Anal. Chem. 2007, 79, 1523-1528
Distortion of Ion Structures by Field Asymmetric Waveform Ion Mobility Spectrometry Alexandre A. Shvartsburg, Fumin Li, Keqi Tang, and Richard D. Smith*
Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352
Field asymmetric waveform ion mobility spectrometry (FAIMS) is emerging as a major analytical tool, especially in conjunction with mass spectrometry (MS), conventional ion mobility spectrometry (IMS), or both. In particular, FAIMS is used to separate protein or peptide conformers prior to characterization by IMS, MS/MS, or H/D exchange. High electric fields in FAIMS induce ion heating, previously estimated at 10 unresolved isomers, as revealed by comparison of measured IMS peak widths with those determined by instrumental resolution.25,26 The peak widths often indicate multiple conformers present even for relatively small peptide ions, such as bradykinin.27 Some species unresolved by conventional IMS may be distinguished28 by field asymmetric waveform IMS (FAIMS)6,29-32 based on the differences between their mobilities at high and low E. In FAIMS, a gas flow pulls ions through the gap between two electrodes carrying a periodic asymmetric waveform, VD(t), of some frequency, wc, and amplitude (“dispersion voltage”), Vmax. Most FAIMS systems (including the Selectra used here) employ a bisinusoidal form
VD(t) ) [2 sin(2πwct) + sin(4πwct - π/2)]Vmax/3 (2) that provides the optimum32 (high E)/(low E) ratio of 2. This waveform creates a strong oscillatory electric field across the gap, ED(t) with the amplitude of Emax, causing loss of ions to one of the electrodes.29-31 A small dc compensation voltage (CV) superposed on VD(t) may offset the time-averaged drift due to ED(t) for a given species, allowing it to traverse the FAIMS device; scanning CV reveals the spectrum of the sampled ion mixture. The values of a function and its derivative are not correlated a priori; hence, FAIMS and IMS separations can be largely complementary. Experimentally, that is the case for peptides and many small ions.33,34 This has prompted us to couple FAIMS to IMS, establishing 2-D gas-phase ion separations.28,35 Analyses of tryptic peptides35 have confirmed a strong orthogonality between the two dimensions, increasing the total peak capacity by an order of magnitude (24) Light-Wahl, K. J.; Schwartz, B. L.; Smith, R. D. J. Am. Chem. Soc. 1994, 116, 5271. (25) Li, J.; Taraszka, J. A.; Counterman, A. E.; Clemmer, D. E. Int. J. Mass. Spectrom. 1999, 185/186/187, 37. (26) Hudgins, R. R.; Woenckhaus, J.; Jarrold, M. F. Int. J. Mass Spectrom. Ion Processes 1997, 165/166, 497. (27) Counterman, A. E.; Valentine, S. J.; Srebalus, C. A.; Henderson, S. C.; Hoaglund, C. S.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1998, 9, 743. (28) Shvartsburg, A. A.; Li, F.; Tang, K.; Smith, R. D. Anal. Chem. 2006, 78, 3304; ibid, 8575. (29) Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. K. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143. (30) Guevremont, R. J. Chromatogr., A 2004, 1058, 3. (31) Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2004, 15, 1487. (32) Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2005, 16, 2. (33) Guevremont, R.; Barnett, D. A.; Purves, R. W.; Vandermey, J. Anal. Chem. 2000, 72, 4577. (34) Shvartsburg, A. A.; Mashkevich, S. V.; Smith, R. D. J. Phys. Chem. A 2006, 110, 2663. (35) Tang, K.; Li, F.; Shvartsburg, A. A.; Strittmatter, E. F.; Smith, R. D. Anal. Chem. 2005, 77, 6381.
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compared to 1-D IMS. For both peptides and proteins, FAIMS can distinguish conformers not resolved by IMS, including four bradykinin (2+) isomers6 and multiple unfolded geometries for higher charge states of ubiquitin and cytochrome c (Cyt c).28 Those findings establish the utility of FAIMS “fractionation” of ions prior to their structural characterization by IMS or IMS/ MS. Similarly, FAIMS may be used in conjunction with other separation or structural elucidation methods, such as H/D exchange,36,37 tandem mass spectrometry (especially involving a nonergodic dissociation by electron capture38 or transfer39), photoelectron40 or photodissociation41 spectroscopy, and combinations of these techniques.42,43 A key issue with FAIMS separations preceding structural characterization is the extent of ion geometry disruption. By definition, FAIMS operates in the high-field regime where ions are collisionally heated above the gas temperature, which may drive ions to isomerize. The extent of heating (∆T) may be evaluated using the two-temperature treatment:19,21
∆T(E) ) Tion(E) - T ) M [K(E)E]2/(3kB)
(3)
where Tion is the ion temperature. In FAIMS, E and thus ∆T are time-dependent; one may compute maximum ∆T (∆Tmax) at E ) Emax or mean ∆T (〈∆T〉) by integrating eq 3 over ED(t). Clearly, ∆T depends on the ion: species with higher K are more extensively heated. Isomerization due to heating in FAIMS should be most significant for fragile ions such as proteins, which have numerous low-energy conformers separated by small barriers. Isomerization in FAIMS has been theoretically considered for ubiquitin44 and Cyt c37scommon model proteins in biophysics and mass spectrometry that were also studied by FAIMS.28,36,37,44-46 For a reasonable |Vmax| (4.2 or 4.4 kV), the values calculated using eq 3 were ∆Tmax ) 31 °C, 〈∆T〉 ) 7 °C for ubiquitin44 (z ) 11-15) and ∆Tmax ) 47 °C, 〈∆T〉 ) 10 °C for Cyt c37 (z ) 16). Based on 〈∆T〉 ∼10 °C, changes to the 3-D protein structure were expected to be minimal. However, isomerization in FAIMS may be controlled by ∆Tmax rather than 〈∆T〉. As an analogy, egg white protein that denatures (for example) at 100 °C may obviously be denatured by boiling in several steps separated by longer periods in cold water, though (36) Robinson, E. W.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2005, 16, 1427. (37) Purves, R. W.; Ells, B.; Barnett, D. A.; Guevremont, R. Can. J. Chem. 2005, 83, 1961. (38) Adams, C. M.; Kjeldsen, F.; Zubarev, R. A.; Budnik, B. A.; Haselmann, K. F. J. Am. Soc. Mass Spectrom. 2004, 15, 1087. (39) O’Connor, P. B.; Cournoyer, J. J.; Pitteri, S. J.; Chrisman, P. A.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2006, 17, 15. (40) Hoffmann, M. A.; Wrigge, G.; von Issendorff, B.; Muller, J.; Gantefor, G.; Haberland, H. Eur. Phys. J. D 2001, 16, 9. (41) Fielicke, A.; Ratsch, C.; von Helden, G.; Meijer, G. J. Chem. Phys. 2005, 122, 091105. (42) Fromherz, R.; Gantefor, G.; Shvartsburg, A. A. Phys. Rev. Lett. 2002, 89, 083001. (43) Hagman, C.; Tsybin, Y. O.; Hakansson, P. Rapid Commun. Mass Spectrom. 2006, 20, 661. (44) Purves, R. W.; Barnett, D. A.; Ells, B.; Guevremont, R. J. Am. Soc. Mass Spectrom. 2001, 12, 894. (45) Purves, R. W.; Barnett, D. A.; Ells, B.; Guevremont, R. J. Am. Soc. Mass Spectrom. 2000, 11, 738. (46) Purves, R. W.; Barnett, D. A.; Guevremont, R. Int. J. Mass Spectrom. 2000, 197, 163.
〈∆T〉 is 10 °C only. Provided each heating step is long enough for a jump to a nearby basin on the energy surface, the height of possible jumps and thus the accessible conformational space may be limited by the maximum rather than the mean temperature. For the waveform of eq 2, E > 0.95Emax and thus ∆T > 0.9∆Tmax over 7.2% of the FAIMS cycle. Hence, for wc ) 750 kHz used here, each heating step (at >90% of peak intensity) lasts ∼0.1 µs, which exceeds the molecular vibration periods that set the time scale of elementary conformational transitions by many orders of magnitude. Then, with the typical ion residence time in FAIMS (tres) of ∼150-300 ms,31 near-maximum heating may apply for ∼10-20 ms. This may induce substantial protein unfolding (as seen, for example, in IMS data for ubiquitin47 and Cyt c15 ions trapped for similar times after generation by ESI). From these considerations, FAIMS may significantly affect macroion conformations. Here we study this issue for ubiquitin using a (FAIMS)/IMS/MS instrument, where the IMS stage allows comparing the 3-D structures of ions that have and have not passed FAIMS. We find that a FAIMS stage causes substantial protein ion unfolding, consistent with that produced via thermal heating by calculated ∆Tmax values. EXPERIMENTAL METHODS AND RESULTS Experiments were performed employing a previously described FAIMS/IMS/MS system,28,35 comprising the Selectra cylindrical FAIMS device (Thermo Electron), a custom IMS drift tube, and a modified time-of-flight MS (Sciex Q-Star). All three stages are coupled using electrodynamic ion funnels that provide an effective ion transmission through the instrument and thus enable practical 2-D FAIMS/IMS analyses in conjunction with MS. The experimental procedure and the FAIMS/IMS data for ubiquitin (z ) 6-13) ions have been reported.28 Those involved the composite IMS spectra, obtained by integration of CV-selected IMS spectra over the CV distribution. Here we present the direct IMS spectra measured using the ESI-IMS/MS configuration48 without FAIMS, all other conditions held constant (Figure 1). Experiments were performed at 20 °C, but as usual, the data were converted to reduced mobilities19 (K0) for standard N (at 0 °C). Ions in ion funnels are collisionally heated by the focusing rf field, and the heating magnitude scales approximately as the peak rf voltage (Urf) squared.28 That heating may cause ions to isomerize while stored in the ion funnel preceding IMS: the unfolding for ubiquitin ions appears28 at Urf ∼20 V and becomes pronounced at 40 V. Hence, to clearly separate the isomerization in FAIMS from that in the front IMS funnel, we compare direct and composite IMS spectra at Urf of 10 (that causes no observable unfolding for any z) and 40 V (that produces maximum unfolding).28 At either Urf, the composite spectra for each z (Figure 1) exhibit features at somewhat greater Ω than direct spectra, which indicates a universal protein unfolding caused by the FAIMS stage. To quantify this, we define a parameter reflecting the displacement of whole spectrumsthe relative Ω shift equal to (〈Ω〉composite/ 〈Ω〉direct - 1), where 〈Ω〉 is the mean of Ω distribution. The magnitude of that shift and its dependence on z are similar at low (47) Myung, S.; Badman, E.; Lee, Y. J.; Clemmer, D. E. J. Phys. Chem. A 2002, 106, 9976. (48) Tang, K.; Shvartsburg, A. A.; Lee, H. N.; Prior, D. C.; Buschbach, M. A.; Li, F.; Tolmachev, A. V.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2005, 77, 3330.
Figure 1. IMS spectra for bovine ubiquitin (z ) 6-13 as labeled), directly measured using ESI-IMS/MS (solid lines) and composite from ESI-FAIMS/IMS/MS data28 (dashed lines) at Urf ) 10 (top panel) and 40 V (bottom panel).
Figure 2. Relative shifts of mean ubiquitin ion cross sections induced by FAIMS, at Urf ) 10 (solid line) and 40 V (dashed line).
and high Urf (Figure 2), which also suggests that this unfolding is not due to the heating in the ion funnel. At both Urf, the shift is 1-3% for the lowest and highest z but reaches 9% at intermediate z. This behavior resembles that observed when ubiquitin25,28,49 or other28,50 proteins lacking disulfide bridges are thermally or collisionally heated prior to IMS analyses and has a clear physical basis. For low z (for ubiquitin, z e 6), compact geometries are stable and cannot be unfolded much by minor heating, while for high z (here z g 11), the proteins are already mostly unfolded by Coulomb repulsion and heating cannot have a large effect. However, for intermediate z (here z ) 7-10 and especially 7+ and 8+), compact folds are barely stable and minor excitation may have major structural consequences.25,28 The curve in Figure 2 shifts toward lower z at higher Urf, presumably because of the cumulative effect of heating in FAIMS and in the funnel. In most cases of multiple isomers present in direct IMS spectra, FAIMS unfolds only a fraction of more compact ions (Figure 1). However, direct IMS reveals some compact conformers not seen by FAIMS/IMS.28 This is particularly the case for 8+ with the (49) Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1997, 8, 954. (50) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240.
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Figure 3. Cross sections of ubiquitin ion conformers as a function of charge state: species distinguished by FAIMS/IMS28 (filled circles) and additional isomers found by direct IMS here (empty circles).
greatest difference between composite and direct IMS spectra (at low Urf): the latter has intense features at 1/K0 ) 0.77 - 0.85 V‚s/cm2 while the former contains no species with 1/K0 < ∼0.85 V‚s/cm2. The direct spectra for z ) 9-12 also exhibit (minor) conformers at lower Ω than any found by FAIMS/IMS. The cross sections for ubiquitin ion conformers distinguished by both FAIMS/IMS28 and direct IMS here are plotted in Figure 3. For each z, the direct IMS (at low Urf) reveals species about as compact as any seen in all previous measurements using both IMS47,49 and energy loss methods,44,45 including those starting from a pseudonative (∼90% aqueous) ESI solution (summarized in Figure 8b of ref 28). In particular, we have found an abundant compact structure for 8+ that was previously observed using pseudonative solution only.25,47 This (i) demonstrates that, despite some inevitable rf heating of ions, the ion funnel interface for ESIIMS may be at least as “soft” as any other design and (ii) further verifies that the protein unfolding isolated in Figure 2 is induced by FAIMS and not elsewhere in the ESI-IMS interface. DISCUSSION Understanding how protein unfolding in FAIMS compares to expectations based on eq 3 is important to clarify the unfolding mechanism and gain the capability to predict isomerization under various FAIMS conditions. Experiment and theory may be linked by comparing the isomerization in FAIMS to that induced by controlled thermal heating of same species. Fully or partly folded ubiquitin ions (z ) 6-10) unfold when heated from 25 to 132 °C (with intermediate points of 68, 76, 97, and 117 °C) in a gas prior to IMS analyses25 (Figure 4). At low Urf, the unfolding in Figure 1 is less than that at 97 °C (with compact geometries reduced to secondary isomers for z ) 6-8 and destroyed completely for 9+ and 10+) and looks broadly comparable to that at 68-76 °C (with slight shifts to longer tD for 6+ and 7+, a distinct “unfolded” feature appearing for 8+, a similar change in the fractions of two isomers for 9+, and the more compact structure for 10+ reduced to a ledge of the major peak). Hence, the effect of FAIMS on ubiquitin ions is similar to their heating by ∼50-55 °C above room temperature. For z ) 6-10, the compact conformers that unfold in FAIMS have a narrow 1/K0 range of 0.78-0.96 V‚s/cm2, because at higher z the increase of mobility for a given Ω by eq 1 is offset by unfolding that increases Ω (Figure 3). Adjusting those values to 1526
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Figure 4. IMS spectra for ubiquitin ions (z ) 6-11) measured at various temperatures of the heated capillary at the ESI-IMS interface over the 25-132 °C range, as labeled.25
293 K and substituting them into eq 3 yields ∆Tmax ) 56-85 °C, 〈∆T〉 ) 12-18 °C, which exceed previously computed44 ∆T by a factor of ∼2-2.5 despite a lower absolute Vmax in this work (4.0 vs 4.4 kV). The discrepancy is due to the earlier calculation44 assuming K0 ) 0.7 (cm2/V‚s), the average mobility of ubiquitin ions (z ) 5-11) in N2 measured51 at 150-200 °C. However, (i) K0 for a given Ω is proportional to T-1/2 (eq 1) and K0 at ∼20 °C in FAIMS is higher than that at 150-200 °C by 20-27%, (ii) ubiquitin ions for z g 6 unfold at >150 °C resulting in a lower average K (Figure 4), and (iii) the mobilities relevant to the present issue are for compact geometries that may unfold rather than a lower average K of all isomers. The highest ∆T values are for most compact isomers of z ) 8 and should be excluded from comparisons with temperature-dependent IMS data,25 where those geometries were not observed, which reduces 〈∆T〉 to 12-16 °C and ∆Tmax to 56-71 °C. The experimental estimate for heating of ubiquitin ions in FAIMS (∼50-55 °C) is far higher than 〈∆T〉 but in an excellent agreement with ∆Tmax, especially as that applies only instantaneously at VD(t) ) Vmax and the maximum effective ion heating (that is, available over some finite time needed for isomerization) is slightly lower. Hence, the measured conformational change of ubiquitin ions in FAIMS is consistent with unfolding in a multitude of steps by hopping between nearby energy basins during the peak FAIMS voltage. Such steps are reversible in principle, but as for the unfolding of multiply charged proteins in other regimes, the Coulomb repulsion between protonated sites determines the preferred direction of hops. (51) Guevremont, R.; Siu, K. W. M.; Wang, J.; Ding, L. Anal. Chem. 1997, 69, 3959.
Figure 5. IMS spectra for ubiquitin ions (z ) 6-8) measured as a function of storage time in the preceding ion trap over the 20-1000ms range, as labeled.47
Central to the topic of ion isomerization in FAIMS is the “selfcleaning” process.28,37 In general, isomers have different CVs. If two geometries are fully resolved by FAIMS (that is, the CV difference exceeds the baseline instrumental peak width), a transition from one to the other would normally remove the ion by FAIMS filtering. This is inherent to the scanning techniques such as quadrupole MS and FAIMS: ions may change conformation in conventional IMS (a dispersion technique) with no effect on detection. Some isomers may have similar CVs and be separated by FAIMS only partly if at all (for example, o- and m-phthalates in N2).4 However, the CVs of compact and most unfolded ubiquitin conformers for all z ) 6-10 differ by ∼3-6 V (vs the instrumental peak width of ∼1 V)28,45 and full unfolding in FAIMS should result in ion loss. Expected exceptions are transitions occurring within a period of time after injection into or before exiting FAIMS shorter than that needed to filter out ions with “wrong” CV (tfil). Based on simulations31 and rapid analyses using a micromachined FAIMS device,52 tfil is proportional to the CV offset between the ion and FAIMS transmission window and amounts to a few milliseconds (at most). Thus, the probability of an ion isomerizing within tfil before exiting (defined by tfil/tres) is small,
