Probing Electrospray Ionization Dynamics Using Differential Mobility

Aug 17, 2012 - by: (1) the presence of distinct peaks in the DMS ionogram, (2) the observed ... system, (3) the observed 13C NMR chemical shifts arisi...
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Probing Electrospray Ionization Dynamics Using Differential Mobility Spectrometry: The Curious Case of 4‑Aminobenzoic Acid J. Larry Campbell,* J. C. Yves Le Blanc, and Bradley B. Schneider AB SCIEX, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada S Supporting Information *

ABSTRACT: Here, we present the separation of two ions that differ only by the site of protonation of the analyte molecule using differential mobility spectrometry (DMS). Protonated 4-aminobenzoic acid molecules (4-ABA) generated by positivemode electrospray ionization [ESI(+)] can exist with the proton residing on either the amine nitrogen (N-protonated) or the carboxylic acid oxygen (O-protonated), and the protonation site can differ on the basis of the solvent system used. In this study, we demonstrate the identification and separation of N- and O-protonated 4-ABA using DMS, with structural assignments verified by: (1) the presence of distinct peaks in the DMS ionogram, (2) the observed effects resulting from altering the ESI(+) solvent system, (3) the observed 13C NMR chemical shifts arising from altering the solvent system, (4) the observation of distinct MS/MS fragmentation patterns for the two DMS-separated ions, (5) the unique hydrogen−deuterium exchange behavior for these ions, and (6) the fundamental behavior of these two ions within the DMS cell, linked back to the structural differences between the two protonated forms.

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generated.1,2 These ratios were estimated using action spectroscopy, tandem mass spectrometry (MS/MS), and hydrogen− deuterium exchange (HDX) experiments. However, the mass spectrometers employed could not separate the two isomeric 4-ABA ion populations based upon mass analysis, and so, the ratios of the two sites of protonation were only estimates. Here, we demonstrate the ability to separate and characterize the two sites of protonation for 4-ABA using an atmosphericpressure differential mobility spectrometry (DMS) technique.

enerally, when a molecule is subjected to positive-mode electrospray ionization mass spectrometry (ESI-MS), it is ionized via protonation, and the added proton(s) (i.e., charge) is presumed to reside at the molecule’s site of greatest gas-phase basicity. However, this assumption has been challenged by recent research wherein the choice of ESI solvent has influenced the observed site of protonation1,2 or deprotonation3−6 (for negativemode ESI). One particular case involves 4-aminobenzoic acid (4ABA) (Scheme 1), wherein ESI(+) of a methanol/water solution provides only O-protonated molecules (i.e., the carboxylic acid group is protonated). However, when the ESI solvent is changed to acetonitrile/water, a ∼70/30 mixture of O-protonated to N-protonated (i.e., the amino group is protonated) molecules is © 2012 American Chemical Society

Received: June 7, 2012 Accepted: August 17, 2012 Published: August 17, 2012 7857

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and nitrogen was used as the curtain gas (3.5 L/min), throttle gas (0 or 0.7 L/min), and target gas (∼3 mTorr) for the MS/MS experiments. In each experiment, an analyte solution (10 ng/mL) was infused into the ESI source at a rate of 20 μL/min. The fundamental behavior of DMS devices is described elsewhere,14−17 and a brief description is provided in the Supporting Information section. For the DMS experiments conducted in this study, one of two operational modes was employed. In the first mode, the separation voltage (SV) was held at an optimum value (+3500 V, 116 Td) while the compensation voltage (CV) was scanned from −15 V to +15 V in 0.1-V increments. At each incremental value of CV, an MS or MS/MS spectrum of protonated 4-ABA was recorded (vide infra). These data were plotted in the form of ionograms. For the second mode of operation, both the SV and CV were scanned synchronously. For example, as SV was stepped from 0 to 3500 V (in 250-V increments), CV is scanned from −15 V to +15 V at each incremental SV value; again, at each increment of CV, an MS/MS spectrum of protonated 4-ABA was recorded. These data were plotted as dispersion plots,10,18 with SV as the y-axis, CV as the x-axis, and the intensity of the trace as the abundance of the specified ions. Tandem mass spectrometry (MS/MS) experiments were conducted using the enhanced product ion (EPI) scan mode19 of the QTRAP mass spectrometer. Each MS/MS experiment was conducted under identical instrumental conditions: precursor ion of m/z 138, 10 ms fill time, collision energy of 30 eVLab, and ∼3 mTorr of N2 target gas. Fragment and residual precursor ions were mass analyzed over the range of m/z 50−145 by massselective axial ejection (MSAE).20 To perform hydrogen−deuterium exchange (HDX) experiments, we admitted the deuterating reagent, deuterium oxide (D2O), into the junction chamber between the end of the DMS cell and the orifice of the MS. To do this, we allowed the throttle gas (Figure 1) to sample the headspace of a bottle containing 99% D2O. Although the exact concentration of D2O in this region was not determined, conditions were held constant during the HDX experiments for each protonated 4-ABA molecule. With the SV fixed at +3500 V, the CV was scanned from −15 V to +15 V in 0.1-V increments; at each incremental value of CV, ions were analyzed using an enhanced mass spectra (EMS) scan for ions of m/z 135−145.

Scheme 1. Structures of the Two Most Energetically Favored Sites of Protonation for 4-Aminobenzoic Acid Formed by Positive-Mode Electrospray Ionization

DMS and other related techniques (such as field asymmetric waveform ion mobility spectrometry or FAIMS)7 are well-known for their abilities to separate analyte ions from the less-desired chemical noise associated with ESI. These techniques have also demonstrated the ability to distinguish ions that are structural isomers,8,9 stereoisomers,10 and even isotopomers.11 However, to our knowledge, this is the first example of a DMS-based separation of isomeric species that differ only in their sites of protonation.

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EXPERIMENTAL SECTION Materials. Three isomers of aminobenzoic acid (2-, 3-, and 4-ABA), as well as deuterated water (99%), were obtained from Sigma-Aldrich (St. Louis, MO) and used without further purification. HPLC-grade acetonitrile (Caledon Laboratory Chemicals, Georgetown, ON) and HPLC-grade methanol (J.T. Baker, Avantor Performance Chemicals, Center Valley, PA) were also used without further purification. Distilled deionized water (15 MΩ) was produced in-house using a Millipore (Billerica, MA) Integral 10 water purification system. Differential Mobility Spectrometry-Mass Spectrometry. A differential mobility spectrometer (SelexION, AB SCIEX, Concord, ON) system10 was mounted on a 5500 QTRAP system (AB SCIEX),12,13 between a TurboV ESI source and the mass spectrometer’s sampling orifice (Figure 2). The ESI probe was maintained at a voltage of 4800 V, with a source temperature of 150 °C, nebulizing gas pressure of 30 psi, and auxiliary gas pressure of 20 psi. The DMS temperature was maintained at 150 °C,

Figure 1. Cross-sectional view of the DMS, depicting the relevant components and gas flows (Adapted from ref 10). While volatile chemical modifiers can also be added to the curtain gas, HDX reagents were used only in the throttle gas. 7858

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Figure 2. Results for three individual ABA isomers analyzed by ESI(+)-DMS-MS. Only 4-ABA yields two distinct peaks upon analysis.

Theoretical Methods. All calculations were performed using Gaussian 09 (revision A.1),21 and the results were visualized using GaussView 5.0.9. Geometry optimization was performed at the density functional (DFT) level of theory with functionals of the “pure”DFT type; both of these used the gradient-corrected exchange functional of Becke combined with the gradient corrected correlation functional of Lee, Yang, and Parr22−25 (B3LYP) using the cc-pVTZ basis set.26

produced by ESI(+) as a function of the organic solvents used. These differences lend support to the generation of 4-ABA molecules protonated at two distinct locations and their separation using DMS. For example, we analyzed 4-ABA solutions prepared in one of three different solvent systems: (1) 100% water, (2) 75% methanol/25% water, and (3) 50% acetonitrile/50% water (Figure S1, Supporting Information). Solvent systems (2) and (3) are similar to those used in earlier 4-ABA studies.1,2 In each case, two dominant peaks were observed at CV = −7.5 V and CV = −1.5 V, which we will henceforth label as “A” and “B”, respectively. These two species were present at the same CVs regardless of the organic solvent used in the ESI(+) solution; only the relative abundance of each peak changed. According to the findings of the Kass group,1,2 when MeOH/ H2O was used as the ESI solvent system, only O-protonated 4-ABA was formed. In our analysis of a similar 4-ABA solution with the same solvent composition [Figure S1b, Supporting Information], we observed the “B” 4-ABA ion in vast excess (∼92%) to the “A” 4-ABA ion (∼8%). When the Kass group analyzed 4-ABA from an aqueous acetonitrile solvent system (50/50), they reported the formation of a ∼70/30 mixture of O- and N-protonated 4-ABA. Interestingly, when acetonitrile was used in the ESI(+) solvent of our DMS experiments [Figure S1c, Supporting Information], we observed a similar ratio of “B” to “A” 4-ABA ions (∼78% “B” versus ∼22% “A”). These results lend support to the generation and transmission of N-protonated and O-protonated 4-ABA. We also examined other organic-solvent-to-water ratios to understand the influence of methanol and acetonitrile on the DMS behavior of 4-ABA. In addition to analyzing a 100% water 4-ABA solution [Figure S1a, Supporting Information], we also analyzed solutions containing increasing percentages of methanol [Figure 3a−c], as well as increasing acetonitrile/water ratios [Figure 3d−g]. Increasing the relative amount of methanol in the ESI(+) solvent maintained a high percentage of the “B” 4-ABA ions (O-protonated 4-ABA), while increasing acetonitrile concentrations favored formation of “A” 4-ABA ions transmitted (N-protonated 4-ABA). The enhanced formation of N-protonated 4-ABA when high percentages of aprotic acetonitrile

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RESULTS AND DISCUSSION Electrosprayed 4-ABA Molecules Produce Two Peaks in a DMS Ionogram. Typically, when analytes of MW < 1000 Da are ionized by ESI(+) and analyzed using DMS, the ions are transmitted at one optimum CV (at a given SV), providing one peak in the DMS ionogram. However, this was not the case for 4-ABA solutions, which yielded two distinct peaks centered at CV = −7.5 V and −1.5 V (SV fixed at +3500 V) in the DMS ionogram (Figure 2, blue trace). At these two CV values, we recorded MS/MS spectra for the protonated 4-ABA ions (m/z 138). While the fragmentation patterns were consistent with library MS/MS of protonated 4-ABA, we observed distinct differences between the two data sets (vide infra). However, one possible explanation for the multiple peaks in the DMS ionogram existed: the 4-ABA sample could have been contaminated with either the 2- or the 3-ABA isomers. Either of these species could provide similar MS/MS spectra from precursor ions with an identical m/z value and isomeric structures to 4-ABA. To test this hypothesis, we analyzed 2-ABA and 3-ABA samples independently using the same ESI(+)-DMS experiment parameters as the 4-ABA sample. As displayed in Figure 2, the 2-ABA (orange trace, CV = −2.9 V) and the 3-ABA (pink trace, CV = −4.9 V) yielded ionograms containing only one peak, with each peak appearing at unique CV values to the 4-ABA data. In addition, we verified the structure and purity of the 4-ABA sample using 13C NMR spectroscopy (Table S2, Supporting Information). Influence of the Organic Solvents Used during ESIDMS-MS/MS. Similar to previous studies,1,2 we observed differences in the behavior of protonated 4-ABA molecules 7859

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Figure 3. Observed differences in the DMS behavior of protonated 4-ABA produced by ESI(+) as a function of the organic solvents used (as labeled in the figure).

and the “A” 4-ABA ions at CV=-7.5 V shows several key differences between the two MS/MS spectra: (1) a much larger amount of residual precursor ion (m/z 138) for the “A” ions, (2) a very abundant m/z 120 peak (corresponding to loss of water, −18 Da) for the “B” ions, (3) a very abundant fragment ion of m/z 92 (loss of water and carbon monoxide, −46 Da) for the “B” ions, and (4) unique ions of m/z 121 (loss of ammonia, −17 Da) and m/z 103 (loss of ammonia and water, −35 Da) for the “A” ions. The MS/MS spectra obtained here show similar features to those previously reported for N- and O-protonated 4-ABA.1,2 For example, the larger amount of residual precursor ion (m/z 138) present in the MS/MS spectrum of the “A” 4-ABA ions matches the reported poorer fragmentation behavior of N-protonated 4-ABA. The lack of any residual precursor ion present in the MS/MS spectrum for the “B” 4-ABA ions matches the reported fragmentation behavior of the O-protonated analogue. In addition, while fragment ions indicating water loss from protonated 4-ABA (m/z 120) was reported in both the MeOH and ACN ESI MS/MS data, loss of ammonia (m/z 121), which was previously reported only for N-protonated 4-ABA,1 was observed here only for the “A” 4-ABA ions. Additionally, the unique fragment ion at m/z 103, corresponding to loss of ammonia and water, is present only for the “A” ions and can be rationalized as forming after the initial loss of ammonia to

was employed may be the result of the stabilization of the most stable site of protonation in the liquid phase: the amino group. Conversely, the use of methanol and water (protic solvent systems) facilitated stabilization of O-protonated 4-ABA upon ESI(+).2 MS/MS Fragmentation Patterns Confirm the Presence of Two Different Ion Structures. Besides the differences observed when altering the ESI(+) solvent systems, we also observed different fragmentation behavior for the 4-ABA ions as a function of their optimal transmission through the DMS. Like the stability of the CV values, the fragmentation patterns in the MS/MS spectra were unchanged at each specific CV value when ESI(+) solvent systems were altered. This revealed that the “A” and “B” 4-ABA ions had structural differences consistent with different sites of protonation and that formation of these structures was reproducible. Upon interpretation of these spectra and comparison to similar studies’ results,1,2 we find support in assigning an N-protonated structure to the “A” 4-ABA molecules and an O-protonated structure to the “B” 4-ABA ions. As described earlier, MS/MS fragmentation spectra were collected for protonated 4-ABA ions as they were transmitted through the DMS and Q1 mass filter, using identical instrumental conditions (e.g., same collision energy, target gas pressure, etc.). An inspection of the MS/MS spectra (Figure 4, Table S1, Supporting Information) obtained for the “B” 4-ABA ions of m/z 138 transmitted through the DMS cell at CV = −1.5 V (Figure 4) 7860

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Figure 4. MS/MS spectra (CE = 30 eVLab) obtained for Q1-selected ions of m/z 138 ([M + H] + of 4-ABA) transmitted at CV = −1.5 V (“B” ions) and at CV = −7.5 V (“A” ions).

However, interactions between ions and neutral molecules added prior to the DMS cell can promote enhanced separation by altering the clustering between these species.28,29 In these experiments, we performed the HDX experiments post-DMS and focused on the use of the DMS to separate the 4-ABA ions based upon their native ESI(+) conditions. No HDX reagent− ion clustering interactions were promoted using this configuration. Lastly, while we did not have an accurate assessment of the true number density of D2O in the post-DMS region, we estimated that the amount of D2O each 4-ABA ion encountered was equivalent over the time period of the DMS experiments. Examining the DMS Behavior of Each Ionic Isomer as They Relate to Ion Structure. To examine the fundamental behavior of the protonated 4-ABA molecules in the DMS, we generated dispersion plots10,18 from the MS/MS data. To create these plots, both the SV and CV were scanned synchronously while electrospraying 4-ABA solution and subsequently collecting MS/MS spectra for the precursor ion (m/z 138) (Figure 6). Several of the fragment ions that were unique to the “A” and “B” ions come from protonated 4-ABA molecules that behave very differently in the DMS dispersion plots. While CV has little effect on the separation of ions at low SV values, as SV is increased, the differences between the high- and low-field mobilities

form the 4-dehydrobenzoic acid cation that subsequently loses water. Hydrogen−Deuterium Exchange Behavior Reveals the Two Different 4-ABA Ion Structures. Similar to previous reports, we observed differences between the HDX behavior of the two 4-ABA ions as a function of their optimal DMS transmission conditions (Figure 5). These HDX reactivities also matched the reactivities previously assigned to the two 4-ABA protonation sites.1,2 For example, the “A” 4-ABA ions of m/z 138 showed very little incorporation of deuterium [Figure 5b], in contrast to the “B” 4-ABA ions, which displayed a much greater level of deuterium incorporation [Figure 5c]. Given the identical reaction times and HDX reagent concentrations available to these ions, one can infer that the “B” ions incorporated deuterium at a much faster rate than the “A” ions. Previous research2 demonstrated that the N-protonated species underwent HDX at a much slower rate (∼25× slower) than the O-protonated analogue. For the HDX experiments, we employed the throttle gas line (Figure 1) to introduce the deuterating reagent, deuterium oxide (D2O) vapor, allowing the 4-ABA ions to be separated by the DMS before undergoing HDX. Using the atmospheric pressure regions within the ion source for HDX27 has the benefit of fast reaction times and ease of implementation. 7861

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Figure 5. (a) DMS ionogram obtained before and after an HDX experiment on protonated 4-ABA formed by ESI(+); (b) ESI-MS recorded with CV = −7.5 V before (pink) and after (blue) HDX; (c) ESI-MS recorded with CV = −1.5 V before (pink) and after (blue) HDX.

trends toward negative CV values for increasing SV, but ultimately shifts more positive with increasing SV. The Type A behavior is associated with N-protonated 4-ABA [Figure 6g], which has no resonance stabilization of the positive charge by the rest of the molecule. The calculated dipole moment for this ion (13.07 D) is a result of the charge localization on the protonated amino group. Conversely, the calculated structure for O-protonated 4-ABA [Figure 6h] revealed a resonance-stabilized structure involving the aromatic ring and the amino group. Accordingly, the calculated dipole moment of O-protonated 4-ABA is much smaller (1.47 D) than the N-protonated analogue. We propose that, when the positive charge is localized at the amino group (N-protonated), the clustering of residual ESI solvent and/or ambient N2 gas molecules occurs mainly at the charged ammonium group. The stronger dipole moment for N-protonated 4-ABA is expected to provide more of a polarization effect to the transport gas, promoting clustering between the ion and the background gas. Clustering suppresses the low-field mobility for these ions and leads to Type A behavior due to the dynamic clustering/declustering of these species.15 For O-protonated 4-ABA, its resonance-stabilized structure leads to a smaller calculated dipole moment, which we postulate reduces the clustering interactions of the ion with the transport gas within the DMS cell. With increasing SV (i.e., where the high-field mobility regime dominates), the behavior of these ions exhibits more “hard sphere” characteristics as the clustering interactions with solvent and background gas play a much reduced role, hence the observed Type B character for this ion.

of N- and O-protonated 4-ABA become more pronounced [Figure 6b−f]. By evaluating the patterns present in the dispersion plots, we discovered two distinct mobility behaviors from the protonated 4-ABA populations. For example, in Figure 6b−d, the optimal CV for transmission through the DMS shifts to more negative CV values as SV is increased, ultimately settling at CV = −7.5 V for the maximum SV employed (+3500 V). These species correspond to fragment ions of N-protonated 4-ABA (m/z 138), including ions of m/z 138, intact precursor ions remaining after MS/MS [Figure 6b]; ions of m/z 121, which correspond to the loss of NH3 from the precursor ion [Figure 6c]; and ions of m/z 103, produced from the loss of NH3 and H2O from the 4-ABA precursor ions [Figure 6d]. In contrast, O-protonated 4-ABA demonstrated much different DMS mobility behavior. For example, the dispersion plot for 4-ABA ions that fragment via loss of H2O to form ions of m/z 120 [Figure 6e] show similar curvature as the dispersion plot for 4-ABA ions that fragment to form ions of m/z 92 (loss of H2O and CO) [Figure 6f]. In the analysis of the MS/MS spectra (vide supra), these fragment ions were associated with the 4-ABA ions transmitted at CV = −1.5 V, O-protonated 4-ABA. These ions show an initial shift toward negative CV values with increasing SV and then curve back to more positive CV values. We postulate that the differences in DMS mobility behavior can be associated with the structures of the two protonated 4ABA molecules. The dispersion plots presented in Figure 6b,c,d display the behavior of ions exhibiting Type A behavior,7,10 wherein the optimal CV becomes more negative with ever increasing SV. Conversely, the dispersion plots in Figure 6e,f demonstrate Type B behavior, where the optimal CV initially 7862

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Figure 6. (a) Ionogram recorded with SV = +3500 V showing the optimal separation of the two 4-ABA ions; (b−f) Dispersion plots collected for the 4-ABA ions, displaying two distinct SV vs CV patterns for the N- and O-protonated 4-ABA species. Calculated structures of the (g) N-protonated and (h) O-protonated forms of 4-ABA, with the calculated dipole moments for each ion.

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CONCLUSIONS By combining the results of various MS-based experiments along with computational theory, we have demonstrated that two ions differing by only their sites of protonation can be separated using DMS. The evidence for this capability derives from: (1) the presence of distinct peaks in the DMS ionogram, (2) the observed effects

resulting from altering the ESI(+) solvent system, (3) the observation of distinct MS/MS fragmentation patterns for the two DMSseparated ions, (4) the unique HDX behavior for these ions, and (5) the fundamental behavior of these two ions within the DMS cell. In addition to the capabilities of the DMS instrumentation outlined here, another ion mobility technique (traveling wave ion 7863

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(12) Guna, M.; Biesenthal, T. A. J. Am. Soc. Mass Spectrom. 2009, 20, 1132−1140. (13) Collings, B. A.; Romaschin, M. A. J. Am. Soc. Mass Spectrom. 2009, 20, 1714−1717. (14) Eiceman, G.; Karpas, Z. Ion Mobility Spectrometry, 2nd ed.; CRC Press: Boca Raton, FL, 2005. (15) Krylov, E. V.; Nazarov, E. G.; Miller, R. A. Int. J. Mass Spectrom. 2007, 226, 76−85. (16) Shvartsburg, A. A. Differential Ion Mobility Spectrometry: Nonlinear Ion Transport and Fundamentals of FAIMS; CRC Press: Boca Raton, FL, 2009. (17) Krylov, E. V.; Coy, S. L.; Vandermey, J.; Schneider, B. B.; Covey, T. R.; Nazarov, E. G. Rev. Sci. Instrum. 2010, 81, 024101. (18) Levin, D. S.; Miller, R. A.; Nazarov, E. G.; Vouros, P. Anal. Chem. 2006, 78, 5443−5452. (19) Hager, J. W.; Le Blanc, J. C. Y. Rapid Commun. Mass Spectrom. 2003, 17, 1056−1064. (20) Londry, F. A.; Hager, J. W. J. Am. Soc. Mass Spectrom. 2003, 14, 1130−1147. (21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.1; Gaussian, Inc., Wallingford CT, 2009. (22) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652. (23) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785−789. (24) Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. J. Phys. Chem. 1994, 98, 11623−11627. (25) Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 1200− 1211. (26) Kendall, R. A.; Dunning, T. H., Jr.; Harrison, R. J. J. Chem. Phys. 1992, 96, 6796−6806. (27) Takáts, Z.; Schlosser, G.; Vékey, K. Int. J. Mass Spectrom. 2003, 228, 729−741. (28) Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Eur. J. Mass Spectrom. 2010, 16, 57−71. (29) Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Anal. Chem. 2010, 82, 1867−1880. (30) (a) Lalli, P. M.; Iglesias, B. A.; Toma, H. E.; de Sa, G. F.; Daroda, R. J.; Silva Filho, J. C.; Szulejko, J. E.; Araki, K.; Eberlin, M. N. J. Mass Spectrom. 2012, 47, 712−719. (b) Karpas, Z.; Berant, Z.; Stimac, R. M. Struct. Chem. 1990, 1, 201. (31) Joyce, J. R.; Richards, D. S. J. Am. Soc. Mass Spectrom. 2011, 22, 360−368.

mobility spectrometry or TWIMS) has recently been employed to separate ions based upon their sites of protonation30a using an unconventional polar carrier gas (i.e., carbon dioxide) in the TWIMS cell. Interestingly, this study provided more conclusive separation and identification for two forms of protonated aniline than an earlier drift-time IMS study30b had provided. The data presented here also highlight the importance for understanding the potential influence of somewhat subtle effects (i.e., ESI solvents, sites of protonation, etc.) on the outcomes of DMS analyses. However, once an understanding of such effects has been acquired, these factors can be exploited to promote better S/N though selective manipulation of the optimal DMS voltage settings. For example, if chemical noise consisting of ions isobaric to protonated 4-ABA were present at CV = −1.5 V, the ESI(+) solvent conditions could be modified (e.g., greater percentage of ACN) to promote formation of N-protonated 4-ABA, with optimal DMS transmission at CV = −7.5 V instead. With this in mind, future research will examine the DMS behavior of other ions that can protonate or deprotonate at different and/or multiple sites. Some research has shown that fragmentation patterns for peptides and proteins and even drug molecules31 can be influenced by the site of protonation. As well, sites of deprotonation for acidic species [e.g., deprotonated tyrosine,3,5 4-hydroxybenzoic acid4] will be examined for the potential role of solvent systems in the ESI-DMS behaviors of these ions.

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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 Drs. Rob Nieckarz, Tom Covey, Ron Bonner, Steve Tate, and David Cox of AB SCIEX for invaluable insights and Dr. Howard Hunter of York University’s NMR facility for sample analysis and helpful discussions.

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dx.doi.org/10.1021/ac301529w | Anal. Chem. 2012, 84, 7857−7864