Simultaneous Mass Analysis of Positive and Negative Ions Using a

Positive and negative ions produced from matrix-assisted laser desorption/ionization (MALDI) were simultaneously measured using a newly developed ...
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Anal. Chem. 2006, 78, 7729-7734

Simultaneous Mass Analysis of Positive and Negative Ions Using a Dual-Polarity Time-of-Flight Mass Spectrometer Shang-Ting Tsai, Chiu Wen Chen, Ling Chu Lora Huang, Min-Chia Huang, Chung-Hsuan Chen, and Yi-Sheng Wang*

Genomics Research Center, Academia Sinica, 128, Academia Road, Section 2, Nankang District, Taipei 115, Taiwan, R. O. C.

Positive and negative ions produced from matrix-assisted laser desorption/ionization (MALDI) were simultaneously measured using a newly developed dual-polarity time-offlight mass spectrometer. This instrument is effective not only for express and comprehensive mass analysis but also for studying the ionization mechanisms of biomolecules. It comprises two identical time-of-flight mass analyzers located symmetrically about a MALDI ion source. The ion optics are arranged to be able to extract positive and negative ions synchronously with equal efficiency to each corresponding mass analyzer. Mass spectra of various proteins with molecular weights as large as that of myoglobin monomer and dimer were obtained. The spectral patterns obtained in this work are approximately mirror images with opposite polarities. Since the invention of the matrix-assisted laser desorption/ ionization (MALDI) method in the late 1980s,1 mass spectrometry (MS) has advanced in the study of biological problems.2-5 Peptide mass fingerprinting is one of the most well-established approaches of MS-based proteomics, which is a database search algorithm based on the results of MALDI interfaced time-of-flight (TOF) mass spectrometry.6-10 The combination of MALDI and TOF has the greatest speed and sensitivity among all mass spectrometers; it has therefore spurred constant development in the field of biological technology, such as chip-based surface-enhanced laser desorption/ionization11-14 and imaging mass spectrometry.15-17 * To whom correspondence should be addressed. E-mail: wer@ gate.sinica.edu.tw. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Aebersold, R.; Goodlett, D. Chem. Rev. 2001, 101, 269-295. (3) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (4) Patterson, S.; Aebersold, R. Nat. Genet. 2003, 33, 311-323. (5) Tyers, M.; Mann, M. Nature 2003, 422, 193-197. (6) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 5011-5015. (7) James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Biochem. Biophys. Res. Commun. 1993, 195, 58-64. (8) Mann, M.; Hojrup, P.; Roepstorff, P. Biol. Mass Spectrom. 1993, 22, 338345. (9) Pappin, D.; Hojrup, P.; Bleasby, A. Curr. Biol. 1993, 3, 327-332. (10) Yates, J.; Specicher, S.; Griffin, P.; Hunkapiller, T. Anal. Biochem. 1993, 214, 397-408. (11) Shiwa, M.; Nishimura, Y.; Wakatabe, R.; Fukawa, A.; Arikuni, H.; Ota, H.; Kato, Y.; Yamori, T. Biochem. Biophys. Res. Commun. 2003, 309, 18-25. (12) Rawel, H.; Rohn, S.; Kroll, J.; Schweigert, F. Mol. Nutr. Food Res. 2005, 49, 1104-1111. 10.1021/ac061213v CCC: $33.50 Published on Web 10/17/2006

© 2006 American Chemical Society

Although having superior features, the development of MALDITOF MS for biological analysis is restricted in many respects. The puzzle of the ionization mechanism is probably one of the major barriers to the broad use of MALDI, which impedes the development of mass spectrometric technology for polysaccharides, deoxyribonucleic acids, lipids, and organic polymers. Additionally, the difficulty of switching the instrument rapidly between positive and negative ion modes limits the usefulness of MALDI-TOF MS because the analytes might exist in either polarity. Therefore, MALDI-TOF experiments on highly complex samples require a considerable period of parameter trials. In fact, cations and anions are generated simultaneously in the MALDI source, the population of which depends on their gasphase basicity, proton affinity, temperature, and other factors.18 A general picture of MALDI is as follows: the biological analytes undergo protonation and deprotonation reactions via gas-phase proton-transfer reactions when they collide with highly excited matrix molecules. These reactions are currently believed to be initiated by rapid laser heating and evaporation. Consequently, the electric field of the ion optics determines the polarity of ions to be extracted toward the mass analyzer in most conventional mass spectrometers. For instance, the desorbed cations are detected if the sample plate is positively biased in relation to the extraction. In contrast, the same field will pull the anions back to the sample surface and finally cause them to vanish. Accordingly, most mass spectrometers detect only half of the generated ions in the source region and sacrifice the substantial information associated with the other half. The losses in temporal and compositional information are critical in the analysis of infinitesimal, complex, and instantaneously existing samples (from HPLC, for example). Examples of such analyses include the diagnosis of disease in physiological fluids, the examination of air pollution in ambient aerosols, and biohazardous dust. (13) Carrette, O.; Demalte, I.; Scherl, A.; Yalkinoglu, O.; Corthals, G.; Burkhard, P.; Hochstrasser, D.; Sanchez, J. Proteomics 2003, 3, 1486-1494. (14) Jain, K. Curr. Opin. Mol. Ther. 2002, 4, 203-209. (15) Reyzer, M.; Caprioli, R. J. Proteome. Res. 2005, 4, 1138-1142. (16) Maddalo, G.; Petrucci, F.; Iezzi, M.; Pannellini, T.; Del Boccio, P.; Ciavardelli, D.; Biroccio, A.; Forli, F.; Di Ilio, C.; Ballone, E.; Urbani, A.; Federici, G. Clin. Chim. Acta 2005, 357, 210-218. (17) Altelaar, A.; van Minnen, J.; Jimenez, C.; Heeren, R.; Piersma, S. Anal. Chem. 2005, 77, 735-741. (18) Karas, M.; Gluckmann, M.; Schafer, J. J. Mass Spectrom. 2000, 35, 1-12.

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Figure 1. Schematic diagram of the dual-polarity time-of-flight mass spectrometer. It consists of a duplex MALDI ion source and two TOF mass analyzers located symmetrically about it. The inset depicts the steric view of the duplex source and the laboratory frame axes.

Electrospray ionization (ESI) source interfaced ion trap instruments have been optimized to switch alternatively between cationic and anionic modes in a few milliseconds. They are suitable for the analysis of both proton-accepting and proton-donating analytes coeluted from liquid chromatography (LC) systems.19,20 Such rapid polarity switching is feasible for ESI ion traps because the electronic devices all operate in the range of a few thousand volts. The switching speed suffices to match the running speeds of most commercially available LC systems. Indeed, considerable effort has been made by instrument manufacturers to increase further the acquisition speed of LC-MS platforms.21,22 However, the development of similar functionality in MALDI-TOF instruments has not been reported. In 1996, Hinz et al. developed the first dual time-of-flight mass spectrometer for the analysis of airborne particles.23 It consists of both cation and anion analyzers facing opposite directions about a beam generator. In 1997, Prather and co-workers announced a portable aerosol time-of-flight mass spectrometer (ATOFMS) with extended sizing function and mass range.24 It can select an aerosol particle of specific size and examine its chemical composition by photonization in real time. The critical requirement for getting both the cations and the anions successfully into the mass analyzers is that the aerosol particle must be kept electrically neutral as it enters the TOF acceleration region; this innovative design, however, is not applicable to the biological ions produced from conventional ion sources, such as ESI and MALDI. In 2005, Russell et al. used a collison nebulizer to generate bioaerosols from the nozzle of an ATOFMS,25 demonstrating a so-called bioaerosol mass spectrometer for the analysis of biological samples. The bioaerosol consists of a biomolecular core with a matrix coating around a bulk sphere as it enters the TOF region. Photoexcitation causes the biomolecules to undergo protonation or deprotonation by way of interactions with matrix molecules, a behavior similar to that occurring in the conventional MALDI (19) Yin, O.; Lam, S.; Chow, M. Rapid Commun. Mass Spectrom. 2005, 19, 767774. (20) Tolstikov, V.; Fiehn, O. Anal. Biochem. 2002, 301, 298-307. (21) O’Connor, D.; Mortishire-Smith, R. Anal. Bioanal. Chem. 2006, 385, 114121. (22) Wu, Y.; Engen, J.; Hobbins, W. J. Am. Soc. Mass Spectrom. 2006, 17, 163167. (23) Hinz, K.; Kaufmann, R.; Spengler, B. Aerosol Sci. Technol. 1996, 24, 233242. (24) Gard, E.; Mayer, J.; Morrical, B.; Dienes, T.; Fergenson, D.; Prather, K. Anal. Chem. 1997, 69, 4083-4091. (25) Russell, S.; Czerwieniec, G.; Lebrilla, C.; Steele, P.; Riot, V.; Coffee, K.; Frank, M.; Gard, E. Anal. Chem. 2005, 77, 4734-4741.

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reaction. However, the practical mass working range of such aerosol mass spectrometers still remains under 20 000 amu.26 Thus, the development of an alternative mass spectrometer is highly desirable for the routine mass analysis of biological samples of both polarities This work develops an innovative dual-polarity time-of-flight mass spectrometer (DTOFMS) with a conventional MALDI ion source. As in the ATOFMS, detecting cations and anions simultaneously is made possible by using two independent TOF analyzers that share one duplex MALDI ion source, but the configuration of the source has been carefully designed. It comprises a supporting electrode in the center of the TOF acceleration region to accommodate the MALDI target, which is kept at the electric potential of the midpoint of the entire mass spectrometer. All ion optics and electric potentials of the two polarities are applied symmetrically about the target. Since it simultaneously monitors all ions generated from the complex reactions, the mechanism can be determined efficiently from the comprehensive mass information obtained from all experimental events. DTOFMS enables more detailed investigation of biological problems than could be made by MALDI-TOF in the past, including the ionization mechanism, quantitative analysis, and the fragmentation reaction. EXPERIMENTAL SECTION The entire apparatus were made in-house, except for the microchannel plate (MCP) assemblies. Figure 1 schematic depicts the DTOF mass spectrometer, which comprises five main components; (1) the duplex MALDI ion source, (2) the cation flight tube, (3) the cation MCP detector, (4) the anion flight tube, and (5) the anion MCP detector. The duplex MALDI source comprises one source electrode and two sets of binary extraction plates located symmetrically about it. The five electrodes are aligned parallel to each other to form two acceleration stages for each polarity. After they have been extracted from the source region, cations and anions are mass-separated and detected in the flight tubes and by the chaveron-type MCP detectors (C-701/25, Jordan TOF Products, Inc.), respectively. This setup is an analogue of the combination of two Wiley and McLaren time-of-flight mass spectrometers.27 The duplex MALDI ion source is enclosed in a custom-made, 8-in., six-way cube chamber. The source chamber is pumped using (26) van Wuijckhuijse, A.; Stowers, M.; Kleefsman, W.; van Baar, B.; Kientz, C.; Marijnissen, J. J. Aerosol Sci. 2005, 36, 677-687. (27) Wiley: W.; Mclaren, I. Rev. Sci. Instrum. 1955, 26, 1150-1157.

Figure 2. Five electrodes of the duplex MALDI ion source. The sample electrode consists of a target surface on a side of the rectangular slot and a clearance in front of the target surface for laser examination.

a 360 L/s turbomolecular pump (Turbovac 360, Leybold GmbH) to 5 × 10-7 mbar. The center of the MALDI target surface is defined as the origin of the laboratory frame. The normal of the target surface and the propagation directions of cations and anions are along the z, the +x, and the -x axis, respectively, as presented in the steric view of Figure 1. Figure 2 presents the details of components of the duplex MALDI ion source, which consists of five 40 × 100 mm parallel stainless steel electric plates that are equally spaced at 6 mm from each other. The sample electrode is the one at the center of the source with a thickness of 6 mm. The MALDI target surface is along the longer side of a 26 × 3 mm rectangular open slot, which is 18 mm away from the front of the sample electrode. The MALDI target is delivered from the back of the sample electrode and is propagated along a tunnel on the z axis until it reaches the rectangular open slot. Another rectangular slot opens a clearance on top of the target surface, which allows the laser beam to examine the target surface perpendicularly. This configuration enables the cations and anions to be extracted toward the +x and -x directions after generation, respectively. On both the +x and -x sides of the sample electrode, the binary extraction plates are all 3 mm thick and are arranged symmetrically about the sample electrode. On every plate, a circular clearance with a diameter of 5 mm allows ions to propagate into the next stage. Notably, the clearances of the first and second plates are centered 1.5 and 2.5 mm, respectively, away from the x axis in the +z direction. All of the electrodes are fixed on an acryl base block to isolate them electrically from the others. The structure and the electric potentials were optimized by computer simulation (Simion 3D ver 7.0, Bechtel BWXT Idaho, LLC). The two flight tubes both have an internal diameter of 32 mm, a length of 1123 mm, and are located 4.5 mm behind the last extraction plates. Except for the first 150-mm section, which adheres to the source chamber, the tubes are enclosed in two differentially pumped regions which are coevacuated using a 360 L/s turbomolecular pump (Turbovac 360, Leybold GmbH). The pressure in this region is always kept below 3 × 10-7 mbar during measurement. Both tubes are offset 2.5 mm from the x axis and in the +z direction. The MCP detectors are ∼25 mm away from the flight tubes without further differential pumping stages. The configurations of the electrical optics for the two polarities are identical, and the electric potentials are applied symmetrically about that of the sample electrode, as was shown in Figure 1. No

Figure 3. Diagram of the dc decoupling circuit. The box on the left represents a protection component.

delayed ion extraction was used herein. In this measurement, +5.9 kV was applied as the reference potential of the sample electrode because of the limitation on the electric potential applied to the MCPs. The potentials of the adjacent plates in the +x and -x directions are respectively +2.5 and +9.3 kV, which produce a strong field gradient to extract positive ions toward the +x and negative ions toward the -x directions. Along the cation and anion beams, the potentials of the flight tubes are respectively 0 and +11.8 kV, whereas those of the second extraction plates are respectively +3.8 and +8 kV. Notably, the potentials of the second extraction plates are set higher than the first to produce the ionfocusing effect, markedly enhancing the transmission efficiency. Although the configurations of the two polarities are identical and both are installed symmetrically about the source electrode, a significant difference exists between the circuits of the two MCP detectors. On the cation side (or +x side) the MCP is driven by a conventional circuit, such that the entrance, the exit, and the anode of the MCP are floated at -2200, -200, and 0 V, respectively (a chevron-type MCP). However, the entrance on the anion side MCP must be kept at +14 kV to yield the same ion impact energy as that on the cation side, making the final potentials of the exit and the anode +16 and +16.2 kV, respectively. The large bias voltages on the MCP for detecting anions must be applied with caution because they may cause voltage breakdown across the nearby low-voltage electrodes and the vacuum chamber. The MCP assembly was used as is, but isolated at 67 mm from the vacuum chamber using an 8-in. acryl flange adaptor to simplify the design and to prevent MCP and electronic devices from arcing. The frame of the detector assembly was biased by applying +14 kV to reduce the voltage differences around the electrodes, preventing the anion detector from highvoltage breakdown during the operation. The grids in front of the MCP detectors minimize the field penetration into the flight tubes. Another important refinement is the readout circuit for the anion MCP. Since the acquisition electronics only accept signals of a few volts, the +16.2-kV biased signal from the output of anion MCP must be isolated carefully. Figure 3 presents the capacitor dc decoupling circuit that is used to read out the anion signal. It is wired based on the standard circuit, except we replace the two capacitors with those of a higher voltage rating (2 nF, 40 kV, N4700, Murata Manufacturing Co., Ltd.) and modify the overall capacitances. Additionally, the circuit is enclosed inside a glass housing, which is electrically isolated from the ambient environment. Most of the conducting wires at the high-voltage side of the capacitors are silicone-jacked with a maximal voltage rating of 100 kV. Notably, they are not shielded with a grounding jacket, to prevent the circuit from unexpectedly shorting. Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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After it passes through the dc decoupling circuit, the anion MCP signal is terminated by an external 50-Ω resistance before it is fed into a digital storage oscilloscope (Waverunner 6050A, 500 MHz, LeCroy Inc.). The cation MCP signal is directly terminated by another external 50-Ω resistance and fed into the oscilloscope. The oscilloscope is triggered by the output signal of a photodiode (818-BB-21, Newport Co.) that senses the scattering light of the laser output. The spectra of the cations and anions are obtained simultaneously using two independent channels with a time resolution of 20 ns. A pulsed frequency-triplet Nd:YAG laser (355 nm, LS-2134UTF, Lotis TII Ltd.) is utilized to initiate the MALDI process. It is triggered internally and is Q-switched at 3 Hz. Two polarizers are placed in front of the laser output to attenuate the laser power to 2-10 µJ, depending on the sample to be examined. The laser beam is focused using a fused-silica, plan-convex lens with a focal length of 250 mm before it passes through a fused-silica vacuum window. It irradiates the MALDI target surface perpendicularly with a final spot size of roughly 100 µm in diameter, which corresponds to an irradiance of roughly 106-107 W/cm2. The biological samples used herein include insulin chain B (MW ) 3495.9), equine skeletal muscle myoglobin (MW ) 16951.5), and calibration protein mixture, which includes angiotensin I (MW ) 1296.7), adrenocorticotropic hormone (ACTH) clip 1-17 (MW ) 2093.1), ACTH clip 18-39 (MW ) 2465.2), ACTH clip 7-38 (MW ) 3657.9), and insulin (MW ) 5730.6). Since the structure of the analyte and matrix cocrystal markedly influences the spectral quality, 2,4,6-trihydroxyacetophenone (THAP; MW ) 186.2) and R-cyano-4-hydroxycinnajmic acid (CHCA; MW ) 189.2) were chosen as the matrixes because they form uniform crystals. The samples were all purchased from Sigma-Aldrich Co., except the calibration protein mixture, which was from Applied Biosystems, and they were all used without further purification. The samples were dissolved in deionized water to a concentration of 50 pmol/µL, and the matrix was dissolved in 50% aqueous acetonitrile to a concentration of 0.1 M. To prepare the MALDI target, the biological sample and matrix were premixed in equal volumes and deposited onto the target probe for vacuum-drying. The analyte-to-matrix molar ratio was always kept at 1:2000 to evaluate the effect of molecular weight on overall efficiency. RESULTS AND DISCUSSION Ion Optics Simulation. Figure 4 presents a sectional view of the simulated electrodes, the potential contours, and the ion trajectories in the region of the duplex MALDI source. The initial kinetic energy of the ions in the +z direction was defined by the empirical results in the literature.28-30 Since no additional focusing device is installed in the field-free drift tubes, the configuration of the duplex source exhibits many unique features for optimizing the transmission efficiency, reducing the beam divergence, and extending the mass working range. According to the predictions for the current configuration, the appropriate initial ionic energy is below 5 eV; for ions of higher initial energy, such as heavy biomolecules, a higher extraction potential is recommended. (28) Fernandez-Lima, F.; Collado, V.; Ponciano, C.; Farenzena, L.; Pedrero, E.; da Silveira, E. Braz. J. Phys. 2005, 35, 170-174. (29) Gluckmann, M.; Karas, M. J. Mass Spectrom. 1999, 34, 467-477. (30) Karas, M.; Bahr, U.; Fournier, I.; Gluckmann, M.; Pfenninger, A. Int. J. Mass Spectrom. 2003, 226, 239-248.

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Figure 4. Simulated electric contour map and the ion trajectories in the duplex MALDI ion source. The inset shows the potential at the sample electrode generated by the adjacent electrodes, in which the positive and the negative ions propagate along the potential gradient as shown by the black curves.

The first unique characteristics of the duplex source is that it deflects the ions desorbed from the target surface by 90°, in the electric field produced by the adjacent extraction plates. The design is similar to that of a binary orthogonal TOF extraction source except that the instrument is operated under dc mode. The second feature is the rectangular open-slot envelope that surrounds the target surface. A rectangular slot is superior to either circular or wide-open structures because the resulting field gradient is less distorted along the y axis and is better shaped. For example, deflecting the ion efficiently from the z to the x direction requires a good potential surface with little distortion in the y direction. According to the simulation herein, the elongated clearances in the y direction from the target center cause no field change along the y axis; in contrast, the ions are extracted diversely if they are present in a three-dimensional parabolic potential gradient that is produced by a circular hole. However, if a fully open space is used instead of a slot in front of the target surface, then the field gradient does not suffice for successful ion extraction. Third, the center of the circular clearances on the extraction plates are offset from the x axis in the +z direction by 1.5 and 2.5 mm, respectively, as displayed in Figure 4. Checking the ion trajectory closely reveals that the ions initially travel straight along the z direction and then bend toward the x axis. They will escape the sample plate near the forward sides of the open slot and will be overturned by the steep potential at its edge, making them fly across the first extraction region that is tilted ∼10° from the x axis. The offset of the circular clearances on the extraction plates can countersteer the ions sequentially back to the x direction with sufficient transmission efficiency. Notably, the potential change of the second extraction plates causes an ion-focusing effect in this region and can roughly double the transmission efficiency. Experimental Observations and Statistical Analysis. The test measurements are made on proteins and mixture of proteins

Figure 5. Observed spectra of (a) insulin B chain and (b) myoglobin. The cation and anion spectra are shown respectively at the top and the bottom of the m/z axes.

Figure 7. Cation and anion spectra of protein calibration mixture, which includes angiotensin I, ACTH clip 1-17, ACTH clip 18-39, ACTH clip 7-38, and insulin. Figure 6. Change of ion intensities of cationic and anionic insulin B chain versus the radial positions of the laser on the target surface. The origin of the plot represents the origin of the laboratory frame.

of various molecular weights. The cation and anion spectra are presented at the top and the bottom of the m/z axis, and the signal polarity of the cation side is reversed for ease of comparison. Possible cross contamination of spectral features from the opposite polarity is negligible as confirmed by measurements of alkali metal halides of distinct patterns. Parts a and b in Figure 5 present the cation and anion spectra of the insulin B chain and the myoglobin, both of which were prepared in a final amount of 50 pmol on the target, but with THAP and CHCA as the matrixes, respectively. The spectra are the average of 200 laser events for the insulin B chain and 1000 for myoglobin. Figure 5a presents unambiguously the singly charged insulin B chain monomer and dimer in both cation and anion modes, but the doubly charged monomer can only be found in the cation mode. On the other hand, Figure 5b shows the features of singly changed myoglobin monomers and dimers, as well as the doubly charged monomers in both cation and anion modes. However, the weak triply charged monomer presents only in cation mode. Although the spectral features of cationic and anionic monomers and dimers are similar, the cation signal is slightly stronger than that of the anions in both cases, and the ion intensities of

multiply charged cations exceed those of the corresponding anions. To examine whether the differences result from errors in manufacture or assembly, the polarities of all electric potentials were reversed and the measurements repeated; this reversal causes the cations to fly in the -x direction and the anions to fly in the +x direction. Therefore, any significant variation in spectral pattern from the original setting indicates the presence of conformational defects. Fortunately, the spectral patterns of both polarities were the same; thus, any defect in the configuration is assumed to be negligible in our instrument. Hence, differences between spectral features may reveal differences between the production yields of ions. Notably, the reversed voltage setting is unfavorable because a highly negative voltage is prone to cause breakdown during operation, especially when very many charged particles are generated by the laser. However, the absolute ion signal varied considerably with the position irradiated by the laser. As displayed in Figure 6, both cation and anion spectra were systematically analyzed in relation to the laser irradiation position using the singly charged insulin B chain to elucidate their correlation. In the figure, the origin of the laser position is the center of the target surface (and the origin of the instrument). The spectra were obtained as the laser was scanned from -1 to +1 mm along the x axis and the intensity profiles were both bell-shaped, except for a fluctuation at the Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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Figure 8. Statistical analysis of the spectral pattern obtained from calibration mixture. (a) Analyte-to-insulin intensity ratios of the calibration mixture; (b) cation-to-anion intensity ratio of every analyte.

scanning edge near the attraction end. Interestingly, this result agrees reasonably with the ion trajectories predicted by computer simulation: for ions with an initial kinetic energy of 1 eV, the applicable laser irradiation position for cations ranges from -0.9 to +0.8 mm, and the intensity profiles drop steeply after +0.8 mm. The anions behave similarly but symmetrically opposite to the cations. Fitting curves to the two observed intensity profiles, except for the two data points of the cation at the +x edge and the three data points of the anion at the -x edge, yields two Gaussian curves. According to the fitting, the cation signal begins to rise at around -0.9 mm along the Gaussian profile and peaks at +0.09 mm. The intensity profile of the anion is just opposite to that of the cation, except in that the observed steep drop at the traveling edge is replaced by an oscillating and slow decline. The narrow working window is associated with mainly the change in the ion trajectories with the change in the potential field along the target surface. For example, when the ions are generated far from the mass analyzer (where cations are generated at -x side), then they are affected by the repulsive field and strike the sample plate before escaping from the sample plate region. However, when they are generated near their corresponding mass analyzer (such that the cations are generated at the +x side), the ions would be accelerated by the field before they propagate into an optimal field region and fail to escape from the edge of the slot. In the present study, the laser is aligned to the center of the target surface to enable subsequent measurement to obtain dualpolarity mass spectra with the greatest possible reliability. Measurements of a protein calibration mixture are made and the intensity ratios are statistically analyzed to evaluate the stability and the reproducibility of the DTOFMS. Figure 7 presents the spectra of the protein mixture of 20 pmol of angiotensin I, 20 pmol of ACTH clip 1-17, 15 pmol of ACTH clip 18-39, 30 pmol of ACTH clip 7-38, and 35 pmol of insulin, with THAP as the matrix. All of the proteins can be identified unambiguously in both polarities, although the relative intensities of proteins differ slightly. Panels a and b in Figure 8 display the analyte-to-insulin ratios of each polarity and the cation-to-anion ratios (IC/IA) of all analytes, respectively. Insulin is utilized as the reference compound in Figure 8a because it yielded the most reproducible results of all analytes. The figure shows no apparent correlation between the intensity and the neutral abundance, which implies that the analytes have different ionization efficiencies. Notably, the intensity variation is 10-33% in the cation mode and 14-23% in the anion 7734

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mode, suggesting that the reproducibility in the anion mode is superior to that in the cation mode. However, a trend is evident in the plot of the cation-to-anion ratio, which increases as the molecular weight decreases. As displayed in Figure 8b, the ratio remains at 1-1.4 for analytes that are larger than 2400 amu, but increases almost exponentially to 2.3 and 6.4 for ACTH clip 1-17 and angiotensin I, respectively. Statistical analysis demonstrates that the error in most of the measurements is between 7 and 18% except for that of angiotensin I, which is 28%. Hence, the spectral reproducibility of the DTOF MS remains better than 70%. CONCLUSION The simultaneous mass analysis of MALDI-generated cations and anions was successfully conducted using a combination of the duplex MALDI ion source and two time-of-flight mass analyzers. According to results obtained by computer simulations, MALDI-generated cations and anions can be extracted perpendicularly into the analyzer using a well-designed potential gradient. Since the potential field critically determines the ion trajectories, the extraction and transmission efficiencies of the ions depend strongly on the position of the laser interaction. The results of systematic analysis demonstrate that the observed cation and anion intensities are comparable and reliably determined when the laser is interrogating the center of the target. Observations of the cationic and anionic analytes reveal similar spectral features, except that the cation mode is more intense and exhibits more features of multiply charged proteins. The overall reproducibility/stability of the instrument exceeds ∼70%, based on a statistical analysis of the cation-to-anion ratios of all proteins. This work presented an innovative mass spectrometer for comprehensively and rapidly elucidating compositions of substances. The unique feature supports bioanalysis and thorough studies of the ionization reactions involved in MALDI. Associated topics will be discussed in subsequent reports. ACKNOWLEDGMENT The authors thank Dr. Chau-Chung Han and Dr. Jim Jr-Min Lin for their technical support and Dr. Yuan-Tseh Lee for his guidance. This work is supported by the Genomics Research Center, Academia Sinica of Taiwan, Republic of China.

Received for review July 5, 2006. Accepted August 15, 2006. AC061213V