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Macromolecular ion accelerator mass spectrometer† Cite this: Analyst, 2013, 138, 7384
Yun-Fei Hsu,a Jung-Lee Lin,a Ming-Lee Chu,b Yi-Sheng Wanga and Chung-Hsuan Chen*a We present a newly developed macromolecular ion accelerator mass spectrometer that combines a dualion-trap device and a macromolecular ion accelerator (MIA) to achieve the capability of analyzing samples with a mixture of large biomolecules. MIA greatly increases detection efficiency. The dual ion trap includes a quadrupole ion trap (QIT) and a linear ion trap (LIT) in tandem. The dual ion trap is mounted ahead of the MIA. The QIT is used to store multiple species, and the LIT is employed to capture the ions that are sequentially ejected out of the QIT. Subsequent to their capture, the ions inside of the LIT are extracted and transferred to the MIA. The synchronization between the QIT and MIA is bridged by the LIT. A
Received 13th August 2013 Accepted 2nd October 2013
sample containing a mixture of several large biomolecules was employed to examine the performance of this new type of mass spectrometer. The result reveals that larger biomolecules show a comparable signal to smaller biomolecules, even though the mixture contains equal quantities of each type of
DOI: 10.1039/c3an01534c
protein. The overall assembly produces a nearly constant detection efficiency over a broad mass range.
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Thus, this device provides an alternative platform to analyze complex large-protein mixtures.
Introduction Progress in proteomics analysis using mass spectrometry has mostly occurred through continuous improvements in instrumentation. During the past couple of decades, combinations of different types of mass analyzers have produced advantages over each individual device.1,2 In addition, ion traps and ion trap-based instruments have been broadly employed as analytical platforms. To satisfy the growing demand for characterization with high throughput, a great many techniques have been applied for the manipulation of ion traps. The strategic control of trapping and ejection of ions inside of the ion traps enables a range of novel analytical methods. For example, various ejection methods have been developed to analyze a wide mass range of biomolecules.3–5 The robustness, fast-scan-speed, and MS/MS sensitivity of ion traps have made them popular over the past decade. Ion traps can be classied into two types: the 3D quadrupole ion trap (QIT) or the 2D linear (or rectilinear) ion trap. The 3D ion trap (or Paul trap) mass spectrometer is simple and convenient to use6–8 but has some disadvantages such as a space charge effect,9,10 low trapping capacity,11 and low resolution. Combining a linear quadrupole ion guide12–15 with a 3D ion trap can help overcome some of these limitations.16–18
a
The Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan. E-mail: [email protected]; Fax: +011886-2-27899923; Tel: +011-886-2-27871200
b
Institute of Physics, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan † Electronic supplementary 10.1039/c3an01534c
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7384 | Analyst, 2013, 138, 7384–7391
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The major application of ion guides has been to interface with the electrospray (ESI)19 source. Furthermore, the use of quadrupole or octupole guides20 rather than traps removes the ability to concentrate the ions of interest. In addition, only small molecular ions have been tested; the detection of large biomolecules has not been demonstrated due to the low detection efficiency. 2D ion traps (typically called linear ion traps) are known for having a better trapping capacity and a lower space charge effect than 3D ion traps.21,22 Moreover, their high degree of exibility allows them to integrate with other mass analyzers and has made them an indispensable element in the evolution of hybrid instruments.23–26 In general, hybrid designs that incorporate the same types of ion traps are built to conduct MS/MS and/or MSn (n $ 3) analyses.27–29 Double or multiple ion traps have also been reported to execute different analyses.30–32 Among these reports, the transfer efficiency poses the biggest challenge and limits the further evolution of these assemblies. Moreover, novel analytical techniques are required to serve as complementary strategies for proteomics research. Matrix-assisted laser desorption/ionization (MALDI) has been broadly used for the ionization of large molecules. MALDI is operated with a time-of-ight (TOF) spectrometer to detect large proteins. Electron amplication devices, such as electromultipliers, channeltrons and microchannel plates (MCPs), are widely used as detectors by producing secondary electrons. The efficiencies of these electron amplication devices strongly depend on the velocity of the ions. The detection efficiency approaches zero when the velocity of an ion is signicantly lower than 104 m s1. For large biomolecular ions, the detection efficiencies are very low due to the low velocity with the same kinetic energies. A cryogenic detector has been used for the
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detection of large biomolecules. However, the response time is slow for cryogenic detectors. The macromolecular ion accelerator (MIA)36 offers a novel method by directly enhancing the ion energy to lead to high detection efficiency. Recently, we successfully demonstrated the combination of a 3D ion trap (i.e., QIT) with a 2D linear ion trap (LIT) for biomolecule samples with matrix-assisted laser desorption/ ionization (MALDI).33 For molecular weight calculation of a step frequency scan of the QIT, it follows an equation that was derived from the Mathieu equation: m¼
8eV : qz ðr0 þ 2Z0 2 ÞU2 2
The parameters represent molecular weight m, charge number e, radio frequency potential V applied to the ring of QIT, ring electrode of radius r0, and electrode space between two end-cap 2z0, trapping parameter qz (value of 0.908 under unstable ejection conditions) and angular frequency U which is equal to 2pf, where f is the radio frequency in Hertz. In this step frequency scan case, the amplitude of radio frequency was constant but only the frequency changed. Therefore, m/z is related to the radio frequency. For frequency scanning, the smaller the step for the frequency scan, the higher the resolution. In addition, the throughput can possibly be improved if the rst quadrupole ion trap is replaced by a linear ion trap. For samples with a mixtures of biomolecules, MALDI analysis shows a distinct advantage over ESI due to the low number of peaks.34 In this work, a dual-ion-trap is employed as an ion source and a mass analyzer for biomolecular ions that are subsequently accelerated35 to produce high detection efficiency. Because our dual ion traps exhibit good transmission efficiency and the macromolecular ion accelerator (MIA) gives an effective improvement of the detection efficiency,36 we incorporated the accelerator into the dual ion traps to achieve the mass analysis of biomolecule mixtures. A large portion of ions with a broad time-spread exited the QIT and was mostly captured by the LIT. Hence, the integration of a dual-ion trap with the accelerator is an advance that can lead to a new type of mass spectrometer: the macromolecular ion accelerator mass spectrometer (MIA). In this study, the details of combining the QIT, LIT and accelerator are examined and results are reported.
secretory IgA (sIgA) (20 mM) were mixed together and cocrystallized with the matrix solution. Every analyte was prepared at a volume of 1 mL and mixed with 9 mL of the matrix solution before being placed upon the probe tip. This sample was then allowed to air-dry before being introduced into the chamber. Instrumentation setup Fig. 1 is the schematic of the dual ion trap-accelerator mass spectrometer. A QIT–LIT combination is employed. In the test of this dual-trap device, ions were stored in the QIT device and then they were ejected out of the QIT. The LIT was used to capture the ejected ions. To obtain ions of the analyte, a pulsed laser for MALDI was phase-locked relative to the RF frequency of the QIT. The phase was adjusted to optimize the trapping of ions, and these ions were subsequently cooled by collision with helium in the QIT. The frequency step scan was designed to sequentially execute trapping, laser ring, frequency step scan and data sampling. Typically, the desorbed ions were rst accumulated in the QIT by exposing them to several shots of the laser. Once the accumulation was accomplished, the step scan was triggered to eject selected ions from the QIT; subsequently, these ions were stored in the LIT. It was found that the transfer efficiency from the QIT to the LIT was 60%. More details on the dual-ion trap will be published separately.33 In this work, helium was introduced into the QIT and LIT as a buffer gas in the pressure ranges of 30–50 mtorr and 70–80 mtorr, respectively. The rst acceleration plate was positioned at 54 mm away from the exit endcap of the LIT. This spacing was adopted to provide a compartment between the dual-ion trap and the accelerator. Between the remainder of the plates, an equal spacing of 25 mm was employed. Only 8 out of the 12 plates are shown in this gure. Aer the 12 plates, a channeltron detector (CEM 5900 M, Photonics) was mounted and oated at 1850 V to allow ion detection. Electronics A step scan was performed in mass-selective axial instability mode for the QIT. A TTL signal was started at the same time that the step scan was initiated. The TTL signal was used to trigger and time the pulses on the z-electrodes of the LIT as well as the subsequent acceleration. Typical wiring of the step scan, pulse
Experimental details Sample preparation Sinapinic acid (SA) was prepared at a concentration of 100 nmol mL1 in 60 : 40 acetonitrile/0.1% TFA. Peroxidase, BSA, IgG, brinogen and sIgA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). These chemicals were prepared at a concentration of 20 pmol mL1. To create a single compound sample, 1 mL of a protein sample, which included 20 pmol of biomolecules and 4 mL of matrix solution (400 nmol), was deposited onto a stainless steel probe and was allowed to air-dry. To create a sample containing a mixture of compounds, equal amounts of bovine serum albumin (BSA), IgG and
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Fig. 1 Schematic of the dual-ion-trap accelerator mass spectrometer. The black arrow indicates the direction along which ions travel and are directed towards the channeltron. The cutaway view shows only 8 of the 12 acceleration plates. The 1st acceleration plate is positioned at 54 mm after the exit endcap of the LIT. A channeltron is positioned at the end of the accelerator.
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Analyst and waveform on both endcaps are displayed in Fig. 2. To synchronize the moment of extraction out of the LIT with the 1st stage of acceleration, a synchronization trigger was generated at the same time extraction begun. This synchronization trigger was used to initiate the whole acceleration sequence aer an appropriate delay. Aer acceleration, ions were directed towards the channeltron for detection. The step scan offers the opportunity for each type of compound in the sample to be ejected separately. Molecular ions of different masses can be ejected one by one during the frequency step scan in the QIT. Ions with different masses are captured by the LIT one aer the other and are then sequentially accelerated. As a result, a mass spectrum can be readily obtained containing distinct signals that correspond to different species. Data acquisition As the frequency step scan was initiated by a home-made control program to eject the ions, a TTL signal was generated simultaneously to trigger the pulses at both endcaps of the LIT and the acceleration. The acceleration event was synchronized to the extraction of the LIT by wiring between an arbitrary waveform generator (AWG) (WW2572, Tabor Electronics Ltd. Tel Hana Israel) and a broadband power amplier (5/80, TREK). The pulse(s) of acceleration can be timed by synchronization of the output trigger of the AWG. To optimize the signals, we employed empirical ne-tuning of the pulses throughout the entire work. The signals were averaged by accumulating 60–80 laser shots, and the spectra were displayed using a LeCroy Waverunner 64Xi oscilloscope (LeCroy Corp., Chestnut Ridge, NY). A spectrum showing intensity versus time was acquired and
Fig. 2 The electronics of the dual-ion-trap accelerator mass spectrometer. To time the acceleration pulses, a synchronization trigger is generated on the basis of the timing of the extraction at Z1. Therefore, the acceleration is determined by the timing of the extraction. Definite timing is established by the applied voltage. Upon successful demonstration of the acceleration, acceleration pulses were applied at the 1st, 3rd, 5th, . 11th plates while keeping the 2nd, 4th, 6th, . 12th plates grounded for simplicity. Each pulse is exerted at 20 kV; therefore, the maximum ion energy is 120 kV. The signal is generated at the oscilloscope as the ions impact upon the channeltron.
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Paper transferred through a general-purpose interface bus (GPIB) to a personal computer (PC) for further processing. Either oscilloscope screenshots or recorded traces in the American Standard Code for Information Interchange (ASCII) format were taken using the ScopeExplorer soware (LeCroy). During step scanning to eject various masses, the traces recorded from the oscilloscope indicated the intensity vs. ejection frequency and, hence, a mass spectrum was obtained.
Trajectory simulation A simulation was conducted to study the integration between the dual ion traps and the accelerator. Fig. 3 shows a typical result of the simulation using IgG ions. A side view shows the interior of the LIT and the plates for acceleration (Fig. 3A). It is noteworthy that the length of the rods in the LIT is equal to the spacing between adjacent plates of the accelerator. This design is employed for the purpose of not only assuring a sufficient trapping capacity of the LIT37 but also restricting the ions within the distance of 25 mm. This spatial restriction in the LIT is intended to allow the ions to fully exist in the gap between adjacent plates. If the length of the rods exceeds 25 mm, the ions ejected out of the LIT cannot be completely included within the spacing between two plates.
Fig. 3 Simulation of the dual-ion-trap accelerator mass spectrometer. (a) For simplicity, the ions were initiated inside the LIT. Ions were extracted by placing 1000 V upon the entrance endcap of the LIT. Subsequent to the extraction, acceleration was applied to the ions as the blue trajectories display. (b) The potential energy view of the accelerated ions. The bidirectional arrows indicate the 1st and 2nd plates. Before ions reach the 1st acceleration plate, they have the same kinetic energy (the black trajectories). The energies begin to vary after they experience the 1st stage of acceleration.
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Paper For simplicity, only 4 plates were assumed in this simulation, and the conguration was identical to that in Fig. 1. To account for the practical situation, the initial position of the ions was set-up across the axial direction within the LIT while the RF potential was maintained at 930 Vpp. The original kinetic energy of the ions, in principle, was determined by the competition between collisional cooling with the buffer gas (70 mtorr) and RF heating from the trapping eld. The initial kinetic energy of the ions was close to a thermal distribution at room temperature based on the evidence of a successful capture. The extraction of ions from inside of the LIT was achieved by applying a pulse voltage of 1000 V upon the Z1 endcap. Subsequent to the extraction, the 1st stage of acceleration was applied to the ions as shown in the blue trajectories. The potential energy view in Fig. 3b depicts the underlying details of the movements of the ions and especially those occurring between the 1st and 2nd plates. The pulsing electric eld in particular is removed from this gure to increase the ease of observation of the trajectories. While being extracted out of the LIT, the ions' motion is designated by the black trajectories, which display a uniform energy status. Moreover, the trajectories show the inuence of the RF potential upon the motion of the ions. The inuence is reected by the oscillation of their trajectories, which are caused by RF interference through the hole at the center of the endcap. The 1st acceleration pulse was switched on just as all of the ions arrived at the 1st plate. Apparently, the spatial distribution of the ions can be conned between the 1st and 2nd plates as expected. When the 1st pulse was applied, the ions were accelerated by 20 kV as shown by the 9 vertical lines orthogonal to the potential energy surface. However, the energy distributions were broadened as
Analyst designated by the trajectories with different colors. Each energy status, on average, differs from another by 9 eV; therefore, the largest difference of energy among these ions is 70 eV aer they have undergone the 1st stage of acceleration. Although this value is only 0.35% of one pulse voltage, this calculation along with the spatial distribution evidently demonstrates the necessity of empirically adjusting the pulses rather than following pre-programmed calculations. Consequently, the subsequent experimental results employing acceleration from more than one pulse were conducted by stepwise ne-tuning of the pulse timings.
Results and discussion The results of the dual-ion-trap accelerator device Fig. 4 presents a typical result acquired from the integration of the dual trap and the accelerator. Fig. 4a shows the results using peroxidase38 ions with one or two stages of acceleration, which are designated by the black and red traces, respectively. A dramatic increase of signal intensity is noticeable and is highly reproducible. This result was conrmed by at least 10 replicate measurements. Fig. 4b depicts the results acquired under onestage acceleration (i.e., 20 kV). In this gure, the onset of acceleration was begun at different times relative to the initiation of extraction in the LIT. The 1st acceleration plate is positioned at 72 mm past the exit endcap of the LIT in this case. From the bottom to the top, time delays of 450 ms, 550 ms, 600 ms, 650 ms and 700 ms post-extraction were applied accordingly. No signal was obtained at a delay of 450 ms. Signals were observed for delay times from 550 to 700 ms. For a delay of 750 ms or more, signals were again not observed. These results
Fig. 4 Results of integrating the dual ion traps with the accelerator. Peroxidase (molecular weight 43 kDa) was selected to examine the integration of the dual-trap and the accelerator. (a) The black and red traces represent the results of applying one and two stages of acceleration, respectively. A significant enhancement of the signal intensity is observed for the cumulative acceleration. (b) All results were obtained under one stage of acceleration (i.e., 20 kV), but the acceleration was started at different times. From the bottom to the top, acceleration was started at time lags of 450 ms, 550 ms, 600 ms, 650 ms and 700 ms subsequent to extraction. It is found that the acceleration can be applied over a spread time of 150 ms. This result further implies that ions within the LIT possess a spatial spread of 25 mm (i.e., the length of the rods) and a temporal spray of 150 ms.
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Analyst imply that ions within the LIT possess a longitudinally spatial distribution of 25 mm, which is the maximal length of the rods. Furthermore, a temporal distribution of 150 ms is observed. These results lead to an empirical choice of timing to optimize the acceleration. Fig. 5 displays the optimized result for IgG ions. Fig. 5a shows the result when IgG ions experience a step scan from 30 kHz to 18 kHz before being captured inside of the LIT for 1 ms. They are then extracted and further accelerated by a voltage of 60 kV. Fig. 5b displays the relative timings of the pulses among the endcaps (Z1 and Z2) and the accelerator. Herein, the pulse on Z2 shows an output amplitude of 100 V with a pulse duration of 1 ms (the black trace). The waveform on the Z1 endcap exhibits a gating of 110 ms subsequent to the Z2 pulse, which allows the entrance of ejected ions from the QIT (as the red trace and the green arrow indicate). The acceleration pulses are enlarged in Fig. 5c. In this gure, the relative timing between these pulses and the Z1 waveform is clearly shown. The acceleration was initiated 432.3 ms aer the extraction, which was a value determined by empirical ne-tuning. The initiation of the 1st acceleration pulse was regarded as t0 for the purposes
Paper of evaluating the acceleration efficiency; this will be discussed further in the following section. A series of successful results revealed that the integration between a dual trap and an accelerator can work quite well. The signals are much better than those in which only a single ion trap is integrated into the accelerator (data not shown). The LIT truly builds a bridge between the QIT and the accelerator. Review of the effects of acceleration In the MIA, the efficiency of sequential acceleration was thoroughly investigated and will be demonstrated and discussed briey. Fig. 6 depicts the results for IgG ions undergoing acceleration at different energies. Fig. 6A displays signals from IgG ions that were captured for 1 ms and then accelerated by 40, 60, or 80 kV. The start of the 1st pulse was referred to as t0 (with an electronics jitter39 of 10 ns) and, thus, the evolution of signals at different accelerations is readily observed. This time jitter is not taken into account throughout this work because it exerts almost no inuence on the acceleration efficiency. The movement of the ions occurs on a time scale of microseconds, and this level is at least 1000-fold slower than the jitter. The improvement of the signals is reected in the fact that each series of peaks has higher signal intensities than the one before it. This fact implies that an ever-boosted ion velocity is obtained by ever-increasing the number of steps of acceleration. In addition to the peak shape, the temporal evolution displays good agreement with the applied energy. Fig. 6b displays signals from IgG ions that were captured for 2 ms and then accelerated by 20, 40, or 60 kV. A two-fold longer capture time creates no attenuation of the signals. This result shows that the signals remain strong throughout a long capture time. The integration between the capture and the acceleration is not affected by an elongated capture time. However, it was experimentally conrmed that the capture time cannot be shorter than 1 ms. Otherwise, ions cannot efficiently thermalize, and this leads to the unsuccessful acceleration or metastable decay of ions. In this case, the signals cannot be obtained. Moreover, it is noteworthy that IgG ions cannot be detected by applying only 20 kV of acceleration voltage. This result must be compared with the results shown in Fig. 4, in which peroxidase ions could be detected at 20 kV. This difference is attributed to the fact that the velocity of IgG ions is nearly one-half of that of peroxidase ions at 20 kV. Thus, more energy input is needed to increase the kinetic energy of IgG ions. Measurements of brinogen ions
Fig. 5 Typical results of the integration between the dual-ion trap and the accelerator by examining IgG molecular ions. (a) The step scan was adopted by frequency hopping from 30 to 18 kHz on the ring electrode of the QIT. This signal was acquired by trapping the ions inside the LIT for 1 ms, extracting and then accelerating. (b) Three overlapping traces display the relationship among the Z1 and Z2 endcaps and the pulses for acceleration. The black, red, and blue traces represent the pulse at Z2, the waveform at Z1 and multiple acceleration pulses, respectively. (c) The enlarged view of the traces of Z1 and of the acceleration pulses. The blue trace contains 3 pulses of acceleration, which are initiated at 432.3 ms subsequent to the start of extraction. Here, the initiation of the 1st acceleration pulse is defined as t0 (with a jitter of 10 ns) to examine the acceleration efficiency upon the IgG ions. Results are shown in Fig. 6.
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Fig. 7 reveals the results for brinogen ions. Frequency stepping was conducting from 14 kHz to 9 kHz in the QIT; the ions were captured by the LIT with an RF at 80 kHz; and the ion motion was synchronized with a total acceleration voltage of 120 kV. The capture time for brinogen in these tests was 3 ms. Although the signals also exhibited no attenuation at longer times, it was again found that the shortest capture time cannot be less than 1 ms. In general, synchronization between the dual ion traps and the accelerator has been successfully validated on the basis of
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Fig. 6 Results of IgG ions undergoing acceleration with different energies. The noise prior to 50 ms results from the pick-up of the high voltage pulses in the accelerator. (a) IgG ions were captured for 1 ms and then accelerated by 2, 3, or 4 pulses (the black, red and blue traces, respectively). The evolution of the signals is in good agreement with the acceleration energy. The initiation of the 1st acceleration pulse was defined as t0 (with a jitter of 10 ns), and the efficiency of the acceleration is thus clearly characterized. (b) IgG ions were captured for 2 ms and then accelerated by 1, 2, or 3 pulses (the olive, black and red traces, respectively). Unlike peroxidase ions (the results in Fig. 4), IgG ions cannot be detected by applying only 20 kV (olive color). The kinetic energy is not sufficient to cause the IgG ions to efficiently impinge upon the channeltron detector because the detector was only floated at 1600 V in this experiment. A dramatic improvement of the signal was observed when 40 or 60 keV of acceleration energy was supplied.
Fig. 8 A typical spectrum obtained by the dual-ion-trap accelerator mass spectrometer. The step scan was divided into 20 steps from 35 kHz to 8 kHz to sequentially eject BSA+, IgG+ and IgA+. The acceleration energies upon each species were 80, 120 and 120 keV, respectively.
Fig. 7 Typical results acquired via the dual-ion-trap accelerator mass spectrometer. (a) The signal recorded from the scope while using fibrinogen ions was obtained by step scanning from 14 kHz to 9 kHz in the QIT, capturing the ions in the LIT with the RF at 80 kHz and synchronizing the total acceleration voltage of 120 kV. The RF voltages applied upon the QIT and LIT were 760 Vpp and 930 Vpp, respectively. (b) A typical waveform applied to the Z1 endcap. (c) The pulses were synchronized to the waveform with a proper delay for efficient acceleration. The inset represents an enlarged picture of the pulses.
the evidence shown above. In the following section, an attempt is made to utilize the combination device to conduct an analysis of samples containing multiple compounds.
Mixture analysis A typical demonstration that employs the dual-ion-trap accelerator mass spectrometer is shown in Fig. 8. A step scan was adopted by dividing the range from 35 to 8 kHz into 20 steps. BSA+, IgG+ and IgA+ ions were gradually ejected one aer another. It is noteworthy that larger biomolecules can be measured with a signal-to-noise ratio comparable to that of
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smaller ones even though the sample contains equal quantities of each type of molecule. This result neatly demonstrates an appealing feature; our mass spectrometer exhibits a constant detection efficiency throughout a broad mass range. The reason for this can be attributed to the integration of our MIA. The pulse duration of the acceleration for high-mass ions shows a better compatibility with the instrument electronics, especially the switches. When accelerating high-mass ions, the duration can be sufficiently long to prevent the need for an extremely fast rise/fall of the switch. This performance causes the switches to work optimally and results in a more efficient acceleration. Therefore, Fig. 8 shows an example of good detection efficiency that would not be observed with conventional mass spectrometers; conventional models always display signicantly diminished signals for the high-mass ions with respect to low-mass ions using a channeltron or microchannel plate as the detector. Moreover, the almost total lack of noise demonstrates the lack of interference upon these signals from the analytes. The reason can be attributed to the high mass selectivity that the accelerator possesses. In the dual ion trap, the results were directly recorded aer the extraction of ions from the LIT. Although the analysis of a mixture was demonstrated, low detection efficiency of large biomolecules is still a concern. With the integration of the accelerator, the pulses are preset on the basis of a certain mass value; thus, temporal and spatial connement is
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Analyst established over the course of acceleration. As a result, noise can be discriminated from the signals. The resolutions of the BSA+, IgG+ and IgA+ peaks are estimated at 3.7, 8.3, and 7, respectively. Comparable resolutions between the IgG+ and IgA+ peaks lead us to consider attempting an analysis of biomolecular ions with even higher molecular weights in the future. This appears to be feasible because there is no dramatic decrease in resolution. However, the resolutions, in general, are not high because of the presence of buffer gas in the course of ejection. More sophisticated designs are needed to improve the resolution. Because the ejected ions from the MALDI were stored in the QIT, most metastable ions produced in the MALDI-TOF device are expected to be quenched. In this work, few metastable ions were observed. Therefore, this device can further simplify mass spectra by eliminating metastable ions.
Conclusion The rst design of a dual-ion-trap accelerator mass spectrometer has been accomplished. This device exhibits not only high detection efficiency but also the capability for mixture analysis. The high detection efficiency is achieved by the accelerator, and the storage and selectivity of various ions are accomplished by the dual-ion-trap device. Moreover, the enrichment of the selected ions is one of the special advantages of this device. The overall combination in our instrument results in a desk-top accelerator with the capability of a mass spectrometer.
Acknowledgements We thank the former President of Academia Sinica, Yuan T. Lee, for his valuable discussions and suggestions. Financial support for this work from the Academia Sinica, the National Science Council (grant no. NSC 99-2113-M-001-002-MY3) and the National Health Research Institutes (grant no. NHRI-EX1019803EI) of Taiwan, R.O.C, is gratefully acknowledged.
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Macromolecular ion accelerator mass spectrometer† Cite this: Analyst, 2013, 138, 7384
Yun-Fei Hsu,a Jung-Lee Lin,a Ming-Lee Chu,b Yi-Sheng Wanga and Chung-Hsuan Chen*a We present a newly developed macromolecular ion accelerator mass spectrometer that combines a dualion-trap device and a macromolecular ion accelerator (MIA) to achieve the capability of analyzing samples with a mixture of large biomolecules. MIA greatly increases detection efficiency. The dual ion trap includes a quadrupole ion trap (QIT) and a linear ion trap (LIT) in tandem. The dual ion trap is mounted ahead of the MIA. The QIT is used to store multiple species, and the LIT is employed to capture the ions that are sequentially ejected out of the QIT. Subsequent to their capture, the ions inside of the LIT are extracted and transferred to the MIA. The synchronization between the QIT and MIA is bridged by the LIT. A
Received 13th August 2013 Accepted 2nd October 2013
sample containing a mixture of several large biomolecules was employed to examine the performance of this new type of mass spectrometer. The result reveals that larger biomolecules show a comparable signal to smaller biomolecules, even though the mixture contains equal quantities of each type of
DOI: 10.1039/c3an01534c
protein. The overall assembly produces a nearly constant detection efficiency over a broad mass range.
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Thus, this device provides an alternative platform to analyze complex large-protein mixtures.
Introduction Progress in proteomics analysis using mass spectrometry has mostly occurred through continuous improvements in instrumentation. During the past couple of decades, combinations of different types of mass analyzers have produced advantages over each individual device.1,2 In addition, ion traps and ion trap-based instruments have been broadly employed as analytical platforms. To satisfy the growing demand for characterization with high throughput, a great many techniques have been applied for the manipulation of ion traps. The strategic control of trapping and ejection of ions inside of the ion traps enables a range of novel analytical methods. For example, various ejection methods have been developed to analyze a wide mass range of biomolecules.3–5 The robustness, fast-scan-speed, and MS/MS sensitivity of ion traps have made them popular over the past decade. Ion traps can be classied into two types: the 3D quadrupole ion trap (QIT) or the 2D linear (or rectilinear) ion trap. The 3D ion trap (or Paul trap) mass spectrometer is simple and convenient to use6–8 but has some disadvantages such as a space charge effect,9,10 low trapping capacity,11 and low resolution. Combining a linear quadrupole ion guide12–15 with a 3D ion trap can help overcome some of these limitations.16–18
a
The Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan. E-mail: [email protected]; Fax: +011886-2-27899923; Tel: +011-886-2-27871200
b
Institute of Physics, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan † Electronic supplementary 10.1039/c3an01534c
information
7384 | Analyst, 2013, 138, 7384–7391
(ESI)
available.
See
DOI:
The major application of ion guides has been to interface with the electrospray (ESI)19 source. Furthermore, the use of quadrupole or octupole guides20 rather than traps removes the ability to concentrate the ions of interest. In addition, only small molecular ions have been tested; the detection of large biomolecules has not been demonstrated due to the low detection efficiency. 2D ion traps (typically called linear ion traps) are known for having a better trapping capacity and a lower space charge effect than 3D ion traps.21,22 Moreover, their high degree of exibility allows them to integrate with other mass analyzers and has made them an indispensable element in the evolution of hybrid instruments.23–26 In general, hybrid designs that incorporate the same types of ion traps are built to conduct MS/MS and/or MSn (n $ 3) analyses.27–29 Double or multiple ion traps have also been reported to execute different analyses.30–32 Among these reports, the transfer efficiency poses the biggest challenge and limits the further evolution of these assemblies. Moreover, novel analytical techniques are required to serve as complementary strategies for proteomics research. Matrix-assisted laser desorption/ionization (MALDI) has been broadly used for the ionization of large molecules. MALDI is operated with a time-of-ight (TOF) spectrometer to detect large proteins. Electron amplication devices, such as electromultipliers, channeltrons and microchannel plates (MCPs), are widely used as detectors by producing secondary electrons. The efficiencies of these electron amplication devices strongly depend on the velocity of the ions. The detection efficiency approaches zero when the velocity of an ion is signicantly lower than 104 m s1. For large biomolecular ions, the detection efficiencies are very low due to the low velocity with the same kinetic energies. A cryogenic detector has been used for the
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detection of large biomolecules. However, the response time is slow for cryogenic detectors. The macromolecular ion accelerator (MIA)36 offers a novel method by directly enhancing the ion energy to lead to high detection efficiency. Recently, we successfully demonstrated the combination of a 3D ion trap (i.e., QIT) with a 2D linear ion trap (LIT) for biomolecule samples with matrix-assisted laser desorption/ ionization (MALDI).33 For molecular weight calculation of a step frequency scan of the QIT, it follows an equation that was derived from the Mathieu equation: m¼
8eV : qz ðr0 þ 2Z0 2 ÞU2 2
The parameters represent molecular weight m, charge number e, radio frequency potential V applied to the ring of QIT, ring electrode of radius r0, and electrode space between two end-cap 2z0, trapping parameter qz (value of 0.908 under unstable ejection conditions) and angular frequency U which is equal to 2pf, where f is the radio frequency in Hertz. In this step frequency scan case, the amplitude of radio frequency was constant but only the frequency changed. Therefore, m/z is related to the radio frequency. For frequency scanning, the smaller the step for the frequency scan, the higher the resolution. In addition, the throughput can possibly be improved if the rst quadrupole ion trap is replaced by a linear ion trap. For samples with a mixtures of biomolecules, MALDI analysis shows a distinct advantage over ESI due to the low number of peaks.34 In this work, a dual-ion-trap is employed as an ion source and a mass analyzer for biomolecular ions that are subsequently accelerated35 to produce high detection efficiency. Because our dual ion traps exhibit good transmission efficiency and the macromolecular ion accelerator (MIA) gives an effective improvement of the detection efficiency,36 we incorporated the accelerator into the dual ion traps to achieve the mass analysis of biomolecule mixtures. A large portion of ions with a broad time-spread exited the QIT and was mostly captured by the LIT. Hence, the integration of a dual-ion trap with the accelerator is an advance that can lead to a new type of mass spectrometer: the macromolecular ion accelerator mass spectrometer (MIA). In this study, the details of combining the QIT, LIT and accelerator are examined and results are reported.
secretory IgA (sIgA) (20 mM) were mixed together and cocrystallized with the matrix solution. Every analyte was prepared at a volume of 1 mL and mixed with 9 mL of the matrix solution before being placed upon the probe tip. This sample was then allowed to air-dry before being introduced into the chamber. Instrumentation setup Fig. 1 is the schematic of the dual ion trap-accelerator mass spectrometer. A QIT–LIT combination is employed. In the test of this dual-trap device, ions were stored in the QIT device and then they were ejected out of the QIT. The LIT was used to capture the ejected ions. To obtain ions of the analyte, a pulsed laser for MALDI was phase-locked relative to the RF frequency of the QIT. The phase was adjusted to optimize the trapping of ions, and these ions were subsequently cooled by collision with helium in the QIT. The frequency step scan was designed to sequentially execute trapping, laser ring, frequency step scan and data sampling. Typically, the desorbed ions were rst accumulated in the QIT by exposing them to several shots of the laser. Once the accumulation was accomplished, the step scan was triggered to eject selected ions from the QIT; subsequently, these ions were stored in the LIT. It was found that the transfer efficiency from the QIT to the LIT was 60%. More details on the dual-ion trap will be published separately.33 In this work, helium was introduced into the QIT and LIT as a buffer gas in the pressure ranges of 30–50 mtorr and 70–80 mtorr, respectively. The rst acceleration plate was positioned at 54 mm away from the exit endcap of the LIT. This spacing was adopted to provide a compartment between the dual-ion trap and the accelerator. Between the remainder of the plates, an equal spacing of 25 mm was employed. Only 8 out of the 12 plates are shown in this gure. Aer the 12 plates, a channeltron detector (CEM 5900 M, Photonics) was mounted and oated at 1850 V to allow ion detection. Electronics A step scan was performed in mass-selective axial instability mode for the QIT. A TTL signal was started at the same time that the step scan was initiated. The TTL signal was used to trigger and time the pulses on the z-electrodes of the LIT as well as the subsequent acceleration. Typical wiring of the step scan, pulse
Experimental details Sample preparation Sinapinic acid (SA) was prepared at a concentration of 100 nmol mL1 in 60 : 40 acetonitrile/0.1% TFA. Peroxidase, BSA, IgG, brinogen and sIgA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). These chemicals were prepared at a concentration of 20 pmol mL1. To create a single compound sample, 1 mL of a protein sample, which included 20 pmol of biomolecules and 4 mL of matrix solution (400 nmol), was deposited onto a stainless steel probe and was allowed to air-dry. To create a sample containing a mixture of compounds, equal amounts of bovine serum albumin (BSA), IgG and
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Fig. 1 Schematic of the dual-ion-trap accelerator mass spectrometer. The black arrow indicates the direction along which ions travel and are directed towards the channeltron. The cutaway view shows only 8 of the 12 acceleration plates. The 1st acceleration plate is positioned at 54 mm after the exit endcap of the LIT. A channeltron is positioned at the end of the accelerator.
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Analyst and waveform on both endcaps are displayed in Fig. 2. To synchronize the moment of extraction out of the LIT with the 1st stage of acceleration, a synchronization trigger was generated at the same time extraction begun. This synchronization trigger was used to initiate the whole acceleration sequence aer an appropriate delay. Aer acceleration, ions were directed towards the channeltron for detection. The step scan offers the opportunity for each type of compound in the sample to be ejected separately. Molecular ions of different masses can be ejected one by one during the frequency step scan in the QIT. Ions with different masses are captured by the LIT one aer the other and are then sequentially accelerated. As a result, a mass spectrum can be readily obtained containing distinct signals that correspond to different species. Data acquisition As the frequency step scan was initiated by a home-made control program to eject the ions, a TTL signal was generated simultaneously to trigger the pulses at both endcaps of the LIT and the acceleration. The acceleration event was synchronized to the extraction of the LIT by wiring between an arbitrary waveform generator (AWG) (WW2572, Tabor Electronics Ltd. Tel Hana Israel) and a broadband power amplier (5/80, TREK). The pulse(s) of acceleration can be timed by synchronization of the output trigger of the AWG. To optimize the signals, we employed empirical ne-tuning of the pulses throughout the entire work. The signals were averaged by accumulating 60–80 laser shots, and the spectra were displayed using a LeCroy Waverunner 64Xi oscilloscope (LeCroy Corp., Chestnut Ridge, NY). A spectrum showing intensity versus time was acquired and
Fig. 2 The electronics of the dual-ion-trap accelerator mass spectrometer. To time the acceleration pulses, a synchronization trigger is generated on the basis of the timing of the extraction at Z1. Therefore, the acceleration is determined by the timing of the extraction. Definite timing is established by the applied voltage. Upon successful demonstration of the acceleration, acceleration pulses were applied at the 1st, 3rd, 5th, . 11th plates while keeping the 2nd, 4th, 6th, . 12th plates grounded for simplicity. Each pulse is exerted at 20 kV; therefore, the maximum ion energy is 120 kV. The signal is generated at the oscilloscope as the ions impact upon the channeltron.
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Paper transferred through a general-purpose interface bus (GPIB) to a personal computer (PC) for further processing. Either oscilloscope screenshots or recorded traces in the American Standard Code for Information Interchange (ASCII) format were taken using the ScopeExplorer soware (LeCroy). During step scanning to eject various masses, the traces recorded from the oscilloscope indicated the intensity vs. ejection frequency and, hence, a mass spectrum was obtained.
Trajectory simulation A simulation was conducted to study the integration between the dual ion traps and the accelerator. Fig. 3 shows a typical result of the simulation using IgG ions. A side view shows the interior of the LIT and the plates for acceleration (Fig. 3A). It is noteworthy that the length of the rods in the LIT is equal to the spacing between adjacent plates of the accelerator. This design is employed for the purpose of not only assuring a sufficient trapping capacity of the LIT37 but also restricting the ions within the distance of 25 mm. This spatial restriction in the LIT is intended to allow the ions to fully exist in the gap between adjacent plates. If the length of the rods exceeds 25 mm, the ions ejected out of the LIT cannot be completely included within the spacing between two plates.
Fig. 3 Simulation of the dual-ion-trap accelerator mass spectrometer. (a) For simplicity, the ions were initiated inside the LIT. Ions were extracted by placing 1000 V upon the entrance endcap of the LIT. Subsequent to the extraction, acceleration was applied to the ions as the blue trajectories display. (b) The potential energy view of the accelerated ions. The bidirectional arrows indicate the 1st and 2nd plates. Before ions reach the 1st acceleration plate, they have the same kinetic energy (the black trajectories). The energies begin to vary after they experience the 1st stage of acceleration.
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Paper For simplicity, only 4 plates were assumed in this simulation, and the conguration was identical to that in Fig. 1. To account for the practical situation, the initial position of the ions was set-up across the axial direction within the LIT while the RF potential was maintained at 930 Vpp. The original kinetic energy of the ions, in principle, was determined by the competition between collisional cooling with the buffer gas (70 mtorr) and RF heating from the trapping eld. The initial kinetic energy of the ions was close to a thermal distribution at room temperature based on the evidence of a successful capture. The extraction of ions from inside of the LIT was achieved by applying a pulse voltage of 1000 V upon the Z1 endcap. Subsequent to the extraction, the 1st stage of acceleration was applied to the ions as shown in the blue trajectories. The potential energy view in Fig. 3b depicts the underlying details of the movements of the ions and especially those occurring between the 1st and 2nd plates. The pulsing electric eld in particular is removed from this gure to increase the ease of observation of the trajectories. While being extracted out of the LIT, the ions' motion is designated by the black trajectories, which display a uniform energy status. Moreover, the trajectories show the inuence of the RF potential upon the motion of the ions. The inuence is reected by the oscillation of their trajectories, which are caused by RF interference through the hole at the center of the endcap. The 1st acceleration pulse was switched on just as all of the ions arrived at the 1st plate. Apparently, the spatial distribution of the ions can be conned between the 1st and 2nd plates as expected. When the 1st pulse was applied, the ions were accelerated by 20 kV as shown by the 9 vertical lines orthogonal to the potential energy surface. However, the energy distributions were broadened as
Analyst designated by the trajectories with different colors. Each energy status, on average, differs from another by 9 eV; therefore, the largest difference of energy among these ions is 70 eV aer they have undergone the 1st stage of acceleration. Although this value is only 0.35% of one pulse voltage, this calculation along with the spatial distribution evidently demonstrates the necessity of empirically adjusting the pulses rather than following pre-programmed calculations. Consequently, the subsequent experimental results employing acceleration from more than one pulse were conducted by stepwise ne-tuning of the pulse timings.
Results and discussion The results of the dual-ion-trap accelerator device Fig. 4 presents a typical result acquired from the integration of the dual trap and the accelerator. Fig. 4a shows the results using peroxidase38 ions with one or two stages of acceleration, which are designated by the black and red traces, respectively. A dramatic increase of signal intensity is noticeable and is highly reproducible. This result was conrmed by at least 10 replicate measurements. Fig. 4b depicts the results acquired under onestage acceleration (i.e., 20 kV). In this gure, the onset of acceleration was begun at different times relative to the initiation of extraction in the LIT. The 1st acceleration plate is positioned at 72 mm past the exit endcap of the LIT in this case. From the bottom to the top, time delays of 450 ms, 550 ms, 600 ms, 650 ms and 700 ms post-extraction were applied accordingly. No signal was obtained at a delay of 450 ms. Signals were observed for delay times from 550 to 700 ms. For a delay of 750 ms or more, signals were again not observed. These results
Fig. 4 Results of integrating the dual ion traps with the accelerator. Peroxidase (molecular weight 43 kDa) was selected to examine the integration of the dual-trap and the accelerator. (a) The black and red traces represent the results of applying one and two stages of acceleration, respectively. A significant enhancement of the signal intensity is observed for the cumulative acceleration. (b) All results were obtained under one stage of acceleration (i.e., 20 kV), but the acceleration was started at different times. From the bottom to the top, acceleration was started at time lags of 450 ms, 550 ms, 600 ms, 650 ms and 700 ms subsequent to extraction. It is found that the acceleration can be applied over a spread time of 150 ms. This result further implies that ions within the LIT possess a spatial spread of 25 mm (i.e., the length of the rods) and a temporal spray of 150 ms.
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Analyst imply that ions within the LIT possess a longitudinally spatial distribution of 25 mm, which is the maximal length of the rods. Furthermore, a temporal distribution of 150 ms is observed. These results lead to an empirical choice of timing to optimize the acceleration. Fig. 5 displays the optimized result for IgG ions. Fig. 5a shows the result when IgG ions experience a step scan from 30 kHz to 18 kHz before being captured inside of the LIT for 1 ms. They are then extracted and further accelerated by a voltage of 60 kV. Fig. 5b displays the relative timings of the pulses among the endcaps (Z1 and Z2) and the accelerator. Herein, the pulse on Z2 shows an output amplitude of 100 V with a pulse duration of 1 ms (the black trace). The waveform on the Z1 endcap exhibits a gating of 110 ms subsequent to the Z2 pulse, which allows the entrance of ejected ions from the QIT (as the red trace and the green arrow indicate). The acceleration pulses are enlarged in Fig. 5c. In this gure, the relative timing between these pulses and the Z1 waveform is clearly shown. The acceleration was initiated 432.3 ms aer the extraction, which was a value determined by empirical ne-tuning. The initiation of the 1st acceleration pulse was regarded as t0 for the purposes
Paper of evaluating the acceleration efficiency; this will be discussed further in the following section. A series of successful results revealed that the integration between a dual trap and an accelerator can work quite well. The signals are much better than those in which only a single ion trap is integrated into the accelerator (data not shown). The LIT truly builds a bridge between the QIT and the accelerator. Review of the effects of acceleration In the MIA, the efficiency of sequential acceleration was thoroughly investigated and will be demonstrated and discussed briey. Fig. 6 depicts the results for IgG ions undergoing acceleration at different energies. Fig. 6A displays signals from IgG ions that were captured for 1 ms and then accelerated by 40, 60, or 80 kV. The start of the 1st pulse was referred to as t0 (with an electronics jitter39 of 10 ns) and, thus, the evolution of signals at different accelerations is readily observed. This time jitter is not taken into account throughout this work because it exerts almost no inuence on the acceleration efficiency. The movement of the ions occurs on a time scale of microseconds, and this level is at least 1000-fold slower than the jitter. The improvement of the signals is reected in the fact that each series of peaks has higher signal intensities than the one before it. This fact implies that an ever-boosted ion velocity is obtained by ever-increasing the number of steps of acceleration. In addition to the peak shape, the temporal evolution displays good agreement with the applied energy. Fig. 6b displays signals from IgG ions that were captured for 2 ms and then accelerated by 20, 40, or 60 kV. A two-fold longer capture time creates no attenuation of the signals. This result shows that the signals remain strong throughout a long capture time. The integration between the capture and the acceleration is not affected by an elongated capture time. However, it was experimentally conrmed that the capture time cannot be shorter than 1 ms. Otherwise, ions cannot efficiently thermalize, and this leads to the unsuccessful acceleration or metastable decay of ions. In this case, the signals cannot be obtained. Moreover, it is noteworthy that IgG ions cannot be detected by applying only 20 kV of acceleration voltage. This result must be compared with the results shown in Fig. 4, in which peroxidase ions could be detected at 20 kV. This difference is attributed to the fact that the velocity of IgG ions is nearly one-half of that of peroxidase ions at 20 kV. Thus, more energy input is needed to increase the kinetic energy of IgG ions. Measurements of brinogen ions
Fig. 5 Typical results of the integration between the dual-ion trap and the accelerator by examining IgG molecular ions. (a) The step scan was adopted by frequency hopping from 30 to 18 kHz on the ring electrode of the QIT. This signal was acquired by trapping the ions inside the LIT for 1 ms, extracting and then accelerating. (b) Three overlapping traces display the relationship among the Z1 and Z2 endcaps and the pulses for acceleration. The black, red, and blue traces represent the pulse at Z2, the waveform at Z1 and multiple acceleration pulses, respectively. (c) The enlarged view of the traces of Z1 and of the acceleration pulses. The blue trace contains 3 pulses of acceleration, which are initiated at 432.3 ms subsequent to the start of extraction. Here, the initiation of the 1st acceleration pulse is defined as t0 (with a jitter of 10 ns) to examine the acceleration efficiency upon the IgG ions. Results are shown in Fig. 6.
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Fig. 7 reveals the results for brinogen ions. Frequency stepping was conducting from 14 kHz to 9 kHz in the QIT; the ions were captured by the LIT with an RF at 80 kHz; and the ion motion was synchronized with a total acceleration voltage of 120 kV. The capture time for brinogen in these tests was 3 ms. Although the signals also exhibited no attenuation at longer times, it was again found that the shortest capture time cannot be less than 1 ms. In general, synchronization between the dual ion traps and the accelerator has been successfully validated on the basis of
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Fig. 6 Results of IgG ions undergoing acceleration with different energies. The noise prior to 50 ms results from the pick-up of the high voltage pulses in the accelerator. (a) IgG ions were captured for 1 ms and then accelerated by 2, 3, or 4 pulses (the black, red and blue traces, respectively). The evolution of the signals is in good agreement with the acceleration energy. The initiation of the 1st acceleration pulse was defined as t0 (with a jitter of 10 ns), and the efficiency of the acceleration is thus clearly characterized. (b) IgG ions were captured for 2 ms and then accelerated by 1, 2, or 3 pulses (the olive, black and red traces, respectively). Unlike peroxidase ions (the results in Fig. 4), IgG ions cannot be detected by applying only 20 kV (olive color). The kinetic energy is not sufficient to cause the IgG ions to efficiently impinge upon the channeltron detector because the detector was only floated at 1600 V in this experiment. A dramatic improvement of the signal was observed when 40 or 60 keV of acceleration energy was supplied.
Fig. 8 A typical spectrum obtained by the dual-ion-trap accelerator mass spectrometer. The step scan was divided into 20 steps from 35 kHz to 8 kHz to sequentially eject BSA+, IgG+ and IgA+. The acceleration energies upon each species were 80, 120 and 120 keV, respectively.
Fig. 7 Typical results acquired via the dual-ion-trap accelerator mass spectrometer. (a) The signal recorded from the scope while using fibrinogen ions was obtained by step scanning from 14 kHz to 9 kHz in the QIT, capturing the ions in the LIT with the RF at 80 kHz and synchronizing the total acceleration voltage of 120 kV. The RF voltages applied upon the QIT and LIT were 760 Vpp and 930 Vpp, respectively. (b) A typical waveform applied to the Z1 endcap. (c) The pulses were synchronized to the waveform with a proper delay for efficient acceleration. The inset represents an enlarged picture of the pulses.
the evidence shown above. In the following section, an attempt is made to utilize the combination device to conduct an analysis of samples containing multiple compounds.
Mixture analysis A typical demonstration that employs the dual-ion-trap accelerator mass spectrometer is shown in Fig. 8. A step scan was adopted by dividing the range from 35 to 8 kHz into 20 steps. BSA+, IgG+ and IgA+ ions were gradually ejected one aer another. It is noteworthy that larger biomolecules can be measured with a signal-to-noise ratio comparable to that of
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smaller ones even though the sample contains equal quantities of each type of molecule. This result neatly demonstrates an appealing feature; our mass spectrometer exhibits a constant detection efficiency throughout a broad mass range. The reason for this can be attributed to the integration of our MIA. The pulse duration of the acceleration for high-mass ions shows a better compatibility with the instrument electronics, especially the switches. When accelerating high-mass ions, the duration can be sufficiently long to prevent the need for an extremely fast rise/fall of the switch. This performance causes the switches to work optimally and results in a more efficient acceleration. Therefore, Fig. 8 shows an example of good detection efficiency that would not be observed with conventional mass spectrometers; conventional models always display signicantly diminished signals for the high-mass ions with respect to low-mass ions using a channeltron or microchannel plate as the detector. Moreover, the almost total lack of noise demonstrates the lack of interference upon these signals from the analytes. The reason can be attributed to the high mass selectivity that the accelerator possesses. In the dual ion trap, the results were directly recorded aer the extraction of ions from the LIT. Although the analysis of a mixture was demonstrated, low detection efficiency of large biomolecules is still a concern. With the integration of the accelerator, the pulses are preset on the basis of a certain mass value; thus, temporal and spatial connement is
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Analyst established over the course of acceleration. As a result, noise can be discriminated from the signals. The resolutions of the BSA+, IgG+ and IgA+ peaks are estimated at 3.7, 8.3, and 7, respectively. Comparable resolutions between the IgG+ and IgA+ peaks lead us to consider attempting an analysis of biomolecular ions with even higher molecular weights in the future. This appears to be feasible because there is no dramatic decrease in resolution. However, the resolutions, in general, are not high because of the presence of buffer gas in the course of ejection. More sophisticated designs are needed to improve the resolution. Because the ejected ions from the MALDI were stored in the QIT, most metastable ions produced in the MALDI-TOF device are expected to be quenched. In this work, few metastable ions were observed. Therefore, this device can further simplify mass spectra by eliminating metastable ions.
Conclusion The rst design of a dual-ion-trap accelerator mass spectrometer has been accomplished. This device exhibits not only high detection efficiency but also the capability for mixture analysis. The high detection efficiency is achieved by the accelerator, and the storage and selectivity of various ions are accomplished by the dual-ion-trap device. Moreover, the enrichment of the selected ions is one of the special advantages of this device. The overall combination in our instrument results in a desk-top accelerator with the capability of a mass spectrometer.
Acknowledgements We thank the former President of Academia Sinica, Yuan T. Lee, for his valuable discussions and suggestions. Financial support for this work from the Academia Sinica, the National Science Council (grant no. NSC 99-2113-M-001-002-MY3) and the National Health Research Institutes (grant no. NHRI-EX1019803EI) of Taiwan, R.O.C, is gratefully acknowledged.
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