributions of the heme bound holo-Mb monomer were recorded with 7 ≤ z ≤ 10 (17566.8 Da ± 0.7) and additional multimeric species were also observed. In all cases, the most intense peak corresponds to the monomeric [M + 8H]8+ holo-Mb (m/z = 2197.0). At low pH, spectra obtained in helium, neon, nitrogen, and argon provide in all cases a wide charge state distribution (7 ≤ z ≤ 26) of apomyoglobin (16951.5 Da ± 0.5) accompanied by the heme group (mass 616.5 amu) where the high charges states of the protein are characteristic of unfolded conformations in solution.29 Nanoelectrospray ionization mass spectra of myoglobin were also acquired on the Synapt HDMS instrument with nitrogen

obtained from a plot of average arrival time versus pressure/ temperature, and from this, the rotationally averaged collision cross sections for each resolvable species at a given charge state were obtained using eq 2 above. Each experiment was performed in triplicate. The mean CCS for all recorded myoglobin ions are listed in the Supporting Information (ST3). Synapt HDMS Instrument: TW IM-MS. The TW IM-MS instrument was operated in positive electrospray ionization mode, with a capillary voltage set between 0.5 and 1.5 kV and the cone voltage set to 65 V. Source conditions were optimized for signal transmission. Velocity of the traveling wave was set to 250 m·s−1, and varied wave heights were employed to obtain optimal signal. Each drift gas was introduced directly into to the ion mobility region at pressures 0.79 Torr (nitrogen) and 0.93 Torr (neon) as recorded on a Pirani gauge. Each experiment was performed at three different wave heights, in each gas: 13, 14, and 15 V in nitrogen and 12, 13, and 14 V in neon.

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RESULTS AND DISCUSSION Mass Spectrometry. Mass spectra were obtained on the DT IM-MS instrument, for myoglobin sprayed from nearneutral (10 mM ammonium acetate pH 6.8) (Figure 1A,C,E) 8526

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Figure 2. (A) The effects of four different drift gases (helium, neon, nitrogen, and argon) on the collision cross-section of all of the observed charge states of apo-myoglobin obtained on DT IM- MS from pH 1.5 solution at 300K. (B) The effects of three different drift gases (helium, nitrogen, and argon) on the collision cross-section of all of the observed charge states of holo-myoglobin obtained on DT IM- MS from pH 6.8 solution at 300K. Table ST3 in Supporting Information details the data.

cm2·V−1·s−1; Figure 1D). For identical collision cross sections, lower values of K0 are expected in argon and nitrogen; both gases have larger masses than helium, but the relationship between K0 and buffer gas mass is not quite sufficient to explain the fact that K0 for nitrogen is larger than that for argon. Quantitatively (ignoring the protein ion mass), K0 should be greater by (40/28)2 = 1.20 whereas we find a value of 0.88. The reason for this discrepancy could be attributed to the differing polarizabilites of the two gases (Supporting Information Table ST1). Diatomic nitrogen will interact more strongly at short distances with the molecular ion than argon or helium: each nitrogen atom is actually negatively charged, and the center of the N2 molecule is positively charged (based on results from B3LYP DFT using the basis set aug-cc-pVDZ basis set30); molecular nitrogen is also larger than argon. In addition, it may be that the greater effects of momentum transfer into rotational and vibrational energy for the diatomic gas compared to the monatomic gas causes the increase in K0 for nitrogen. The larger, diatomic more polarizable molecule effectively lengthens the drift time, resulting in correspondingly larger values of Ω obtained as reported in Figure 1D for [M + 8H]8+ and in Supporting Information (Table ST3) for all other observed charge states of myoglobin sprayed from buffered solution. We can compare the CCS obtained from monomeric holomyoglobin (7 ≤ z ≤ 11), and for each of these charge states, we find HeΩ < ArΩ < N2Ω (Supporting Information Table ST3). For conformations of myoglobin with charge states that are considered to be “native-like”,31 the dominant factor in determining Ω is the polarizability of the buffer gas. We have calculated the values of E/N for each of the gases under the experimental conditions, and these are reported in Figure 1. The E/N ratio is lowest for helium, but all remain within low

or neon being introduced into the mobility cell (Supporting Information Figure S2). The charge state distributions of myoglobin are comparable with those obtained on the DT IMMS instrument, with the same base peaks in both sets of spectra. The intensity of multimeric species is more pronounced in the spectra acquired on the DT IM-MS instrument, hinting either at a gentler desolvation process in the source relative to that of the Synapt and/or dissociation of the weakly bound multimers in the TW IM-MS instrument. DT Ion Mobility-Mass Spectrometry. Along with mass spectrometry, ion mobility analysis has been performed on myoglobin sprayed from the two different solutions in the presence of different drift gases. Arrival time distributions acquired for the 8+ holo monomer (m/z = 2197.0) at a drift voltage of 30 V are shown in Figure 1B,D,F. A single resolvable species is observed in each of the three buffer gases, although the actual time spent in the drift cell varies, due to the different pressures used, as well as the varying mobility in each gas. There are differences in the width of the arrival time distribution (ATD) for each gas. This is best seen by comparing the ATD in helium (Figure 1B) with that in argon (Figure 1F) where the average drift times are similar (1.47 ms cf 1.46 ms) but the width of the peaks differs; fitting the ATDs yields effective diffusion coefficients of 0.00872 m2·s−1 for helium versus 0.00355 m2·s−1 for argon. This observation may be attributed to different gas pressures in the drift cell and the surrounding chamber and/or the nature of the buffer gas dependent collisions on the conformational spread of the ions. The largest reduced mobility for the [M + 8H]8+ ion is obtained in helium (K0 = 2.4 cm2·V−1·s−1, Figure 1B), and the smallest is in argon (K0 = 0.503 cm2·V−1·s−1, Figure 1F) with nitrogen providing a marginally greater value (K0 = 0.572 8527

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field limits for the reported CCS and charge states and are comparable to that used in other studies.18,32 We have extended the range of charge states over which we can compare the effect of different drift gases on the reported Ω of myoglobin with the use of different solution conditions, while retaining gentle desolvation conditions in our DT IM-MS source. Figure 2A,B reports the CCS of myoglobin versus its charge state for each buffer gas obtained by DT IM-MS from both pH 6.8 and 1.5 solutions. For z = 7, 8, 9, and 10, there is a significant difference between the obtained CCS values (Figure 2A); for example, the [M + 9H]9+ (apo-myoglobin) has two distinguishable conformers, with CCS 1640 Å2 and 2349 Å2 in helium, 2536 Å2 and 2759 Å2 in neon, 2971 Å2 in argon, and 2861 Å2 and 3267 Å2 in nitrogen. The range of values may be attributed to the effect of different buffer gases at different pressures on the conformation of the molecular ion and/or to the inherent interactions between the buffer gas and the molecular ion and highlights the danger in rescaling IM-MS data recorded in different buffer gases. Under buffered conditions where the charge state distribution of holo-myoglobin is centered on a lower m/z range (Figure 1A,C,E), the corresponding values for Ω for 6 ≤ z ≤ 9 are shown in Figure 2B. Again, we find HeΩ < ArΩ < N2Ω, but the difference between the heavier gases is smaller. Where z = 6, the HeΩ shows two conformers, both of which indicate a collapsed state, whereas for z = 7, 8, and 9 there is little change in the values found for HeΩ, although a more extended form appears where z = 9. For the heavier gases, at all other charge states around the dominant [M + 8H]8+ species, there is more than one conformer present. This is an interesting finding, suggesting that the stability of the holo- protein in a single resolvable fold is favored for [M + 8H]8+ and starts to be more disrupted for other charge states. This is not the case for the apo form (Figure 2A and Supporting Information ST3A) where the [M + 8H]8+ species has more than one conformer in both helium and nitrogen. For higher charge states, Ω obtained in each gas are less different (Figure 2A). However, they are not identical, and the correlation of Ω to the polarizability and mass of the buffer gas breaks down. For z > 11, the reported CCS in helium and argon are highly similar and found to be larger than those in neon and smaller than those in nitrogen. So for z > 11, the order of CCS becomes NeΩ < ArΩ ≈ HeΩ < N2Ω. For the three heavy gases, this order is the same as the polarizability. The anomaly here is helium, which one would predict to provide smaller Ω values for an equivalent geometry than that found for the heavier gases. This is the case in all other studies (see, for example, refs 23 and 25), usually on singly charged molecules and in this work where z < 12. The previous reports by Asbury and Hill21 and Beegle et al.23 also show that different ions are affected differently by gases of different polarizabilites, attributable to conformational differences and due to dipole and multipole effects (which will themselves be charge state and conformation dependent). We can hypothesize that the discrepancy here is due to either the interactions that helium has with the partially unfolded high charge states of apomyoglobin or that measurements made in helium cause or potentiate different unfolded geometries to those found with other gases. Helium atoms with their small radii may become trapped in cavities of the protein that are not accessible to the larger gases in the more folded forms, and this may assist the

Coulombically driven unfolding of the protein. This hypothesis could be explored with force field simulations. For equivalent E/N values, field heating effects will be greatest for ions in helium15 although this is a small effect at low fields, and this too could contribute to the sharp substantial increase in CCS for myoglobin from z = 8 to z = 10. The relationship between field heating (ΔT) and mobility is given by the following:15 ΔT = M(KE)2 /3kB

(3)

where M is the buffer gas molecule mass and kB is the Boltzmann constant. According to eq 3, ΔT scales with Ω−2. Using this model, we can estimate field heating for the experiments described herein. These are found to be small, with ΔT ranging between 0.013 and 0.93 K for 8 ≤ z ≤ 17 in helium and 7.88 and 21.69 K for 8 ≤ z ≤ 17 in argon; the effect in argon is greatest due to the lower pressure we have had to employ for this gas. These small values appear insufficient to cause the effects we observe, although some aspects of field heating are not accounted for by the model of equation: the effect of field heating above the gas temperature has been previously shown to give a preference to extended forms of peptides and proteins in helium over nitrogen,24 which is in support of our findings. Here, for the most intense conformer of the [M + 8H]8+, the ion of myoglobin gives HeΩ = 2196 Å2 and N2Ω = 2836 Å2, an increase of ∼30%, and the corresponding increase in ΔT is ∼0.5 K for helium and ∼1.7 K for nitrogen which we would not expect to activate different unfolding channels. We have compared the CCS we obtain in helium with those reported by Clemmer33 and Bush et al.,18 this data is presented in Supporting Information Figure S4. We see broad agreement in the magnitude of the HeΩ values, but there are noticeable differences. This suggests that ion source conditions, ion transmission, and the trapping of ions prior to injection into the drift cell will affect the observable conformations of apomyoglobin as previously shown by Baker et al.24 and by Ruotolo et al.8 who discourage the use of CCS from charge states where z < 13 in calibrating TW IM-MS data. All of these instrumental factors (the latter two of which will be different with the use of different gases) could cause unfolding and compaction along different conformational landscapes with apo-myoglobin. In particular, the effect of ion injection into the drift region must be considered. Ion heating in TW IM-MS instruments has been predominantly attributed to activation during the injection of ions into the traveling wave IMS cell.34,35 Increasing the energy at which ions are injected into DT IM-MS devices is also well-known to alter observed conformations36 and also can be employed to dissociate complexes via thermal activation.37,38 In these experiments, the use of higher mass drift gases would increase the likelihood of collisional activation, although we work at lower pressures for the heavier gases which will decrease this effect. For each of the gases, we worked at the lowest injection energy at which we saw good ion transmission (Supporting Information ST2). When coupled with our CCS data from other gases (Figure 2A,B), we can summarize that the three heavy gases probably have similar effects on the transmission and unfolding/ annealing of myoglobin and that the different reported CCS (Figure 2 and Supporting Information ST1) are due to polarizability effects. In contrast, helium appears to unfold/ anneal the protein differently, most likely mediated by field 8528

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Figure 3. Arrival time distributions (ATD) acquired for the [M + 7H] 7+ holo-myoglobin peak (m/z = 2510.7) taken at a drift voltage of 20 V at 300K (measured to 3 decimal places) obtained on DT IM-MS in (A) helium (zE/N = 25.4), (C) nitrogen (zE/N = 54.2), and (D) argon (zE/N = 122.9); (B) shows the ATD acquired for the [M + 7H] 7+ apo-myoglobin peak (m/z = 2422.6) taken at a drift voltage of 20 V at 300K (zE/N = 62.2). DTD obtained on TW-IM-MS in nitrogen (IMS wave height = 14 V, wave velocity = 250 m/s) or neon (IMS wave height = 12 V, wave velocity 250 m/s) in the mobility cell are presented in (E) and (F), respectively.

the temperatures of the DT IM-MS experiments performed here. Different gases will contribute differently to heating the ion in a given electric field; this will in turn lead to different behavior in the DT IM-MS instruments with injected ions as the one used here, compared with that in a TW IM-MS instrument where fields vary both temporally and spatially. TW versus DT Ion Mobility Mass Spectrometry. The use of different drift gases in ion mobility experiments can alter the resolving power.39 In TW IM-MS, higher resolving powers have been achieved for a given drift tube length with gases that are larger and more polarizable than helium. In addition, more polarizable gases can withstand higher voltages before breakdown occurs.22 Consequently, if the ion residence time in the drift tube is sufficiently long to reduce the bandwidth contributions of the initial pulse width, higher voltage applied across the drift tube will result in better resolution. With higher resolving powers, the geometry of an ion can be determined to a higher degree of detail. Here, Figure 3 presents the arrival time distributions of the 7+ holo-myoglobin peak at m/z 2510.67 (obtained via nESI from buffered conditions). Data were obtained on the DT IM-MS instrument in helium, neon, nitrogen, and argon (Figure 3A−D) and also on a Synapt

heating effects, and we conclude that the geometry of the protein for each m/z value is more different with helium than with the other gases. The data reported here demonstrates that the effects of the interaction between the gases and the ion are significant and must be accounted for in order to fully understand the transport phenomena in drift tubes. Contributions from the complex geometries of ionized proteins are present (protein ion has a complex surface with which the gas in question interacts); furthermore, the strength of the interaction potential also comes into play. Steiner et al.20 as well as Kim et al.14 have reported on the effects of the dipole and quadrupole moments on the scattering of both atomic and molecular gases (including N2) with a series of amines. These dependencies may originate from the geometry of the gas molecule and will be further accentuated for multiply charged electrosprayed proteins, where the location of charge depends on (and influences) the structure of the protein. The presence of these strong attractive forces will complicate individual collisions. In addition to that, the presence of a large polyatomic ion may give rise to a complex potential profile (potential wells might even arise at longer distances) although this not ought to be significant at 8529

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other gases. Helium also seems to facilitate a very dramatic unfolding of the protein between z = 8 and z = 10, perhaps due to stronger field heating on ion injection into the drift field afforded by this lighter gas, whereas the other gases show a lesser change over the same charge state range. The fact that different gases will provide a different CCS for an identical conformation of a given ion is a known known. What is additionally clear from the data we present here is that different gases can affect the conformations presented by molecular ions in the gas-phase, particularly for native forms. A correct interpretation of ion mobility data on any protein must reconcile these effects.

HDMS in nitrogen and neon (Figure 3E,F). For the DT IMMS acquired data, in helium only, one conformation is resolvable (Figure 3A), whereas in all other gases at least three conformational families are present and at differing relative intensities. The intensity of the most compact conformer in nitrogen is the highest in intensity, in contrast with argon where the compact species is the lowest in intensity. This further supports the argument above that care must be taken when comparing CCS data taken in different gases. DT IM-MS data acquired in nitrogen is qualitatively comparable to that taken on the Synapt (Figures 3C,E). In contrast, the DT IM-MS data for neon is different (Figure 3B), and the Synapt neon ATD (Figure 3F) resembles that taken in nitrogen on both instruments. Similar resolving powers can be accomplished with neon as the drift-gas in the mobility cell in the TW IM-MS instruments as with nitrogen. To achieve a comparable separation, the pressure of the neon gas in the mobility cell had to be increased marginally by 0.15 Torr to a final pressure of 0.93 Torr. The data suggest that, in some cases, conformational selectivity can be achieved through a use of a different drift gas. However, the choice of drift gas must strike a balance between interaction potential and transmission efficiency. The design/operation of TW IM-MS instrumentation can also have an effect on the sampled configurations and subsequent reported CCS values owing to potential ion heating; this has been illustrated by De Pauw and Gabelica35 as well as by Williams et al.34 Furthermore, CCS values taken in nitrogen for apo-myoglobin on a rf-confined linear drift tube instrument18 are larger than those we report here, whereas helium values compare well (Supporting Information Figure S3), and it has previously been reported that rf-confinement in funnels can influence charge state distributions due to space charge effects and rf heating,24,40 both of which presumably also will influence gas phase conformations.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Waters MS technologies and the BMSS for financial support of our work. This work has been funded by the award of a BBSRC Strategic Industrial Case studentship to E.J. in collaboration with Waters MS technologies. We thank Alex Shvartsburg for his critical reading of this manuscript.

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REFERENCES

(1) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240−2248. (2) Badman, E. R.; Hoaglund-Hyzer, C. S.; Clemmer, D. E. Anal. Chem. 2001, 73, 6000−6007. (3) Valentine, S. J.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 2002, 13, 506−517. (4) Gidden, J.; Ferzoco, A.; Baker, E. S.; Bowers, M. T. J. Am. Chem. Soc. 2004, 126, 15132−15140. (5) Bernstein, S. L.; Liu, D.; Wyttenbach, T.; Bowers, M. T.; Lee, J. C.; Gray, H. B.; Winkler, J. R. J. Am. Soc. Mass Spectrom. 2004, 15, 1435−1443. (6) Jurneczko, E.; Barran, P. E. Analyst 2011, 136, 20−28. (7) Giles, K.; Williams, J. P.; Campuzano, I. Rapid Commun. Mass Spectrom. 2011, 25, 1559−1566. (8) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S.-J.; Robinson, C. V. Nat. Protoc. 2008, 3, 1139−1152. (9) Leary, J.; Schenauer, M.; Stefanescu, R.; Andaya, A.; Ruotolo, B.; Robinson, C.; Thalassinos, K.; Scrivens, J.; Sokabe, M.; Hershey, J. J. Am. Soc. Mass Spectrom. 2009, 20, 1699−1706. (10) Shvartsburg, A. A.; Jarrold, M. F. Chem. Phys. Lett. 1996, 261, 86−91. (11) Mesleh, M. F.; Hunter, J. M.; Shvartsburg, A. A.; Schatz, G. C.; Jarrold, M. F. J. Phys. Chem. 1996, 100, 16082−16086. (12) Alexandre, A. S.; George, C. S.; Martin, F. J. J. Chem. Phys. 1998, 108, 2416−2423. (13) Campuzano, I.; Bush, M. F.; Robinson, C. V.; Beaumont, C.; Richardson, K.; Kim, H.; Kim, H. I. Anal. Chem. 2012, 84, 1026−1033. (14) Kim, H. I.; Kim, H.; Pang, E. S.; Ryu, E. K.; Beegle, L. W.; Loo, J. A.; Goddard, W. A.; Kanik, I. Anal. Chem. 2009, 81, 8289−8297. (15) Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases; Wiley: New York, 1988. (16) Hogg, A. M.; Kebarle, P. J. Chem. Phys. 1965, 43, 449−456.

CONCLUSIONS AND OUTLOOK We have shown that different buffer gases yield different collision cross sections for each observed charge state of the same protein. For compact globular forms of myoglobin, the determined values for CCS scale with the polarizability of the gas, and this is also the case for higher charge states, where more extended forms of the protein are presented but only for the heavy gases (neon, nitrogen, and argon). Helium gives anomalous results, the CCS for z > 12 are greater in helium than in neon and are very similar to the recorded CCS in argon. We interpret this as being due to the lighter gas potentiating different unfolded forms of this protein. At very low charge states, where the protein is likely to be folded, the CCS in the heavier gases are significantly larger than for helium; this also could be an unfolding or compaction phenomena but can be interpreted as being due to the effects of the polarizable gas. We find that different conformations and populations of conformers are revealed by different drift gases, in DT IM-MS. TW IM-MS provides data that are qualitatively similar in two buffer gases (N2 and Ne) but that differ from that provided by DT IM-MS. Myoglobin undergoes significant change in the recorded values of HeΩ from 1562 Å2 for [M + 8H]8+ to 3644 Å2 for [M + 24H]24+. This change is far greater than that achieved by the other gases and points to a different unfolding landscape in the gas phase for the lighter gas. It appears that, at low charge states, compact (or compacted) forms of myoglobin are present with helium as the buffer gas, and these are not seen for the 8530

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(17) Gatland, I. R.; Viehland, L. A.; Mason, E. A. J. Chem. Phys. 1977, 66, 537−541. (18) Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Robinson, C. V.; Ruotolo, B. T. Anal. Chem. 2010, 82, 9557−9565. (19) Matz, L. M.; Hill, H. H.; Beegle, L. W.; Kanik, I. J. Am. Soc. Mass Spectrom. 2002, 13, 300−307. (20) Steiner, W. E.; English, W. A.; Hill, H. H. J. Phys. Chem. A 2006, 110, 1836−1844. (21) Asbury, G. R.; Hill, H. H. Anal. Chem. 1999, 72, 580−584. (22) Hill, H. H.; Hill, C. H.; Asbury, G. R.; Wu, C.; Matz, L. M.; Ichiye, T. Int. J. Mass Spectrom. 2002, 219, 23−37. (23) Beegle, L. W.; Kanik, I.; Matz, L.; Hill, H. H. Int. J. Mass Spectrom. 2002, 216, 257−268. (24) Baker, E. S.; Clowers, B. H.; Li, F.; Tang, K.; Tolmachev, A. V.; Prior, D. C.; Belov, M. E.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2007, 18, 1176−1187. (25) Brandon, T. R.; John, A. M.; Kent, J. G.; David, H. R. J. Mass Spectrom. 2004, 39, 361−367. (26) Giles, K.; Pringle, S. D.; Worthington, K. R.; Little, D.; Wildgoose, J. L.; Bateman, R. H. Rapid Commun. Mass Spectrom. 2004, 18, 2401−2414. (27) Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.; Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. Int. J. Mass Spectrom. 2007, 261, 1−12. (28) McCullough, B. J.; Kalapothakis, J.; Eastwood, H.; Kemper, P.; MacMillan, D.; Taylor, K.; Dorin, J.; Barran, P. E. Anal. Chem. 2008, 80, 6336−6344. (29) Konermann, L. J. Phys. Chem. B 2007, 111, 6534−6543. (30) Kim, H.; Kim, H. I.; Johnson, P. V.; Beegle, L. W.; Beauchamp, J. L.; Goddard, W. A.; Kanik, I. Anal. Chem. 2008, 80, 1928−1936. (31) Kuprowski, M. C.; Konermann, L. Anal. Chem. 2007, 79, 2499− 2506. (32) http://www.indiana.edu/∼clemmer/Research/ Cross%20Section%20Database/cs_database.php, accessed 9/26/2012. (33) Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 2002, 117, 10141−10142. (34) Merenbloom, S. I.; Flick, T. G.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2012, 23, 553−562. (35) Morsa, D.; Gabelica, V.; De Pauw, E. Anal. Chem. 2011, 83, 5775−5782. (36) Mao, Y.; Woenckhaus, J.; Kolafa, J.; Ratner, M. A.; Jarrold, M. F. J. Am. Chem. Soc. 1999, 121, 2712−2721. (37) Bernstein, S. L.; Dupuis, N. F.; Lazo, N. D.; Wyttenbach, T.; Condron, M. M.; Bitan, G.; Teplow, D. B.; Shea, J.-E.; Ruotolo, B. T.; Robinson, C. V.; Bowers, M. T. Nat. Chem. 2009, 1, 326−331. (38) Han, L.; Hyung, S.-J.; Ruotolo, B. T. Angew. Chem., Int. Ed. 2012, 51, 5692−5695. (39) Ruotolo, B. T.; Gillig, K. J.; Stone, E. G.; Russell, D. H. J. Chromatogr., B 2002, 782, 385−392. (40) Koeniger, S. L.; Merenbloom, S. I.; Valentine, S. J.; Jarrold, M. F.; Udseth, H. R.; Smith, R. D.; Clemmer, D. E. Anal. Chem. 2006, 78, 4161−4174.

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