Letter pubs.acs.org/ac
High Resolution Trapped Ion Mobility Spectrometery of Peptides Joshua A. Silveira, Mark E. Ridgeway, and Melvin A. Park* Bruker Daltonics, 40 Manning Road, Billerica, Massachusetts 01821, United States S Supporting Information *
ABSTRACT: In the present work, we employ trapped ion mobility spectrometry (TIMS) for conformational analysis of several model peptides. The TIMS distributions are extensively compared to recent ion mobility spectrometry (IMS) studies reported in the literature. At a resolving power (R) exceeding 250, many new features, otherwise hidden by lower resolution IMS analyzers, are revealed. Though still principally limited by the plurality of conformational states, at present, TIMS offers R up to ∼3 to 8 times greater than modern drift tube or traveling wave IMS techniques, respectively. Unlike differential IMS, TIMS not only is able to resolve congested conformational features but also can be used to determine information about their relative size, via the ion-neutral collision cross section, offering a powerful new platform to probe the structure and dynamics of biochemical systems in the gas phase.
the rising edge of the electric field gradient against a flow of neutral gas that pushes ions toward the exit. When the magnitude of the electric field gradient is decreased, ions elute according to their mobility (K). Unlike drift tube IMS, R in TIMS is dependent upon user-defined parameters including the voltage scan rate (δ) and the neutral gas velocity (vg).20−22 For proteomics applications requiring high throughput analysis with high sensitivity, δ can be increased (∼1000 V/s) such that modest IMS separation is achieved and the instrument duty cycle (the ratio of accumulation time to the total analysis time) is maximized. In contrast, we present results from an alternate mode of operation wherein TIMS parameters are optimized for high resolution separation of peptides known to display physicochemical-dependent conformational distributions in the gas phase. The work presented below shows that, currently, the TIMS analyzer can be calibrated to yield accurate Ω values at a resolving power exceeding 250 such that many new conformational features are revealed.
I
on mobility spectrometry (IMS) involves the electrophoretic transport of ionic species through a neutral gas medium.1,2 Recently, interest in reduced pressure (∼0.5 to 5 Torr) IMS has renewed, as advances in instrumentation coupling IMS to mass spectrometry have improved the analytical figures-of-merit resulting in extended applicability for a range of chemical3−6 and biological7−11 applications. Contemporary IMS technology emerged from early drift tube IMS experiments wherein a voltage gradient facilitates ion transport through a static buffer gas. In drift tube IMS, theoretical resolving power (R) is given by the following expression12 1/2 1 ⎛ qLE ⎞ R= ⎜ ⎟ 4 ⎝ ln 2k bT ⎠
(1)
where q is the ion charge, T is the temperature, kb is Boltzmann’s constant, L is the length of the drift tube, and E is the electric field. Because determination of the ion-neutral collision cross section (Ω) from first-order principles requires electric field conditions below the low-field limit, modern instrument platforms commonly feature long (≳1 m) drift tubes to achieve high resolution (R ∼ 50 to 120). While R ∼ 70 is sufficient for many applications (i.e., differentiation of chemical classes and determination of Ω values),13 the conformational multiplicity of biomolecules demands higher resolution. Owing to practical limitations of drift tube IMS, a variety of nontraditional approaches have been explored including traveling wave IMS,14 differential IMS,15−17 and pulsed cyclotron IMS.18,19 Here, we present results from an alternate approach, termed trapped IMS (TIMS), whereby R is no longer principally limited by the geometric length of the device and can be easily tuned in accordance with the particular analytical challenge. TIMS utilizes alternating (rf) voltages to confine ions radially and an axial (dc) voltage component to trap ions along © 2014 American Chemical Society
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EXPERIMENTAL SECTION Peptide solutions of bradykinin (BK, RPPGFSPFR, >98% purity), substance P (SP, RPKPQQFFGLM-NH2, >95% purity), and angiotensin I (AT, DRVYIHPFHL, >90% purity) were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Samples were prepared at ∼600 nM in 50:50 water/acetonitrile containing 0.1% formic acid and directly infused at 150 μL/h into an electrospray ionization (ESI)-QqTOF instrument (Impact HD, Bruker Daltonics, Billerica, MA) equipped with a prototype TIMS tunnel. The details of the instrumentation have been previously deReceived: April 7, 2014 Accepted: May 26, 2014 Published: May 26, 2014 5624
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scribed.22,23 Briefly, the TIMS analyzer is composed of three regions: an entrance funnel, TIMS tunnel (axial length = 46 mm), and exit funnel. Eight hundred and fifty kHz rf (180 V peak-to-peak) is applied to each section creating a dipolar field in the funnel regions and a quadrupolar field inside the tunnel. The ion accumulation time in a single analysis cycle was 28 ms. All data were summed for 100 analysis cycles (≲1 s/cycle) yielding analysis time on the order of ∼1.5 min per frame. Though a single analysis frame typically provides adequate ion statistics to represent the entire TIMS distribution, 10 frames were acquired for all peptides studied herein. Determination of ion-nitrogen Ω values by TIMS has been recently described.22 Briefly, the elution voltage (Ve) for each analyte was extrapolated from a plot of the instantaneous voltage (VTIMS) applied across the TIMS tunnel versus 1/δ. Because the electric field gradient profile was scanned linearly with time, the subsequent plot of reduced mobility (K0) versus 1/Ve was linear (R2 = 0.9990). Using this calibration procedure, the voltage axis was converted to K0 (and subsequently Ω) using values for [BK + 3H]3+ previously measured by drift tube IMS in nitrogen gas.24 All resolving power values reported herein were determined from peak fitting features in the TIMS distributions using the expression R = VTIMS/ΔVTIMS = K /ΔK
(2)
Additional details regarding the calibration methods can be found in the Supporting Information.
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Figure 1. Typical TIMS distributions of bradykinin (a,b), angiotensin I (c), and substance P (d). In (a−c), δ was constant (82.7 V/s) whereas in (d) δ was progressively decreased, as indicated in the legend, by increasing the scan time. The trap time prior to TIMS analysis was 14 ms (black trace) unless otherwise specified (blue trace, 155 ms) in the legend. All data were collected at an entrance pressure of 4.3 mbar with the exception of the red trace in (d) that was acquired at 3.4 mbar.
RESULTS AND DISCUSSION Singly, doubly, and triply charged forms of BK have been extensively studied by a variety of mass spectrometry25−29 and IMS techniques.19,30−38 Consistent with drift tube IMS studies, the TIMS distribution for [BK + 3H]3+ shown in Figure 1a contains three sharp features (Ω = 420, 437, and 456 Å2) of similar peak widths, yielding resolving power in the range of ∼120 to 150. These features correspond to the quasiequilibrium structures [BKA + 3H]3+, [BKB + 3H]3+, and [BKC + 3H]3+ reported by Clemmer et al.33,35 Through residue substitution, it has been shown that cis−trans isomerization of the three proline residues are responsible for producing these interconverting states.37 Here, it is noteworthy that appreciable abundances of both [BKA + 3H]3+ and [BKB + 3H]3+ are present, indicating that ion source and TIMS conditions are sufficiently gentle such that an energetically favored gas phase equilibrium distribution (consisting of ∼80% [BKC + 3H]3+) has not yet been reached.33 Similarly, differential IMS coupled with cold ion spectroscopy experiments by Rizzo et al. demonstrated that [BK + 2H]2+ also exhibits conformations that readily convert upon mild collisional activation in the gas phase.39 While differential IMS measurements by Shvartsburg have separated six features for this species,40 drift tube IMS experiments by Bowers,41 Clemmer,33 and Russell42 have unanimously shown that the [BK + 2H]2+ distribution is composed of two conformational families. Similarly, the TIMS distribution for [BK + 2H]2+ shown in Figure 1b contains two well-resolved features occurring at Ω = 339 ([BKA + 2H]2+) and 344 Å2 ([BKB + 2H]2+). The experimentally measured Ω values are in agreement (≤2% error) with previous drift tube IMS measurements (Ω = 332 and 340 Å2).24 However, not all peptide ions display expansive conformational heterogeneity in the gas phase. For example, the TIMS distribution of [AT + 3H]3+ shown in Figure 1c contains only a
single feature at Ω = 480 Å2. The measured Ω is in good agreement (≤2% error) with drift tube IMS measurements (Ω = 473 Å2).24 However, it is noteworthy that, though the peak shape for AT is fairly Gaussian, the measured peak width is considerably larger than the other peptide systems investigated herein, suggesting the absence of structural rigidity and/or that many congested unresolved geometries are present. Very recently, it has been shown that, upon ESI, a kinetically trapped family of desolvated triply charged SP conformations is produced by stepwise evaporation of solvent molecules that are stabilized in the gas phase by an ensemble of intramolecular interactions ([SPA + 3H]3+).43 Upon mild collisional activation, these interactions are disrupted, and energetically favored extended states are produced ([SPB + 3H]3+). On the basis of the peak widths, it was inferred that multiple conformers were present but could not be differentiated by drift tube IMS at R ∼ 70. Figure 1d shows the TIMS distribution of [SP + 3H]3+ ions as a function of several user-defined parameters including pressure, trap time, and δ. At reduced pressure, the TIMS distribution contains two broad features at Ω ∼440 and 500 Å2 that very closely resemble the previously reported drift tube IMS bimodal distribution.43 In an attempt to resolve additional conformational features, the pressure in the entrance funnel was increased to 4.3 mbar. The substantial enhancement in resolution at higher pressure is attributed to utilization of higher electric fields (see eq 1) required to trap ions when vg is increased. It is estimated that, at a pressure of 4.3 mbar, vg ∼100 m/s, assuming continuous flow inside the tunnel; however, 5625
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detailed effects of flow dynamics are still an ongoing topic of further research. Nevertheless, under optimal pressure conditions, δ was decreased from 124 to 70.9 V/s such that several conformational features, especially [SPA3 + 3H]3+, were progressively resolved. The underlying reason for resolution improvement is intuitive considering that the effective path length is increased when δ is reduced (see eq 1). At 70.9 V/s, it is evident that at least six SP conformers (four compact and two elongated) coexist. Interestingly, it is observed that, under these experimental conditions, neither SP nor BK display significant changes in the relative abundance of the TIMS distributions upon significantly extending the time ions spend trapped inside the tunnel prior to analysis (Figure 1a,b), suggesting that rf heating effects are minimal. An advantage of TIMS is the ability to tune the experimental parameters according to the analytical challenge. This concept is further illustrated by reducing the voltage range to selectively trap and analyze ions of interest, thereby yielding additional insight into the conformational landscape of analytes with unresolved features. It is noteworthy that, under the experimental conditions where δ ≲ 30 V/s, we observe that the relative abundance of these states does not reflect that of the nascent distribution. That is, conformers having smaller Ω (larger K) appear suppressed owing to a relative increase in ion losses (presumably arising from scattering collisions) upon extended storage time, since these populations elute from the TIMS tunnel after the population having larger Ω. This result is consistent with previous experiments demonstrating that ion transmission decreases as the scan time is increased.20 For example, the total scan time of [SP + 3H]3+ under these conditions is ∼990 ms. While [SPB1−2 + 3H]3+ elute in less than ∼270 ms, [SPA4 + 3H]3+ only begins to elute after ∼640 ms resulting in an additional ∼370 to 670 ms of analysis time for the [SPA1−4 + 3H]3+ species. Nevertheless, the analytical utility of this approach is demonstrated for [BK + 2H]2+ and [SP + 3H]3+ in Figure 2. It is evident that, in the case of [BK + 2H]2+, resolution improves when δ is reduced to 10 V/s, as [BKA + 2H]2+ and [BKB + 2H]2+ are now completely resolved. Though not obvious from the results in Figure 1b, on the basis of relative peak widths observed in Figure 2a, it is clear that [BKB + 2H]2+ is significantly broader than ([BKA + 2H]2+), indicative that a multiplicity of structural geometries is present beneath this feature. Figure 2b shows that a similar approach is useful in improving the resolution of the six features shown in Figure 1d for [SP + 3H]3+. Note that, owing to the expansive conformational diversity, the scan rate in this case could only be decreased to δ = 27 V/s such that all conformers were trapped in a single experiment. Note that the small feature at Ω = 493 Å2 previously observed as a shoulder is now well-resolved from the neighboring feature at Ω = 498 Å2. Moreover, while the peak widths for [SPA1−3 + 3H]3+ fall in a similar range, yielding R = 154 to 183, the appreciably larger peak width for [SPA4 + 3H]3+ (R = 79) indicates the presence of additional unresolved geometries. Nevertheless, we unambiguously observe that [SPA + 3H]3+ contains at least four conformations, consistent with the notion that this population emerged from kinetic-trapping of an ensemble of desolvated structures.43
Figure 2. TIMS distributions of (a) [BK + 2H]2+ and (b) [SP + 3H]3+ collected at an entrance pressure of 4.3 mbar. For BK and SP, δ = 10 and 27 V/s, respectively.
resolving the conformational landscape of biomolecules. Resolving power exceeding 250 was attained which is comparatively ∼3 to 8 times greater than modern drift tube or traveling wave IMS techniques, respectively. The results provide evidence that at least six conformations (four compact and two elongated) comprise gas phase distribution of [SP + 3H]3+. Moreover, a straightforward calibration method was employed that yielded Ω values in agreement (≤2% error) with Ω values previously measured by drift tube IMS. The TIMS distributions for these analytes closely resemble the overall features of drift tube IMS distributions operating below the socalled low field limit, which suggests that the temperature of ions during TIMS analysis is near thermal.
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ASSOCIATED CONTENT
S Supporting Information *
Calibration curve used for determination of Ω values (Figure S1) and two-dimensional TIMS-MS contour plots showing the isotopic distributions of BK and SP (Figure S2). 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]. Phone: (978) 663-3600.
CONCLUSIONS We have demonstrated the analytical utility of TIMS for ion mobility applications requiring high resolution, as is the case in
Notes
The authors declare no competing financial interest. 5626
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Letter
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ACKNOWLEDGMENTS The authors wish to thank their colleague, Dr. Jeremy Wolff, for proofreading.
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