B American Society for Mass Spectrometry, 2016
J. Am. Soc. Mass Spectrom. (2017) 28:1262Y1270 DOI: 10.1007/s13361-016-1504-z
FOCUS: BIO-ION CHEMISTRY: INTERACTIONS OF BIOLOGICAL IONS WITH IONS, MOLECULES, SURFACES, ELECTRONS, AND LIGHT : RESEARCH ARTICLE
Dual-Polarity Ion Trap Mass Spectrometry: Dynamic Monitoring and Controlling Gas-phase Ion–Ion Reactions Muyi He,1 You Jiang,2 Dan Guo,1 Xingchuang Xiong,2 Xiang Fang,2 Wei Xu1,3,4 1
School of Life Science, Beijing Institute of Technology, Beijing, 100081, China National Institute of Metrology, Beijing, 100013, China 3 State Key Laboratory Explosion Science and Technology, Beijing Institute of Technology, Beijing, 100081, China 4 Key Laboratory of Convergence Medical Engineering System and Healthcare Technology, the Ministry of Industry and Information Technology, Beijing Institute of Technology, Beijing, 100081, China 2
Abstract. A dual-polarity linear ion trap (LIT) mass spectrometer was developed in this study, and the method for simultaneously controlling and detecting cations and anions was proposed and realized in the LIT. With the application of an additional dipolar DC field on the ejection electrodes of an LIT, dual-polarity mass spectra could be obtained, which include both the mass-to-charge (m/z) ratio and charge polarity information of an ion. Compared with conventional method, the ion ejection and detection efficiency could also be improved by about one-fold. Furthermore, ion–ion reactions within the LIT could be dynamically controlled and monitored by manipulating the distributions of ions with opposite charge polarities. This method was then used to control and study the reaction kinetics of ion–ion reactions, including electron transfer dissociation (ETD) and charge inversion reactions. A dual-polarity collision-induced dissociation (CID) experiment was proposed and performed to enhance the sequence coverage of a peptide ion. Ion trajectory simulations were also carried out for concept validation and system optimization. Keywords: Dual-polarity mass spectrometry, Gas phase ion–ion reaction, Linear ion trap, Collision-induced dissociation, Electron transfer dissociation Received: 5 August 2016/Revised: 29 August 2016/Accepted: 8 September 2016/Published Online: 25 May 2017
Introduction
M
ass spectrometry (MS) is an analytical technique that measures the m/z ratios of gas-phase ions. Analytes in samples need to be ionized and then mass-analyzed in a mass spectrometer. After ionization, different molecules might possess charges with different polarities and states, which depend on the chemical property of each molecule and the ionization environment [1]. In many applications, such as in the omics study, it is highly desirable to detect as many analytes as possible in complex samples in a short period of time [2, 3],
Muyi He and You Jiang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10. 1007/s13361-016-1504-z) contains supplementary material, which is available to authorized users. Correspondence to: Xiang Fang; e-mail:
[email protected], Wei Xu; e-mail:
[email protected], URL: http://www.escience.cn/people/weixu
especially when coupling with chromatography techniques [4, 5]. Therefore, samples sometimes need to be analyzed in both positive and negative modes. However, under the conventional single-polarity mode, ions with one polarity could be analyzed while ions with the opposite polarity were then wasted, even if they could be generated at the same time [6, 7]. Since polarity switching is a relatively slow process [8, 9], several “dualpolarity” MS techniques and instruments were developed [10–12]. Dual-polarity electrospray (ESI) and matrix-assisted laser desorption (MALDI) ionization sources could generate cations and anions simultaneously [6, 11, 13–15]. To analyze dual-polarity ions at the same time, two MS instruments with two independent ion optics systems and mass analyzers were built [11, 12, 16]. Cations and anions were separated and analyzed in each MS instrument independently. On the other hand, a mass spectrometer could also perform ion structure analysis through tandem MS. Gas-phase ion/ion reactions are routinely carried out in tandem MS experiments [17, 18], especially in the identification and localization of
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post-translational modification sites of proteins and peptides [19, 20]. Tandem MS methods, such as collision-induced dissociation (CID) [21, 22], electron transfer dissociation (ETD) [23, 24], negative-ion electron transfer dissociation (NETD) [25], surface-induced dissociation (SID) [26, 27], and higherenergy collision dissociation (HCD) [28], have been widely used for ion structure analyses. With the capabilities of storing ions with opposite polarities and performing MSn experiments, quadrupole ion traps have been proven to be very useful vessels for ion–ion reactions [29]. In the above processes, although particles of opposite polarities are normally involved in the reaction process as substrates or products, ions of only one polarity are usually monitored in traditional MS analyses. In this work, a dual-polarity LIT MS method was proposed and realized by applying a dipolar DC signal on the ejection electrodes of the LIT. With this approach, dualpolarity mass spectra could be obtained in MS and MSn experiments, which not only include the m/z ratio information of an ion but also its charge polarity. Different from previous dual-polarity MS instruments, cations and anions can be manipulated and analyzed simultaneously in the same mass analyzer, the LIT. Therefore, ion–ion reaction kinetics could also be controlled and monitored. Dualpolarity gas-phase ion reactions, such as dual-polarity ETD, charge inversion, and CID experiments were carried out and demonstrated. Dynamically controlling and monitoring ion–ion reactions could help the understanding of ion reaction mechanism. Results also suggest that dual-polarity tandem MS method could enhance the sequence coverage of peptide ions.
Experimental Chemicals and Materials Cytochrome c, bradykinin, adenosine-5'-monophosphate disodium salt (AMPNa2), formic acid (FA), ammonium hydroxide, and anthracene-9-carboxylic acid were purchased from Sigma-Aldrich Chemical Co. Ltd. (St. Louis, MO, USA). Methanol (HPLC grade) was purchased from Fisher Scientific (Fairlawn, NJ, USA). Acetic acid (AA) was purchased from Fluka (St. Louis, MO, USA). Perfluoro-1octanol (PFO) was purchased from Alfa Aesar (Ward Hill, MA, USA). The peptides DRVYIHPFHL (angiotensin I) and LIDDNNLNTAGEGGCYPR were synthesized by SBS Genetech Co. LTD. (Beijing, China). All these reagents were used without any further purification. Distilled water was produced by a Milli-Q system (Millipore Inc., Bedford, MA, USA). Sample solutions were prepared in methanol/water (v/v 1:1) as stock solutions at the concentration of 1 mM, and diluted to the final concentration before experiments
Instrumentation A home-built MS apparatus was developed and used for monitoring and controlling gas-phase ion/ion reactions. As shown
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in Figure 1a, the instrument mainly includes two ionization sources, two ion optics systems, a three-section linear ion trap (LIT) with hyperbolic electrodes, two ion detection systems, a vacuum system, and an electronic control system. Cations and/ or anions could be generated and introduced from either side of the instrument to the linear ion trap. Two electrospray ionization (ESI) sources were used in this study (otherwise specified). In each ion optics pathway, a quadrupole mass filter (QMF, Q1, and Q2) with pre- and post-filters was used to transport or mass-selectively transfer ions into the LIT (Figure 1a). The pre-, post-filter rod length, quadrupole rod length, and rod radius were 20, 20, 130, and 6 mm, respectively. The ratio of rod radius over field radius was 1.125. The ion beam was controlled by turning on/off the radio frequency (rf) voltages applied on the quadrupole mass filters. The LIT has three sections with the following dimensions: center to electrode distance x0 = y0 = 5 mm, length of the front and back sections is 12 mm, length of the center section 37 mm. Pressure in the LIT was maintained at ∼2 mTorr by introducing helium as the buffer gas. An electronic control system was developed in-house. Besides the two rf and direct current (DC) signals applied on the two QMFs (Q1, Q2), a dual phase rf signal (frequency 1.128 MHz) was generated to control the LIT. An alternating current (AC) signal was applied on the pair of electrodes with ejection slits (Figure 1b) for mass selectively ion ejection. An rf voltage of 293.5 V0-p was used for ion trapping, and an ejection q of 0.844 was used for mass analysis. A DC bias potential could be applied on each rf electrode (center section) of the LIT, and they could be controlled independently. Two electron multipliers (EM) with dynodes (DeTech 397) were placed on two sides of the LIT with ejection slits. By applying –15 and +15 kV, respectively, on the dynodes of these two detectors, cations and anions could be detected separately. These two detectors shared a common signal collection system. Two DC potentials could be applied on the pair of electrodes with ejection slits (x-electrodes as shown in Figure 1b), and the spatial distributions of cations and anions could be controlled by tuning this DC voltage. Different effects would be obtained if these DC bias potentials were applied in different time stages, such as the ion introduction stage, the reaction stage, the detection stage, etc. Details about the operation parameters and performances can be found in later sections.
Numerical Simulation Numerical simulation was also carried out using a homedeveloped multi-physics simulation program accelerated by GPU parallel computing technology [30, 31]. Ion trajectories and ion cloud spatial distributions were obtained and used to understand the mechanism of dual-polarity ion operation in the LIT. The electric field inside the LIT was computed using COMSOL (COMSOL, Burlington, MA, USA), and the ion motion differential equation was solved by the 4th-order Runge-Kutta integration method. The hard sphere collision model [32] was used to calculate the ion-neutral collision
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Figure 1. Schematic presentation of the experimental apparatus. (a) Structure of instrument; (b) ion detection pathway and electric connections of the LIT
probability, and the elastic collision model [33] was applied to calculate the energy transfer rate during each ion-neutral collision. An ion–ion reaction model developed earlier [34] was also used in the simulation to characterize ion–ion reactions. In the simulation, angiotensin I (with +3 charges, m/z = +433) and azobenzene (with –1 charge, m/z = –182) were used as model ions. Initially, all ions were placed at the center of the LIT. The rf signal applied on the ion trap has a frequency of 1 MHz and amplitude of 200 V0-p; the AC signal applied on the ion trap has a frequency of 157.5 kHz and amplitude of 2 V0-p; ion trajectories were simulated for 10 ms. The ion ejection process was studied by the simulation program, and the results can be found in the Supporting Information. To serve the purpose of understanding the separation mechanism of dipolar DC field instead of optimizing experimental parameters in detail, the parameters in simulation were chosen so that simulation results could represent the practical situation in experiments. For example, the rf signal frequency and amplitude in simulation were chosen to be close to the values in experiments: 1 MHz in simulation, 1.128 MHz in experiments; 200 V0-p in simulation, 293.5 V0-p in experiments. With similar q values and potential well depths, simulation results could be qualitatively used to explain the phenomena in experiments.
Theory After introducing in a quadrupole ion trap, ions will be cooled to ion trap center regardless of their charge polarities, and ions with different charge polarities would overlap and react with each other. The conventional mass selective ion ejection [35,
36] or Fourier transform mass analysis [37–39] in a quadrupole ion trap could measure the m/z ratio of an ion but not its charge polarity. Therefore, either positive or negative mass analysis mode was conventionally used in a single MS analysis. To achieve dual-polarity ion trap MS, the spatial distributions of cations and anions within the LIT are controlled and manipulated using the additional DC voltages applied on x-electrodes. In general, cations will be shifted from the center of the LIT towards the rf electrode with the application of a negative DC potential, and anions will be shifted from the center of the LIT towards the rf electrode with the application of a positive DC potential. Therefore, the overlapping and interaction between cations and anions could be controlled by tuning this DC voltage. Furthermore, if a DC voltage was applied during the ion ejection process, directional ion ejection could be achieved. In other words, cations could be preferentially ejected from the rf electrode with a negative DC potential and detected by the following cation EM detector; anions could be preferentially ejected from the rf electrode with a positive DC potential and detected by the following anion EM detector (Figure 1b). Simulation results in Figure 2 show that the overlapping and separation of cations and anions could be effectively controlled through the application of the DC voltages. Cation and anion clouds were initially placed at the geometric center of the LIT. After applying a 2 V DC bias voltage (+1 V and –1 V on each x-electrode), cations and anions separate from each other, and stable ion distributions could be achieved within ~2 ms (Figure 2a and b). As shown in Figure 2c, ions will shift further away from the trap center with increased DC voltages, and cations (m/z + 433 at a q value of 0.18) have a larger shift compared with that of anions (m/z –182 at a q value of 0.43).
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Figure 2. Simulation results. (a) The spatial distribution of two ion clouds versus time after applying a 2 V DC bias voltage on xelectrodes; (b) spatial distributions of these two ion clouds after stabilized at 10 ms. (c) Separations of the dual-polarity ion clouds at different DC bias voltages applied on x-electrodes; (d) ion storage efficiencies of the dual-polarity ion clouds at different DC bias voltages applied on x-electrodes
The distance that ions shift from the trap center depends on DC amplitudes as well as the rf trapping condition (q value of the Mathieu’s equation). Typically, ions experiencing deeper rf pseudo-potential well depths will have smaller spatial shifts under the same DC electric field. On the other hand, ion trapping efficiency might decrease as ions shift further and further away from the trap center (Figure 2d). Therefore, this DC voltage needs to be carefully chosen, and a balance needs to be achieved between ion cloud separation and ion trapping efficiency.
Results and Discussions A monopolar DC field refers to the application of a DC voltage on one of the x-electrode, whereas a dipolar DC field refers to the application of two DC voltages with the same amplitude but different signs on x-electrodes [40]. The electric field distributions for monopolar and dipolar DC fields with a 2 V potential difference are plotted in Figure 3a and b (simulation parameters can be found in the Supporting Information). Both monopolar and dipolar DC fields could effectively shift ion clouds from the trap center. Nevertheless, superposition of these DC electric fields might also induce mass shifts and ion intensity variations, which were then characterized through experiments. In the experiments, bradykinin (125 μM with 1% AA) was ionized and trapped in the LIT at a q value of ~0.27. The monopolar or dipolar DC fields weree then applied on x-
electrodes after the ion injection period (details about the scan waveform could be found in the Supporting Information). As shown in Figure 3c, ion intensity could be enhanced by more than two times with the dipolar DC field and three times with the monopolar DC field, compared with the conventional ion ejection method (no additional DC voltage was applied, and a single EM was used to collect ion signal). Theoretically, with the directional ion ejection, the ion signal collected from an EM would be double of that using the conventional ion ejection method. The more than 2-fold ion intensity enhancement might be due to the fact that directional ion ejection would also improve ion ejection efficiency. Although a stronger ion signal could be obtained using the monopolar DC field, it will also induce a serious mass shift problem as shown in Figure 3d. On the other hand, a less than 0.5 Th mass shift was observed with the dipolar DC field. The mass shifts observed with monopolar DC are fully expected since the monopolar DC can be treated as a combination of dipolar and quadrupolar DC voltages. The quadrupolar DC component changes where the ions reside in the stability diagram and causes mass shifts (details can be found in the Supporting Information). It should be noted that results in Figure 3c and d were obtained by averaging 50 mass spectra. Applying DC bias voltage showed no significant impact on mass resolution (Figure S4 in Supporting Information). Therefore, the dipolar DC field was used in later experiments for dual-polarity ion manipulation and detection. With the dipolar DC field, ions with different charge polarities could be mass-selectively ejected from opposite directions
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Figure 3. Electric field distributions in x-y plane of (a) the monopolar DC field, and (b) the dipolar DC field with 2 V potential difference. Effects of the monopolar and dipolar DC fields on (c) the relative ion intensities, and (d) the mass shifts of the bradykinin ions with +2 charges (m/z = 531). The DC potential differences range from 0 to 60 V
and detected by the following EMs separately. Therefore, the charge polarity of an ion could be determined. Figure 4a shows a typical dual-polarity mass spectrum, which was collected for bradykinin (125 μM with 1% AA, nESI HV 1500 V) and PFO (400 μM with 1% ammonium hydroxide, nESI HV –1600V). A dipolar DC of ±10 V was applied the x-electrodes of the linear ion trap. Monomer and dimer of PFO (m/z –399 Th and –799 Th) were observed and plotted in blue; doubly charged bradykinin (m/z +531 Th) was observed and plotted in red. A 2-fold signal intensity enhancement was obtained compared with the conventional ion ejection mode (insets in Figure 4a). No cross-talking between two detection pathways were observed. The more than 2-fold ion intensity improvement also indicates that effective ion directional ejections could be achieved for both cations and anions. It should
be noted that ion–ion reactions will happen when trapping both polarity ions in the ion trap. Typically, this is not a big issue for low charge state ions in our experiments, since the proton transfer rate was not high and no significant ion loss was observed (as shown in Figure 7, no strong proton transfer reaction products were observed). Furthermore, the dipolar DC voltage could be applied to the ion trap as soon as oppositely charged ions were introduced into the same trapping section of the ion trap, so that proton transfer reaction could be minimized as shown in Figure S3b in the Supporting Information. Further ion spatial separation could be achieved by increasing the DC voltage; however, a decreased ion trapping efficiency would be observed if the DC voltage was large compared with the pseudo-potential well depth. Under the pseudo-
Figure 4. (a) Dual-polarity mass spectrum of bradykinin and PFO; (b) the relative ion abundances at different DC voltages
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potential approximation (q < ~ 0.7), ions at lower q values would experience shallower pseudo-potential well depths, and smaller DC voltages could effectively shift ions from the trap center. Figure 4b shows the effects of this DC voltage for different ions during the dual-polarity MS analysis. In experiments, under the same rf trapping voltage, ions with m/z –399 Th, +531 Th, and – 799 Th would experience different q values (0.35, 0.27, and 0.18, respectively). The dipolar DC voltage was applied during ion ejection period (Figure S3b in Supporting Information). As we increase the dipolar DC voltage, ion intensities of heavier ions (m/z –799 Th) start to decrease at 40 V, whereas ion intensities of lighter ions start to decrease at ~60 V. It should be noted that PFO dimers might be dissociated by the rf heating effect as they were further shifted away from the trap center [41–43]. As a consequence, an up to 6-fold ion intensity increase was observed for PFO monomer. In later experiments, less than 20 V DC voltages were typically applied. In ion–ion reactions, both cations and anions are introduced into an ion trap, and it is important to control the reaction process and monitor reaction products. Reaction intermediates or products may have opposite charge polarity to the reactant. In the traditional single-polarity MS method, it is difficult to analyze all reaction participants at the same time. As a demonstration, an ETD experiment was performed. In the experiments, angiotensin I (100 μM with 0.1% FA, ESI HV 3500 V) and anthracene9-carboxylic acid (100 μM with 1% ammonium hydroxide, ESI HV –3500 V) were used as ETD reactants [44]. The reaction between triply charged angiotensin I and anthracene anion (CID product of anthracene-9-carboxylic acid) were carried out. Figure 5a shows a typical dual-polarity mass spectrum in which a dipolar DC voltage (±10 V) was applied during the ion ejection period. As shown in Figure 5b and c, the ETD reaction between cation (m/z = +433) and anthracene anion (m/z = –177) actually stops after ~2 s, since the ETD product ion intensities stop increasing (for example c2 fragment, m/z = +289). However, the proton transfer reactions (PTR) still go on, where the precursor ion intensity as well as the ETD product ion intensities decrease with respect to time (Figure 5c). From the dual-polarity mass spectra, it is actually clear that a water molecule would attach to the anthracene anion (m/z = –177), and the product ion (m/z = –195) does not have the ETD reaction activity [45]. Therefore, although PTRs continue to carry on between the anion (m/z –195) and cations, ETD reactions phase out with the decrease of anthracene anion. As another demonstration, a double proton transfer reaction was performed in the dual-polarity LIT, and charge inversion phenomenon was observed through the dual-polarity mass analysis. Reactions between multiply charged cations and singly charged anions may result in multiple proton transfer, which can be expressed as the following reaction equation: ðM þ nHÞnþ þ Y − →ðM þ ðn−mÞH Þðn−mÞþ þ YHðmm−1Þþ ð1Þ In the experiments, AMPNa2 (100 ppm, nESI HV –1700 V) and cytochrome c (100 ppm with 0.1% FA, nESI HV 1600 V)
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were introduced into the LIT as reactants [46]. Before performing ion–ion reactions, anions (AMPNa2) and cations (cytochrome c) were introduced into the LIT separately in two MS scans, and two mass spectra were recorded (Figure 6a) using conventional MS scan. Then they were introduced simultaneously into the LIT for reaction. After reaction, dualpolarity mass analysis was performed by applying ±10 V dipolar DC voltages on x-electrodes (please refer to Supporting Information for scan waveform). As shown in Figure 6c, a double proton transfer reaction was observed, and protonated AMP cations were generated as the reaction products. The charge state distribution of cytochrome c was shifted from +11 ~ +21 to +8 ~ +20, and the center charge state was shifted from +16 to +15 after this proton transfer reaction. With the dual-polarity MS technique, the reaction kinetics curves of both cations and anions could be obtained in a single experiment. Figure 6b plots the reaction kinetics curves of the reaction substrate (AMP anions) and the reaction product (AMP cations). Moreover, the ion–ion reaction rate could be tuned by manipulating the overlapping of cations and anions. As shown in Figure 6d, the reaction process could be suppressed by separating the ion clouds using the dipolar DC voltage during the ion trapping period (reaction time is 600 ms; please refer to Supporting Information for the scan waveform). The reaction could be completely stopped with the application of a 10 V dipolar DC voltage. Thia suggested that the starting and stopping time of an ion–ion reaction can be precisely controlled in the dual-polarity MS operation.
Dual-Polarity CID A lot of biomolecules, such as peptides and proteins, can be ionized in both positive and negative modes. In most cases, the generated cations and anions have different fragmentation patterns. With this dual-polarity setup, the CID mass spectra of both the cations and anions of a peptide can be obtained at the same time. Peptides DRVYIHPFHL (positive ESI: 100 μM with 0.1 FA, HV +3500 V; negative ESI: 100 μM with 0.1% ammonium hydroxide, HV –4800 V) and LIDDNNLNTAGEGGCYPR (positive ESI: 20 μM with 0.1 FA, HV +3500 V; negative ESI: 20 μM with 0.1% ammonium hydroxide, HV –4800 V) were tested as examples. Figure 7a shows the dual-polarity CID mass spectrum of peptide DRVYIHPFHL in which doubly charged cations (m/z +648.6) and anions (m/z –646.6) of DRVYIHPFHL were isolated and fragmented simultaneously to generate fragment ions. A 101 kHz AC excitation signal with a voltage of 0.12 to 0.42 V was applied on top of the dipolar DC voltage for precursor ion excitation and fragmentation. No apparent neutralization or proton transfer reaction products were observed after the 20 ms CID process. Similarly, the triply charged LIDDNNLNTAGEGGCYPR, cations (m/z +641.7) and anions (m/z –639.7), were isolated and fragmented (AC: 101 kHz, 0.12–0.42 V) with the dual-polarity CID mass spectrum shown in Figure 7b. Under the same excitation condition, cations have more fragments than anions, suggesting that anions have more
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Figure 5. ETD reaction in the dual-polarity MS mode. (a) ETD mass spectrum of angiotensin I and anthracene-9-carboxylic acid, inset: mass spectrum after isolation; (b) dynamic monitoring the anions in the ETD reaction; (c) dynamic monitoring the cations in the ETD reaction
Figure 6. Charge inversion reaction in the dual-polarity MS mode. Mass spectrum of (a) AMPNa2 and cytochrome c in the conventional single-polarity method; (b) dynamic monitoring the reaction process; (c) dual-polarity mass spectra of AMPNa2 and cytochrome c after 600 ms reaction time (a 20 V DC was applied during ion ejection); (d) ion intensities after 600 ms reaction time with the application of different dipolar DC voltages during the reaction process
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Figure 7. Dual-polarity CID mass spectrum. (a) dual-polarity CID mass spectra of ±2 DRVYIHPFHL (angiotensin I); (b) dual-polarity CID mass spectra of ±3 LIDDNNLNTAGEGGCYPR
stable structures than cations. Since cations and anions have different fragmentation patterns, more structural information could be obtained in the dual-polarity CID mass spectra. Although conventional mass spectrometers could switch polarities between scans to obtain the tandem mass spectra for both polarities, the buffer solution could not be switched between scans. As is well known, an acidic buffer solution, which is typically used in positive ion mode, can hardly generate multicharged anions in negative ion mode, and vice versa. Therefore, not many fragments could be obtained in conventional polarity switching mode. In the current setup, we could use different buffer solutions to generate the cations and anions so that multi-charged ions in both polarities could be obtained simultaneously. As a result, rich fragment information could be gained.
Conclusion In this work, a dual-polarity ion trap mass spectrometer was developed, where positive and negative ions can be manipulated and analyzed simultaneously using an additional dipolar DC signal. The DC potential applied to the x-axis electrodes of a LIT can provide useful capabilities in a variety of processes associated with an MSn experiment. Both simulation and experiment were carried out to validate this concept. With this approach, ions could not only be distinguished by their m/z
ratios but also their charge polarities in MS and MSn experiments. The improved ion detection efficiency and increased mass spectrometry information were obtained under optimized operating conditions. Ion/ion reaction kinetics process was controlled and monitored in real time.
Acknowledgments The authors acknowledge support for this work by NNSF China (21475010), “1000 plan” in China, MOST instrumentation program of China (2011YQ09000502, 2011YQ09000501, 2011YQ09000507, and 2012YQ040140-07).
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