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Feb 8, 2016 - ABSTRACT: Historically, high pressure ion mobility drift tubes have suffered from low ion duty cycles and this problem is magnified when...
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Augmenting Ion Trap Mass Spectrometers Using a Frequency Modulated Drift Tube Ion Mobility Spectrometer Kelsey A. Morrison, William F. Siems, and Brian H. Clowers* Department of Chemistry, Washington State University, PO Box 644630, Pullman, Washington 99164, United States S Supporting Information *

ABSTRACT: Historically, high pressure ion mobility drift tubes have suffered from low ion duty cycles and this problem is magnified when such instrumentation is coupled with ion trap mass spectrometers. To significantly alleviate these issues, we outline the result from coupling an atmospheric pressure, dual-gate drift tube ion mobility spectrometer (IMS) to a linear ion trap mass spectrometer (LIT-MS) via modulation of the ion beam with a linear frequency chirp. The time-domain ion current, once Fourier transformed, reveals a standard ion mobility drift spectrum that corresponds to the standard mode of mobility analysis. By multiplexing the ion beam, it is possible to successfully obtain drift time spectra for an assortment of simple peptide and protein mixtures using an LIT-MS while showing improved signal intensity versus the more common signal averaging technique. Explored here are the effects of maximum injection time, solution concentration, total experiment time, and frequency swept on signal-to-noise ratios (SNRs) and resolving power. Increased inject time, concentration, and experiment time all generally led to an improvement in SNR, while a greater frequency swept increases the resolving power at the expense of SNR. Overall, chirp multiplexing of a dual-gate IMS system coupled to an LIT-MS improves ion transmission, lowers analyte detection limits, and improves spectral quality. suffers from a poor duty cycle of 30 ms) in a single experiment. With a dual-gate IMS system, Fourier transformation raises the duty cycle per gate to ∼50%, yielding an overall duty cycle of ∼25%. Unlike the second-time scale frequency sweeps performed with FT-IM-MS using TOFMS systems,30 ion trap FT-IM-MS requires the Fourier experiment time length to be extended to a minute time scale to accommodate the slower acquisition rate of the ion trapping system. Here, we report the operating parameters and experimental approach necessary to couple FT-IMS with a linear ion trap mass spectrometer using peptides and a small protein as target molecules to assess signal-to-noise ratio, resolving power, and overall spectral quality.

differing substantially from the later applications of Fourier multiplexing to IMS.23,24 Owing to an absence of digital electronics, the early 20th century experiments by Tyndall used ion gate frequency modulation to couple analog data acquisition approaches with the comparatively rapid gas-phase occurrences. Modulation of two ion gates using a frequency chirp and subsequent Fourier transformation was demonstrated with ion mobility systems in 198525 and showed promise for its use as a chromatographic detector due to the capacity for FTIMS to obtain a drift time spectrum in a matter of seconds. In the following years, FT-IMS was coupled to GC and SFC and was even used to assess the degree of branching and predict polymer microstructure.26,27 However, the theoretical gains in signal-to-noise ratios and resolving power were not realized due to limitations of electronics at the time. As an effort to accommodate the slower processors and low memory of the 1980s and 1990s computers, a transform-in-place method using the original Tukey-Cooley Fast Fourier Transform approach was applied to FT-IMS.28,29 With suboptimal electronics for performing FT-IMS experiments combined with signal losses to apodization, FT-IMS advancement stagnated and the approach was not widely adopted. Subsequent advancements in microprocessor speed and memory capacity now enable such experiments to be implemented including discrete Fourier transformation. Fourier transform IMS coupled to time-of-flight mass spectrometry has recently been shown to provide the predicted gains in sensitivity and selectivity.30 Part of this success comes from the coupling of ion mobility, a millisecond time base separation technique, to time-of-flight mass spectrometry, which functions on the microsecond time scale. While effective, this approach to multiplexed IMS analysis precludes the use of millisecond time scale mass analyzers, such as ion traps, as a detector for an IMS.

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EXPERIMENTAL SETUP Atmospheric Pressure Dual-Gate Ion Mobility Linear Ion Trap Mass Spectrometer. Drift times were measured 3122

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here to be 50 μs for neurotensin, 20 μs for ubiquitin at 180 °C, and 30 μs for ubiquitin at 120 °C. The dwell times on each particular step were 1 s for neurotensin, 6 s for ubiquitin at 180 °C, and 4 s for ubiquitin 120 °C. For comparative purposes, the scan mode experiments were performed for the same time length as the Fourier multiplexing experiments. Unless stated otherwise, all experiments were performed with a minimum of three replicates. Chemicals and Reagents. Protein and peptide samples (Sigma-Aldrich, St. Louis, MO) including neurotensin, ubiquitin, lysozyme, and cytochrome C digested with immobilized TPCK trypsin beads (Pierce Biotechnology) were used to characterize the capabilities of the FT-IM-MS technique, with ubiquitin also used to compare FT-IM-MS to the dual-gate IM-MS scan mode. Individual solutions of neurotensin, ubiquitin, and lysozyme were made without further preparation in a 50:50 mixture of acetonitrile, water, and 0.1% formic acid (FA), respectively. From concentrated stock solutions, working solutions of neurotensin, ubiquitin, and lysozyme individually were diluted to concentration ranges of 1 nM to 1.1 μM, 1 μM, and 110 nM. All neurotensin experiments, aside from those involving variation in concentration, were performed using 110 nM working solutions. The trypsin digest of cytochrome C was performed using 1 mg of cytochrome C diluted in 1 mL of 0.1 M NH4HCO3 digestion buffer and incubated at 37 °C for ∼24 h. Spin columns containing C18 resin (Pierce Biotechnology) were then used to desalt aliquots of the digest, which were then diluted 1:10 in 50:50 acetonitrile (ACN)/water and 0.1% formic acid (FA). For the horse heart cytochrome C used, the sequence coverage of the resulting digest was determined to be ∼48% (Protein Coverage Summarizer, http://omics.pnl.gov/software/proteincoverage-summarizer). Drift time spectra were extracted for the fragments EETLMEYLENPK2+ (747.850 Da), QVSLEVIPDWLDPPQNLLHVR3+ (817.101 Da), GITWKEETLMEYLENPKK 3+ (736.374 Da), and MDPAPSLGCSLKDVK 3+ (544.597 Da). All resulting solutions diluted in 50:50 ACN/ H2O and 0.1% FA were infused by the LTQ stock syringe pump at 3 μL min−1 held at ∼2700 V above the IMS desolvation region entrance plate. Pulse Design and Data Transformation. Frequency encoding of signals and subsequent Fourier transformation has long been applied to other forms of chemical analysis, such as IR spectroscopy or even ion cyclotron resonance mass spectrometry; as an established technique, a number of resources are available in regard to its applications and challenges.32,33 Circumventing the constraints due to the gating mechanism of IMS has, however, required specific modifications to the typical FT experiment. A requirement of FT-IMS is that the BN-gates must be restricted to simply an on or off state due to the binary mechanism by which the gates function. As such, the pulse sequences used for the FT-IMS procedures here utilized a square wave with a linear sweep of increasing frequency during the allotted experiment time. This type of pulsing sequence results in a mobility time series exhibiting a characteristic oscillating signal in the time domain that decays as the frequency increases (e.g., similar to but distinctly different from the free induction decay observed in NMR measurements). However, in contrast to the prior FT-IM-MS work involving a time-of-flight MS,30 the sweep time must be substantially increased to allow for successful application of the FT-IMS method to the slower LIT-MS. Additionally, this specific FT-IMS setup uses a dual-gate IMS system, allowing

using an atmospheric pressure dual-gate ion mobility spectrometer (MA3100, EXCELLIMS, Acton, MA) coupled without hardware modifications to a linear ion trap mass spectrometer (LTQ, Thermo Scientific, San Jose, CA). A schematic of the instrument is shown in Figure 1a. In this instrument, the drift tube is based on a rectangular electrode design wherein metal-plated ceramics generate a homogeneous electric field. The range of operating temperatures for the system is ∼30−250 °C, and the temperature for these experiments was maintained at 180 °C unless otherwise noted. Using the custom ExcellIMS electronics supporting the MA3100 unit, an electric field of ∼440 V cm−1 was applied in all experiments. Dry, purified nitrogen was introduced into the drift tube immediately before the LTQ inlet capillary, flowing at ∼2 L min−1 counter-current to ions introduced to the system at atmospheric pressure (∼690 Torr in Pullman, WA). After ionization with an electrospray ionization source, ions pass through a desolvation region approximately 6.25 cm long before reaching the first Bradbury-Nielson style gate (BNgate). Following their travel through the 10.56 cm drift tube, ions encounter a second BN-gate located immediately in front of the inlet to the LIT-MS. Using the mass spectrometer as the detector, the ion trap was filled using maximum inject times ranging from 10 to 500 ms and m/z ranges of 50−1000, 1000− 2000, 500−1500, and 50−2000 for analysis of neurotensin, lysozyme, ubiquitin, and the cytochrome C digest, respectively. Although automatic gain control (ACG) was left on for all experiments, the sample concentrations were low enough that the inject times for each run matched the set maximum inject time. Both the first and second gates were pulsed (±40 and ±160 V, respectively) simultaneously for the multiplexing experiments, by connecting the two IMS external gate triggers to a waveform generator (DG1062Z, RIGOL, Beijing, China) or an Analog Discovery microcontroller (Digilent, Pullman, WA). It should be noted that the higher closing voltage for the second ion gate was empirically determined to minimize any ion leakage. While not shown in Figure 1a, the second ion gate is located immediately preceding the LTQ inlet which also has a voltage applied. Frequency encoded spectra were generated by pulsing the ion gates with increasing frequency, sweeping from 5 Hz to ending frequencies of 5505, 7505, 10005, 15005, and 20005 Hz. The sweep times used were 2, 4, 8, and 16 min, with the RIGOL waveform generator used for the 2−8 min sweeps and the Analog Discovery used for the 16 min sweeps due to a time limitation of the RIGOL waveform generator. The frequency sweep of the gates and beginning of LIT-MS acquisition are synchronized via the trailing edge of a 1 Hz pulse of +5 V. For illustration purposes, a 1500 ms segment of the pulsing sequences for an 8 min sweep are shown for gate 1, gate 2, and the ion trap in Figure 1b−d, respectively. Scan mode, a manufacturer-installed feature in the dual-gate IM-MS analogous to the common signal-averaged technique, occurs by differential timing of the two gates opening to scan through a specified drift time window; the first gate simply functions as in traditional drift tube IMS by opening for a given gate pulse width, while the second gate opens for a pulse width at a delayed time after the first gate such that only ions with drift times in the selected drift time window are allowed to pass into the mass spectrometer. Here, the scan mode experiments used gate pulse widths of 400 and 300 μs for neurotensin and ubiquitin, respectively. The second gate continues opening with a given delay for a specified dwell time, after which the delay is increased by the scan step size, typically 20−50 μs and chosen 3123

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Figure 2. Neurotensin 2+ and 3+ charge states produce distinct free induction decays (a) according to their respective drift times; the plot in (a) shows a magnified section of the two decay series while the inset shows both in entirety. Increasing inject time of the LIT-MS during FT-IMS experiments with the 3+ charge state of neurotensin produces an SNR improvement (b) and a fairly consistent peak shape (c), although absolute peak intensity (d) can suffer with longer inject times.

of a sweep is increased, with the inverse of the final frequency corresponding to the finest increment of time measurement in seconds.25 Effects of the four parameters were examined primarily using the 3+ charge state of neurotensin and supplemented by examining results of increasing frequency on lysozyme and a tryptic digest of cytochrome C. A typical Fourier transform IMS decay series is shown in Figure 2a for both the 2+ and 3+ charge states of neurotensin. While the full sweep time is show in the upper subplot, the primary axis of Figure 2a displays the normalized data for both ions from 0 to 1 min. The neurotensin3+ ion had a remarkably stable mean SNR across an order of magnitude change in maximum inject time; SNRs remained approximately constant in the range of 190−230 for inject times of 50−500 ms, shown in Figure 2b. However, below inject times of 50 ms, SNRs drop noticeably, indicating that higher ion counts provide more improvement in signal versus increased data acquisition rate of the LIT-MS. As mentioned previously, though AGC was left in the on state, the ion current was sufficiently low that the maximum ion injection time was attained for all of the data shown. Comparing the data from 10, 50, and 500 ms injection times for neurotensin3+, the peak shape is largely retained between 50 and 500 ms (Figure 2c) and peak intensity is raised with shorted inject times (Figure 2d), but SNR appears to reach a maximum at intermediate injection times (Figure 2b). For the data shown in Figure 2, the frequency sweep was maintained at 5−7505 Hz and the total experiment time held at 8 min. Although the ion trap injection time does not explicitly influence the FT multiplexing waveform, the injection time does alter the mass spectral acquisition rate and by extension the spectral quality of the resulting transformed IMS spectra. As a result, a trade off exists between sufficient spectral quality and a sufficient number of mass spectra sampled during the FT-IMMS experiment to provide optimal SNR. The data giving rise to

the method to forego obtaining two FT decay series 180° out of phase. In regard to data sets obtained here, extracted ion chromatograms for ions of interest underwent transformation to obtain each ion’s individual mobility spectrum. Spectra data files originally obtained in the Thermo Scientific RAW format were converted to mzML for processing by a custom Python script, wherein extracted ion chromatograms for specified ions with a tolerance of ±5 Da are exported to a resulting CSV file. These extracted ion chromatograms contain the encoded drift time data for the given m/z range in the form of a Fourier signal decay. The Fourier data sets were then transformed using the Fast Fourier Transform method, a derivative of a primefactor decomposition transformation method developed from the Cooley-Tukey algorithm (IGOR Pro, Wavemetrics, Lake Oswego, OR). Drift times of a given m/z range are then easily recovered from the resulting transformed sweep by dividing the resultant frequency by the frequency sweep rate in Hz s−1.

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RESULTS Signal to Noise Ratio and Resolving Power Evaluation. Evaluations of signal-to-noise ratio (SNR) trends as a function of ion trap inject time, frequency sweep time, solution concentration, and frequency swept were examined for the purpose of exploring the capabilities of FT-IMS. Here, noise was calculated as 3 multiplied by the standard deviation of a drift time range typically considered to be free of ion current (for example, 33−35 ms) plus the average of that range, which was then compared to maximum signal intensity for a given drift time peak of a particular ion. An additional metric of monitoring spectral changes is monitoring change in resolving power and the number of points for a spectrum, both of which are altered upon changing the net frequency sweep. A greater number of points per peak can be obtained if the net frequency 3124

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Figure 3. Improvement in SNR (a) and absolute intensity (b) can be obtained if the sweep time is increased while (c) peak shape is largely maintained even with shorter sweep times.

Figure 4. (a) A linear trend in peak intensity can be seen in FT mode for neurotensin3+ across 1, 10, and 110 nM concentrations, which is also shown as a linear regression plot in Figure S-1a. Comparable experiments were not performed with scan mode due to low ion current obtained from 8 min sample runs; the lowest concentration visible during the 8 min scan mode experiments (110 nM) is shown for comparison to FT mode. (b) Below 100 nM, SNR for 1−55 nM averages just below 100, but for concentrations in the range of 100−1000 nM, the average SNR falls in the range of 250−300. FT mode peaks from concentrations 1 and 10 nM were multiplied by the factors listed in the legend to facilitate their visualization with other peaks on the same scale.

the plots shown in Figure 2 indicate that an optimal level of SNR is obtained for maximum ion trap inject times that range from 50 to 200 ms with no appreciable benefit for inject times ≥500 ms. Figure 3a exhibits how changes in frequency sweep time impact SNR. When doubling frequency sweep time to 8 min from 4 min, the mean SNR for neurotensin3+ peaks increased from ∼230 to ∼580. By lengthening the sweep time, the frequency sweep rate is effectively lowered which permits more data points across a frequency window to be recorded, even on an LIT-MS time scale. Stated differently, this behavior is directly related to the number of LIT-MS fill and scan cycles that occur at each frequency step.31 Consequently, the absolute signal intensity is significantly improved for the longer sweep times, as shown in Figure 3b. The effect of improving SNR appears to level at approximately 8 min (Figure 3a). The data presented in Figure 3b indicate that when using an ion trap fill

time of 100 ms the mobility domain is maximally sampled at approximately 8 min. Further lengthening of the FT sweep time decreases the relative error simply because more samples in the mobility domain are acquired. Comparing the 2 and 8 min frequency sweeps, peak shape is largely retained (Figure 3c), and the SNR of the 2 min FT-IMS experiments are adequate for quantitation at an average of ∼280. These data suggest that even shorter sweep times can be used if rapid analysis is necessary. FT-IMS spectra obtained for the data shown in Figure 3 were obtain using a 100 ms maximum inject time and a frequency sweep from 5 to 7505 Hz. The effects of varying the concentration of neurotensin on the FT-IMS experiment were also examined using a range of concentrations covering 1, 10, 55, 110, 550, and 1100 nM. In the range of 1 to 110 nM, peak heights increased in a fairly linear fashion, as demonstrated with the 3+ charge state of neurotensin in Figure 4a; linear regression analysis for the mean 3125

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Analytical Chemistry peak heights yielded an R2 value of 0.9942 for the 1 to 110 nM range (Figure S-1a), indicating a linear range of approximately 2 orders of magnitude. Linearity across the entire range studied (e.g., 1 to 1100 nM) drops with regard to R2 to 0.9280 (Figure S-1b). It should be noted that the plots provided in the Supporting Information examine signal intensity as a function of concentration while Figure 4 focuses exclusively on SNR. Although there is a deviation from linear, quantitation across 3 orders of magnitude remains a possibility. Instrument response curves were not performed with scan mode due to low ion current; however, for comparative purposes, the lowest concentration with neurotensin3+ visible in this mode, 110 nM, was used to obtain drift time spectra using a total scan time of 8 min (Figure 4a). As shown in Figure 4b, a sharp increase in SNR was found upon raising the concentration from 55 to 110 nM, with the mean SNR remaining approximately the same even up to 1100 nM. SNR measurements taken for concentrations below 1100 nM were found to suffer from high variability with percent relative standard deviations upward of 30%, as compared to a %RSD of ∼10% for 1100 nM. Because variation of the terminal frequency swept can alter not only SNR but also resolving power and the number of points across a drift time peak, the effects of frequency were explored with a total of three sample systems. In addition to neurotensin, lysozyme and a cytochrome C tryptic digest were both subjected to FT-IMS analysis with varying frequency sweep parameters. Frequency sweep times and maximum inject times were maintained at 8 min and 100 ms, respectively, for all three of these experimental series. Dependence of the number of points in a resulting mobility spectrum on frequency swept is demonstrated in Figure 5a with the triply charged neurotensin, examined at both 7.5 and 15 kHz net frequency sweeps. When sweeping from 5 to 7505 Hz, the neurotensin3+ peak consists of approximately six points; in contrast, completing the sweep in the same time length but with doubling the net frequency to reach 15 kHz, the point number across the same peak was doubled to 12, providing

more detail regarding peak shape. Along with an increase in points across a peak, enlarging the net frequency sweep can correspond to a noticeable increase in resolving power, as shown in Figure 5b for neurotensin3+ ion, while Figure 5c shows that SNR falls off as frequency increases. For terminal frequencies of 5.5 and 10 kHz, the mean resolving power was found to exceed that of the theoretically predicted maximum when accounting for diffusion and initial gate pulse width. Previous experimental and theoretical treatments of the FTIMS experiment demonstrate that the minimum effective gate pulse width for such multiplexing experiments corresponds to the inverse of the terminal frequency which serve as the basis for this comparison.25,29 However, given that the number of points across the mobility peak at lower frequencies is reduced, this observation may be an artifact of the fitting procedure. Nevertheless, it is encouraging that the observed maximum resolving powers track well with the values predicted by theory. For terminal frequencies above 10 kHz, the latter portion of the linear sweep produces pulse periods with a 50% duty cycle that ultimately fall below 100 μs in length. The data shown in Figure 5b suggest that any further increases in the terminal frequency above 10 kHz, corresponding to a resolving power of 80, are not met with further increases in resolving power. Stated differently, these data suggest that the minimum effective gating observed for this ion mobility system and target ion is approximately 100 μs. This observation should be tempered with the notion that higher terminal frequencies can provide additional points across the mobility peak and may in fact be effective for certain ion classes (e.g., high mobility). It should be noted that the impacts of extended frequency are also informative when placed in context of ion gate depletion. Recent work by Puton et al. provides a conceptual and theoretical framework to consider the impact of ion gating, both during the opening and closing cycles, that produces ion packets that deviate from the ideal.34 When the waveform period approaches the time necessary to traverse the fields produced by the ion gate, no appreciable ion current can enter the drift space. Monitoring the effects of frequency on charge states of lysozyme further demonstrates that increasing the end frequency not only improves resolving power of peaks but also refines spectral quality. In Figure 6a, the FT-IMS drift time spectrum acquired for lysozyme sweeping 5.5 kHz is presented. The general peak shape for charge states 8+ through 13+ can be seen in the spectrum, but too few points are present to reveal more features of the drift time peaks; this is true for all charge states shown, both lower and higher charge. By comparison, Figure 6b shows the additional features that can be seen by increasing the net swept frequency to 20 kHz. Shoulders present on peaks for charge states 11+ and 12+ have greater definition resulting from the enhanced resolution. Moreover, the additional points allow all of the peaks to become narrower, facilitating peak centroid determination. Plots of SNR versus frequency and resolving power versus frequency for the six charge states of lysozyme analyzed can be found in Figures S-2 and S-3, respectively. As can be seen in Figures S-2 and S-3, the charge states of lysozyme studied follow SNR and resolving power trends similar to neurotensin3+ (Figure 5) as a function of frequency swept. FT-IMS analysis was also performed on a trypsin digest of cytochrome C, with Figure 7a,b showing the change in drift time spectra for the digest in 5.5 kHz versus 20 kHz. The purpose of the digest was to examine ions with differing charges

Figure 5. (a) Larger net frequency sweeps have a clear advantage in providing more points across drift time peaks; for example, the neurotensin3+ peak has twice the points across the peak for 15 kHz versus 7.5 kHz. (b) Although this results in resolving power experiencing a noticeable gain upon transitioning to 10 kHz from 7.5 kHz, (c) SNR drops as progressively larger frequency sweeps are used. 3126

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Figure 6. Drift time peaks for six lysozyme charge states show substantial gains in spectral quality over 5.5 kHz (a) when the net frequency sweep is increased to 20 kHz (b), but at a cost of absolute signal intensity.

SNR for improved resolving power according to sweep frequency remains. While substantiating the effects of varied frequency, the cytochrome C digest also provides an excellent example of an area where FT-IMS requires further exploration to minimize noise and extraneous peaks: echo formation. As can be seen in Figure 7a, no apparent echo peaks can be seen with a 5.5 kHz sweep, but they are readily visible in the 20 kHz sweep (Figure 7b). These ghosts are a result of instability and changes in the frequency sweep velocity;35 this is substantiated by clearly visible echoes in a 7.5 kHz sweep performed using one of the waveform generators used here, which are not apparent in a 7.5 kHz sweep executed using the other waveform generator (Figure S-6), with otherwise identical experimental parameters. The echoes can reach relatively high peak intensities, with an example from the cytochrome C digest: lower intensity peptide fragments were seen at higher m/z than the other fragments mentioned but were excluded from the series because echoes found at higher sweep frequencies approached the peak intensity of real signals. Future implementations of this technique will utilize signal generators with static sweep velocities. Comparison of Scan and FT Modes. Comparative evaluations of the scan and FT modes were also performed to demonstrate how FT-IMS can capture spectral changes that are seen in signal-averaged spectra; the comparisons were completed using the 3+ charge state of neurotensin and the 8+ charge state of ubiquitin. With neurotensin, a large drift time window (3.0−27.1 ms) was covered with scan mode in 8 min for comparison to the arrival time distributions acquired over the course of 8 min with a 5−7505 Hz sweep FT mode series from the frequency variation experiment. To obtain a scan mode drift time spectrum across such a large window, a fairly coarse step size (50 μs) and short dwell time (1 s) were used so that the entire scan would be completed within the 8 min time frame. For this mode comparison, the concentration of neurotensin was held at 110 nM; however, a fairly large gate pulse width of 400 μs was used to ensure that the resulting SNR would exceed 3 in scan mode. When comparing scan and FT mode spectra with the same experimental time (Figure 8a) and concentration, the resulting FT spectrum not only has double the resolving power over scan mode but also has more than an order of magnitude of SNR improvement. To determine the amount of time required to complete a scan mode experiment under a given set of parameters, the net drift time range covered must be

Figure 7. Even a complex mixture, such as a tryptic cytochrome C digest, obtained across a large m/z range (50−2000 m/z) will yield FT-IM-MS spectra. A sweep of 5.5 kHz (a) provides adequate spectra for determining the fragments’ relative drift times, while some additional peak shape detail can be gleaned from a 20 kHz sweep (b). However, some drawbacks of a large net frequency sweep include a reduction in SNR and the potential for echoes to arise in spectra (b).

for evaluating FT-IMS when applied to a complex mixture. Yet again, a higher net frequency sweep led to additional points per peak and more detailed spectra. Of note is the substantially more defined shoulder peak for the 545 m/z triply charged ion when the frequency covered the 5−20 005 Hz range. The Supporting Information contains further plots showing the SNR versus terminal frequency (Figure S-4) and resolving power versus terminal frequency (Figure S-5) for the fragments with nominal m/z values of 817 (3+), 748 (2+), 736 (3+), and 545 (3+). Even in a complex mixture such as a tryptic digest of cytochrome C, the scenario of having to balance diminishing 3127

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Figure 8. When working under the same time constraints, FT-IM-MS provides superior SNR and resolving power over scan mode spectra, as shown for neurotensin3+. FT-IM-MS is also capable of capturing spectral changes that occur with temperature variation, such as promoted formation of various ubiquitin8+ protomers at 180 °C (b) versus 120 °C (c).

this approach is adapted these optimum values must be empirically determined. From a signal processing perspective, the data produced by the FT-IM-IT experiment are comparatively easily transformed into a standard drift time domain and are compatible with any FFT routine. Although chirp multiplexing is ultimately limited by the data acquisition speed in the m/z domain and the minimum effective gating pulse of the two ion gates, this instrumental mode of operation significantly increases SNR compared to signal averaging experiments and reaches resolving power levels that approach the diffusion limited maximum. A direct SNR comparison between the signal-averaged and chirped experiments suggests that the multiplex mode afforded benefits that exceed the theoretically predicted maximum (e.g., Figure 8); however, this discrepancy is largely attributed to the chronic under-sampling of the ion packets in the signal averaging mode when using the IMS-LTQ instrument. Nevertheless, the outlined multiplexing affords a significant SNR gain (up to 10-fold in some cases) and, perhaps more importantly, marked gains in spectral acquisition rates. Because the standard mode of mobility analysis using an ion trapping mass spectrometer requires discrete drift time windows to be examined individually, spectral acquisition is only possible through a brute force approach. The outlined solution is readily adapted to any hybrid ion mobility-ion trap MS combination and significantly lowers the barriers to acquiring informative drift tube mobility spectra for such instrumental combinations. As data acquisition rates improve for ion trapping instruments, the potential for this method to serve as an additional dimension to traditional LC-IT-MS experiments also grows. Improved ion trap fill and scan times may ultimately permit sufficiently fast sweep times that reach into the second time scale and enable the direct coupling with LC separations.

multiplied by the dwell time and divided by the step size. For example, covering a 20 ms net drift time range with a 2 s dwell time and a scan step size of 40 μs requires (20 ms)(1000 μs/ ms)(2 s/40 μs) = 1000 s or approximately 16.7 min to complete the experiment. As such, FT-IM-MS provides comparatively reduced analysis time that can lead to greater sample throughput for ion mobility-linear ion trap mass spectrometry systems. However, SNR improvements may be seen for both FT mode and scan mode as ion trap scan speeds increase. For ubiquitin, the scan mode and FT mode methods were performed at 120 and 180 °C for an individual spectrum per mode and temperature combination, with the corresponding FT spectra obtained with 5−7505 Hz frequency sweeps. Drift time peaks in FT mode show the same shifts in drift time according to temperature as can be seen in Figure 8b,c. Figure 8b,c exhibits changes in peak width with temperature obtained with FT-IMS: at 180 °C, the scan and FT mode peaks are fairly sharp (Figure 8b), while they both then broaden when the drift tube temperature is set to 120 °C (Figure 8c). As can be seen in the comparison (Figure 8b,c), FT is able to capture the increased presence of the ubiquitin 8+ charge state shoulder as the temperature of the drift tube is decreased from 180 to 120 °C, as is also shown in the two scan mode spectra.

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CONCLUSION Through the ion beam modulation by frequency chirps on both ion gates in a dual-gate IM-MS system, we illustrate how ion inject time, frequency sweep time, frequency range, analyte concentration, and temperature impact the spectral quality for drift tube ion mobility-mass spectrometry experiments. Because each of these variables remains integral to the technique, all must be considered when adapting this approach to existing ion trapping systems. Terminal frequency of the applied chirp plays the largest role in determining improvements in resolving power; however, a 4-fold change in terminal frequency only translates into a resolving power improvement of ∼1.5. The impact of varying sweep time and injection time scale SNR follow in a similar fashion. Adjusting these variables toward longer times improves SNR, but a double of one parameter does not yield a linear change in SNR. In fact, for both of these variables, the impact on SNR diminishes at higher values. It is our assertion that for each new ion trapping system for which

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04223. A linear regression plot showing neurotensin3+ peak intensity as a function of concentration; the SNR versus frequency and resolving power versus frequency plots for both charge states of lysozyme analyzed as well as for the 3128

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four fragments of cytochrome C examined; a comparison of the two waveform generators used for this experiment (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 509-335-8867. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based in part upon work supported by the U.S. Department of Homeland Security under Grant Award Number 2012-DN-120-NF0001-02. Funding for K.A.M. was provided by the Defense Threat Reduction Agency under Grant Award Number HDTRA-14-1-0023. We would also like to acknowledge support from ExcellIMS for technical guidance regarding the MA3100 drift tube.

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DOI: 10.1021/acs.analchem.5b04223 Anal. Chem. 2016, 88, 3121−3129