Anal. Chem. 2002, 74, 6224-6229
Pressure-Assisted Capillary Electrophoresis Electrospray Ionization Mass Spectrometry for Analysis of Multivalent Anions Tomoyoshi Soga,*,† Yuki Ueno,† Hisako Naraoka,† Keiko Matsuda,‡ Masaru Tomita,† and Takaaki Nishioka†,‡
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan, and Graduate School of Agricultural Sciences, Kyoto University, Kyoto 606-8502, Japan
We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamideadenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n ) 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 µmol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells. Recently, capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE/ESI-MS) has become a powerful analytical tool for the analysis of charged species. In this marriage of techniques, CE confers rapid analysis and efficient resolution, and MS provides excellent selectivity and sensitivity. The electrospray ionization (ESI) mode has been utilized for CE/ MS because it is sensitive, versatile, and relatively easy to use in * To whom correspondence should be addressed. Phone: (+81) 235 29 0528. Fax: (+81) 235 29 0530. E-mail:
[email protected]. † Keio University. ‡ Kyoto University.
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combination with CE. A number of CE/ESI-MS methods have been reported for various species ranging from small molecules, such as inorganic anions,1 carboxylic acids,2 phenolic compounds,3 amino acids,4 metal species,1,5 tetramines,6 herbicides,7 and drugs and drug metabolites8 to peptides and proteins.9,10 These CE/ESIMS methods proved to be rapid, selective, high-resolution and high-throughput. Ion chromatography or ion-exchange chromatography has been widely used for ion analysis. However, their selectivity and resolution efficiency are often poor. Since appropriate volatile mobile phases are not generally available, coupling these chromatographic techniques to MS is rather difficult. Considering feasibility and resolution obtained, CE/ESI-MS may become a dominant method for the simultaneous analysis of charged species. However, there has been one major difficulty to achieving anion analysis by CE/ESI-MS. Analysis of anions by CE is usually performed in negative mode, in which the inlet of the capillary is at the cathode and the outlet, at the anode. Since CE/ESI-MS does not possess the outlet vial, EOF11 movement toward the cathode (opposite the MS direction) creates a gap in the liquid at the capillary exit, resulting in a current drop.12 For this reason, few papers on anion analysis have been reported by CE/ESI-MS using this configuration. Johnson et al.2 applied an EOF reversal technique for carboxylic acid analysis by CE/ESI-MS, which reverses EOF toward (1) Corr, J. J.; Anacleto, J. F. Anal. Chem. 1996, 68, 2155-2163. (2) Johnson, S. K.; Houk, L. L.; Johnson, D. C.; Houk, R. S. Anal. Chim. Acta 1999, 389, 1-8. (3) Lafont, F.; Aramendia, M. A.; Garcı´a, I.; Borau, V.; Jime´nez, C.; Marinas, J. M.; Urbano, F. J. Rapid Commun. Mass Spectrom. 1999, 13, 562-567. (4) Soga, T.; Heiger, D. N. Anal. Chem. 2000, 72, 1236-1241. (5) Schramal, O.; Michalke, B.; Kettrup, A. J. Chromatogr. A 1998, 819, 231242. (6) Zhao, J.; Thibault, P.; Tazawa, T.; Quilliam, M. A. J. Chromatogr., A 1997, 781, 555-564. (7) Song, X.; Budde, W. L. J. Am. Soc. Mass Spectrom.1996, 7, 981-986. (8) Lu, W.; Poon, G. K.; Carmichael, P. L.; Cole, R. B. Anal. Chem. 1996, 68, 668-674. (9) Cao, P.; Moini, M. J. Am. Soc. Mass Spectrom. 1998, 9, 1081-1088. (10) Kelly, J. F.; Locke, S. J.; Ramaley, L.; Thibault, P. J. Chromatogr., A 1996, 720, 409-427. (11) Li, S. F. C. Capillary ElectrophoresissPrinciples, Practice and Applications; J. Chromatogr. Library Series; Elsevier: Amsterdam, 1992; Vol. 52, Chapter 1. (12) Soga, T.; Ueno, Y.; Naraoka, H.; Ohashi, Y.; Tomita, M.; Nishioka, T. Anal. Chem. 2000, 74, 2233-2239. 10.1021/ac0202684 CCC: $22.00
© 2002 American Chemical Society Published on Web 11/13/2002
the anode by the addition of a cationic surfactant, such as cetyltrimethylammonium bromide, to the buffer.13 However, the current drop was invariably observed within a few minutes after the voltage was applied.12 This is because cationic surfactants on the capillary wall also migrate toward the inlet vial (cathode) so that silanol groups (SiO-) are revealed, and normal EOF is generated toward the cathode. Therefore, permanent EOF reversal was deemed necessary, independent of buffer conditions, in CE/ ESI-MS with negative mode. Permanently reversing EOF was accomplished using a capillary coated with a strong anion exchanger1 or a cationic polymer (Polybrene).12 These capillaries were able to constantly reverse EOF toward the anode without any additives to the buffer and enabled successive anion analysis by CE/ESI-MS without a deleterious current drop. These methods have demonstrated outstanding performance for many kinds of anions; however, considerable broad tailing of several anions, such as SeO32-, ClO4-, IO3-, and citrate were observed.1,12 Moreover, coenzyme A (CoA) compounds were not detected as well-shaped peaks.12 In this study, we propose a new method to enable polyvalent anion analysis by CE/ESI-MS in which anions are electrophoretically separated with the addition of air pressure to produce constant liquid flow toward the anode, subsequently detected by MS. The method was optimized and applied to the analysis of intracellular tricarboxylic acids, nucleotides, dinucleotides, and CoA compounds. EXPERIMENTAL SECTION Chemicals. ADP, GDP, CTP, CDP, CMP, and succinyl CoA were purchased from Sigma (St. Louis, MO); piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) and 3-(N-morpholino)propane sulfonate (MOPS), from Dojindo (Kumamoto, Japan). All other reagents were obtained from Wako (Osaka, Japan). Individual stock solutions of citrate and isocitrate at a concentration of 10 mM and 100 mM were prepared in Milli-Q water. The working mixture standard was prepared by diluting these stock solutions with Milli-Q water just before injection. The chemicals used were of analytical or reagent grade. Water was purified with a Milli-Q purification system (Millipore, Bedford, MA). Instrumentation. All CE/ESI-MS experiments were performed using an Agilent CE Capillary Electrophoresis System equipped with an air pressure pump, an Agilent 1100 series MSD mass spectrometer, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE/MS adapter kit, and a G1607A Agilent CE/ESI-MS sprayer kit (all Agilent Technologies, Waldbronn, Germany). All system control, data acquisition, and MSD data evaluation were performed via a G2201AA Agilent ChemStation software for CE/MSD. The CE/MS adapter kit includes a capillary cassette that facilitates thermostating of the capillary, and the CE/ ESI-MS sprayer kit, which simplifies coupling the CE system with MS systems, was equipped with an electrospray source. The sprayer has an orthogonal flow design to reduce the detrimental effects caused by the charged particles or droplets, as described by Voyksner and Lee.14 Bacterial Strains and Growth Conditions. Bacillus subtilis 168 strain was used in this work and cultured at 37 °C in S6 (13) Tsuda, T. J. High Resolut. Chromatogr. Chromatogr. Commun. 1987, 10, 622-624. (14) Voyksner, R. D.; Lee, H. Anal. Chem. 1999, 71, 1441-1447.
medium (5 mM KH2PO4, 10 mM NH4SO4, 100 mM MOPS, 0.1 mM tryptophan, 1 mM MgCl2‚6H2O, 0.7 mM CaCl2‚2H2O, 50 µM MnCl2‚4H2O, 1 µM ZnCl2, and 5 µM FeCl3‚6H2O) containing 25 mM glucose (S6-glucose).15 Growth was monitored by measuring optical density at 600 nm (OD600), and the cells were grown to OD600 0.85. Sample Preparation. Intracellular metabolites in B. subtilis 168 cells were extracted as follows: Cells were grown in S6glucose medium, and aliquots of 10 mL were withdrawn at OD600 0.85. The media were passed through a 0.45-µm pore-size filter. The cells were washed with 10 mL of Milli-Q water to prevent contamination of the S6 medium and plunged into 1 mL of methanol containing 10 µL of 0.56 mM PIPES (internal standard), where enzymes were rapidly deactivated. After incubation for 5 min at room temperature, 1 mL of chloroform and 390 µL of Milli-Q water were added to the solution, and the mixture was thoroughly mixed. The separated 1 mL of water layer was removed and centrifugally filtered through a Millipore 5-kDa-cutoff filter to remove proteins. The filtrate was lyophilized and dissolved in 20 µL of Milli-Q water prior to CE analysis. PACE/ESI-MS Conditions. Separations were carried out on a commercially available GC capillary, poly(dimethylsiloxane) (DB-1), which was purchased from Yokogawa Analytical Systems (Tokyo, Japan). The capillary dimension were 50 µm i.d. × 100 cm total length. The electrolyte for the CE separation was 50 mM ammonium acetate solution, pH 7.5. Prior to its first use, a new capillary was pretreated with the run electrolyte for 20 min. Before each injection, the capillary was equilibrated for 5 min by flushing with the run electrolyte. The sample was injected with a pressure injection of 50 mbar for 30 s (∼30 nL). The applied voltage was set at -30 kV, and a pressure of 50 mbar was added to the inlet capillary during the run. The capillary temperature was thermostated to 30 °C, and the sample tray was cooled below 5 °C. The Agilent 1100 series pump equipped with a 1:100 splitter was used to deliver 10 µL/min of 5 mM ammonium acetate in 50% (v/v) methanol-water to the CE interface, where it was used as a sheath liquid around the outside of the CE capillary to provide a stable electrical connection between the tip of the capillary and the grounded electrospray needle. ESI-MS was conducted in the negative ion mode, and the capillary voltage was set at 3500 V. A flow rate of heated dry nitrogen gas (heater temperature 300 °C) was maintained at 10 L/min. The spectrometer was scanned from m/z 100 to 1000 at 0.7 s/scan during the separation and detection. In selective ion monitoring mode, deprotonated [M - H]- ions were monitored for metabolites with 20 ms of sampling time for each. RESULTS AND DISCUSSION PACE/ESI-MS. The CE/ESI-MS methods using positively charged capillaries demonstrated impressive performance for anion analysis.2,12 However, considerable decrease in sensitivity for several anions, such as nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) was observed during consecutive analysis by the method. Furthermore, CoA compounds were not determined.12 Since these compounds exhibit characteristics of multivalent anions, it was assumed that they were adsorbed on the positively charged capillary wall by ionexchange interactions. (15) Fujita, Y.; Freese, E. J. Biol. Chem. 1979, 254, 5340-5349.
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Figure 1. Schematic representation of the PACE/ESI-MS system.
To avoid ion-exchange interactions between the anionic species and the capillary surface, a noncharged polymer-coated capillary was used for the CE/ESI-MS. However, in this system, current drop often occurred. It can be seen that residual silanols exist even in the coated capillary so that a gap in the liquid was grown at the capillary end by EOF generation toward the cathode (opposite the MS direction). To overcome the problem, constant liquid flow toward the MS direction was driven by air pressure to the inlet capillary during electrophoresis, as shown in Figure 1. A pressure mobilization technique has been widely used in capillary electric focusing electrophoresis,16,17 but few in other CE modes. This pressureassisted capillary electrophoresis electrospray ionization mass spectrometry (PACE/ESI-MS) could prevent a gap in the liquid generating, which enabled successive anion analysis. Optimization of Analytical Conditions. As previously reported,18 GC capillaries, such as poly(ethylene glycol) (DB-WAX) and poly(dimethylsiloxane) (DB-1) were useful for CE. These capillaries exhibited no or minimal interaction with anions, enabling determination of anions with excellent reproducibility, good linearity, and long-term stability. Thus, the effect of these capillaries on PACE/ESI-MS system was investigated. Applying a constant pressure of 50 mbar, 15 nucleotides and acetyl CoA were analyzed using 50 mM ammonium acetate electrolyte. For reasons that are unclear, migration times of all peaks were gradually reduced by DB-WAX, but reproducible migration times were obtained with DB-1. Therefore, the DB-1 capillary was selected for all subsequent experiments. Since CE separation is affected by electrolyte pH,18-20 the influence of pH was studied over the pH range 7.5-9.0 using 50 mM ammonium acetate buffer. At a higher pH value, the capillary tended to be damaged, whereas little or no difference in migration (16) Hjerten, S.; Zhu, M. D. J. Chromatogr. 1985, 346, 265-270. (17) Kilar, F.; Hjerten, S. Electrophoresis 1989, 10, 23-29. (18) Soga, T.; Inoue, Y.; Ross, G. A. J. Chromatogr., A 1995, 718, 421-428. (19) Wu, C. H.; Lo, Y. S.; Lee, Y.-H.; Lin, T.-I. J. Chromatogr., A 1995, 716, 291-301. (20) Soga, T.; Ross, G. A. J. Chromatogr., A 1997, 767, 223-230.
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time, separation, and peak shape was observed in these pH regions. Hence, 50 mM ammonium acetate at pH 7.5 was chosen as the optimum electrolyte. The effect of varying the applied pressure was studied at 20, 25, 30, and 50 mbar. While changing the pressure had little effect on resolution, increasing the pressure could reduce analysis time, so that 50 mbar was used for applying pressure in this study. The effect of the capillary temperature was also investigated at 15, 20, 25, and 30 °C. Resolution was almost the same, but better sensitivity was obtained at 30 °C. The choice of the sheath liquid parameters is very important in developing a method employing CE/ESI-MS. In this method, 5 mM ammonium acetate in 50% (v/v) methanol-water at a flow rate of 10 µL/min was employed, because it exhibited excellent sensitivity and long-term stability, as previously described.4 Mass spectra of anions of interest were measured in negative ion mode scanning from m/z 100 to 1000. The deprotonated molecular ion, [M - H]-, dominated the mass spectrum for each anion; consequently, the anions could be selectively determined by their deprotonated molecular weights. The PACE/ESI-MS method was applied to the analysis of a standard mixture, and 18 standards, including citrate isomers, nucleotides, nicotinamide-, and flavin-adenine dinucleotides, CoA compounds, and PIPES (internal standard) could be selectively determined by their deprotonated molecular weights. Although migration times of several peaks were closed, every component could be selectively determined at its deprotonated weight. Method Validation. The reproducibility, linearity, and sensitivity of the method were investigated, and the results are listed in Table 1. The RSD values (n ) 6) were better than 0.6% for migration times and between 1.4 and 6.2% for peak areas, and little or no decrease in performance was observed over 100 consecutive runs. The calibration curves for all species were between 0.991 and 0.999 at 10, 20, 50, 100, 200 ,and 500 µmol/L with correlation coefficients. The concentration detection limits for all components were between 0.5 and 3.7 µmol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3,
Figure 2. Peak height reproducibility of AMP by (A) CE/ESI-MS with SMILE(+) and (B) PACE/ESI-MS with DB-1 capillary. Experimental conditions: (A) capillary, SMILE(+) 50 µm i.d. × 100 cm; electrolyte, 50 mM ammonium acetate, pH 7.5; applied potential, -30 kV; applied pressure, 0 mbar; injection, 30 s at 50 mbar; temperature, 20 °C; sheath liquid, 10 µL/min of 5 mM ammonium acetate in 50% (v/v) methanolwater; (B) capillary, DB-1 50 µm i.d. × 100 cm; applied pressure, 50 mbar; temperature, 30 °C; other conditions are the same as (A).
Table 1. Reproducibility, Linearity, and Sensitivity RSD (n ) 6, %)
detection limit
compd
migration time
peak area
linearity correlation
concn (µmol/L)
mass (fmol)
CMP AMP GMP CDP ADP GDP CTP ATP GTP NAD NADP FAD acetyl CoA
0.5 0.5 0.5 0.5 0.6 0.5 0.6 0.6 0.6 0.5 0.5 0.5 0.4
1.4 1.5 1.7 2.1 1.8 3.0 3.0 3.0 5.2 2.9 3.3 5.1 6.2
0.999 0.998 0.999 0.995 0.992 0.994 0.996 0.998 0.991 0.998 0.999 0.999 0.994
1.4 0.9 3.7 0.4 0.4 0.5 1.0 0.8 1.6 0.7 0.7 1.0 2.1
42 26 110 12 13 15 30 24 48 21 21 30 63
which resulted in the mass detection limits ranging from 12 to 110 fmol. To evaluate the developed PACE/ESI-MS, it was compared to the conventional CE/ESI-MS method12 using a SMILE(+),21 Polybrene-coated, capillary in analyzing nucleotides and dinucleotides. The results of AMP determination show that peak height was gradually reduced in a consecutive 10 run by the CE/ESIMS method (Figure 2A). This phenomenon was observed for every nucleotide. It was also found that migration times gradually increased, whereas peak areas were almost constant. It was assumed that nucleotides adsorbed onto the positively charged capillary by ion-exchange interactions, effectively masking the positive charge on the capillary surface and reducing the EOF. This caused an increase in migration times, broadened peak shapes, and resulting decreased peak heights. This assumption was confirmed by the following experiments. When analyzing mono- or divalent organic acids by the method, this phenomenon was not observed, and good reproducibilities of both migration times and peak heights were obtained. These organic acids are mono- or divalent and were not strongly retained on the capillary surface as well as those behaviors on anionexchange chromatography.22 Figure 2B illustrates the peak height reproducibility (n ) 10) of AMP obtained by the PACE/ESI-MS method. The results of (21) Katayama, H.; Ishihama, Y.; Asakawa, N. Anal. Chem. 1998, 70, 52725277.
other nucleotides were almost similar. Peak heights were nearly constant, as were migration times (RSD ) 0.7%, n ) 10). Importantly, sensitivity of the method was several times higher, as compared to those of the CE/ESI-MS method. These results suggest that since the PACE/ESI-MS system utilizes a nonionic polymer-coated capillary, adsorption of nucleotides was prevented. As compared with the normal CE/ESI-MS method, broadening of peaks was expected in PACE/ESI-MS because of the parabolic flow profile in the pressure-driven system.11 Theoretical plates of nucleotides obtained by both the PACE/ESI-MS and CE/ESI-MS methods were compared. Theoretical plates by PACE/ESI-MS were between 21 400 and 41 500, whereas those by CE/ESI-MS were 2- to 3-fold as high. Even though separation efficiencies of anions by the PACE/ESI-MS were inferior to those by the previously described CE/ESI-MS approach, this did not affect the analysis due to the selective nature of the MS detector. Other biologically important multivalent anions were investigated by PACE/ESI-MS. Citrate and CoA compounds, which exhibited broad tailing and poor or split peaks by the CE/ESIMS system,12 behaved well using the PACE/ESI-MS method. Analysis of Citrates, Nucleotides, and CoA Compounds in B. subtilis. Recently, metabolomics, that is, the analysis of all cellular metabolites, has become a powerful new tool for gaining insight into functional biology.23-27 Measurement of the level of numerous metabolites within a cell and tracking their change under different conditions provide not only direct information on metabolic phenotypes but are also complementary to gene expression and proteomic studies. To demonstrate the utility of the developed PACE/ESI-MS method, intracellular citrate isomers, nucleotides and nucleotide related coenzymes, and CoA compounds in B. subtilis were analyzed. B. subtilis 168 cells were grown to the exponential (22) Re´ve´sz, G.; Hajo´s, P.; Csisza´r, H. J. Chromatogr., A 1996, 753, 253-260. (23) Raamsdonk, L. M.; Teusink, B.; Broadhurst, D.; Zhang, N.; Hayes, A.; Walsh, M. C.; Berden, J. A.; Brindle, K. M.; Kell, D. B.; Rowland, J. J.; Westerhoff, H. V.; Dam, K. V.; Oliver, S. G. Nat. Biotech. 2001, 19, 45-50. (24) Spinnler, H. E.; Ginies, C.; Khan, J. A.; Vulfson, E. N. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3373-3376. (25) Covert, M. W.; Schilling, C. H.; Famili, I.; Edwards, J. S.; Goryanin, I. I.; Selkov, E.; Palsson, B. O. Trends Biochem. Sci. 2001, 26, 179-186. (26) Fiehn, O.; Kopka, J.; Do ¨rmann, P.; Altmann, T.; Trethewey, R. N.; Willmitzer, L. Nat. Biotech. 2000, 18, 1157-1161. (27) Ideker, T.; Thorsson, V.; Ranish, J. A.; Christmas, R.; Buhler, J.; Eng, J. K.; Bumgarner, R.; Goodlett, D. R.; Aebersold, R.; Hood, L. Science 2001, 292, 929-934.
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Figure 3. PACE/ESI-MS selected ion electropherograms for citrates, nucleotides, and CoA compounds of B. subtilis 168 cells at OD600 0.85 grown in S6-glucose medium. Experimental conditions are the same as in Figure 2B. The numbers in the upper left corner of each trace are the abundances corresponding to the displayed peak.
growth phase, and metabolites were extracted from the cells, as described in the Experimental Section. Figure 3 shows the results of the analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds of B. subtilis 168 obtained by the PACE/ESI-MS system. Well-defined selected ion electropherograms were obtained without interference. Although some species were not observed, 14 metabolites, including isocitrate, citrate, nine nucleotides, NADP, CoA, and acetyl CoA were successfully determined. To calculate intracellular metabolite concentrations, a standard curve that relates cell number to optical density at 600 nm (OD600) was prepared, and an internal volume of 1.1 × 10-9 µL/cell was assumed.28 The obtained concentration of each anion per cell is listed in Table 2. The results of AMP, ADP, and ATP were compared with those obtained by the HPLC method,29 giving good agreement. The reproducibility of the actual sample was investigated and the %RSD values (n ) 6) of every component were better than 7.2% for quantified results. CONCLUSIONS We described a PACE/ESI-MS method using a noncharged polymer-coated capillary, in which anions were electrophoretically separated with applied air pressure to the inlet capillary and subsequently detected by MS. This method has several advan(28) Whatmore, A. N.; Reed, R. H. J. Gen. Microbiol. 1990, 136, 2521-2526. (29) Matsuno, K.; Blais, T.; Serio, A. W.; Conway, T.; Henkin, T. M.; Sonenshein, A. L. J. Bacteriol. 1999, 181, 3382-3391.
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Table 2. Metabolite Concentration per B. subtilis 168 Cell Grown in S6-Glucose Medium at OD600 0.85 compd
concn (mM)
compd
concn (mM)
isocitrate citrate CMP AMP GMP CDP ADP GDP
25.4 0.4 2.0 10.2 0.5 0.5 5.1 0.2
CTP ATP GTP NAD NADP CoA acetyl CoA
0.6 2.7 0.3 nda 2.1 1.3 1.2
a
Not detected.
tages: (1) polyvalent anions, such as citrate, nucleotides, nicotinamide coenzymes, and CoA compounds can be determined without adsorption; (2) successive analysis is performed without current drop; (3) no derivatization procedure is required; (4) sensitivity and selectivity are relatively high; and (5) the analysis time is fast. Furthermore, the present methodology provides reproducibility, good linearity, and excellent identification capability. Its utility was demonstrated by the simultaneous analysis of metabolites in B. subtilis. These results indicate that the proposed PACE/ESI-MS method can be useful for the simultaneous analysis of multivalent anions, such as citrates, nucleotides, dinucleotide,s and CoA compounds, in biological samples.
ACKNOWLEDGMENT This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project SY-2102), a grant from New Energy and Industrial Technology Development Organization (NEDO) of the Ministry of Economy, Trade and Industry of Japan (Development of a Technological Infrastructure for Industrial Bioprocesses Project), and a grant from Japan Science and Technology Corporation
(Research and Development for Applying Advanced Computational Science and Technology). We also thank Dr. David N. Heiger, Agilent Technologies, for critical reading of the manuscript. Received for review April 25, 2002. Accepted September 26, 2002. AC0202684
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