MS with a Metal

Aug 24, 2009 - which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, an...
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Anal. Chem. 2009, 81, 7766–7772

Polar Anionic Metabolome Analysis by Nano-LC/MS with a Metal Chelating Agent Khin Than Myint, Taisuke Uehara, Ken Aoshima, and Yoshiya Oda* Eisai Co., Ltd., Tsukuba, Ibaraki 300-2635, Japan, and Core Research for Evolutional Science and Technology, Japan Science and Technology, Saitama 332-0012, Japan We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/ plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MTX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5′-triphosphate (UTP), was produced by MTX-treated Hela cells. Metabolomics is the comprehensive analysis of endogenous metabolites in cells, tissues, or organisms. Conventional biological research generally targets predicted metabolites, but untargeted metabolomics may allow researchers to discover unexpected effects. Metabolome profiling can also offer information on the effects of lifestyle and dietary factors in relation to specific diseases and is useful for mode-of-action studies of drugs and drug candidates.1-3 Several analytical methods have been developed, but metabolic profiling of complex biological samples remains a * To whom correspondence should be addressed. E-mail: [email protected]. Tel: +81-29-847-7084. Fax: +81-29-847-7614. (1) Kaddurah-Daouk, R.; Kristal, B. S.; Weinshilboum, R. M. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 653–683.

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challenge due to the wide range of physiochemical properties and wide dynamic range of metabolites. Mass spectrometry (MS) plays a central role in metabolomics, because MS is superior to other technologies in terms of sensitivity, specificity, and structural information. Many clinically relevant biomarkers are of low abundance within interfering matrixes, so various combination methods have been developed, such as nuclear magnetic resonance/mass spectrometry (NMR/MS),4,5 capillary electrophoresis/mass spectrometry (CE/MS),6,7 gas chromatography/mass spectrometry (GC/MS),8,9 and liquid chromatography/mass spectrometry (LC/MS).10,11 Among them, LC/MS is a widely used tool for qualitative and quantitative applications which require relatively high sensitivity; many metabolites are applicable to LC/ MS without derivatization (in contrast to GC/MS), and LC/MS systems are relatively robust. Although improving the sensitivity and mass accuracy of LC/MS is an ongoing process in metabolome profiling, the sensitivity could be enhanced with the use of low-flow-rate nano-LC with electrospray ionization (ESI)/MS detection.Nano-LC/MShasbeenroutinelyappliedforproteomics12,13 but not for metabolomics. Our group has developed a practical nano-LC/MS procedure to measure the cationic polar metabolome in biological samples without sample derivatization.14 So far, however, a comprehensive and simultaneous nano-LC/MS analysis of anionic metabolites, including organic acids and phosphorylated and sulfated compounds, has not been reported. In this study, we have developed a practical analysis of the anionic (2) Nordstro ¨m, A.; Lewensohn, R. J. Neuroimmune Pharmacol. 2009, in press. (3) Schnackenberg, L. K.; Kaput, J.; Beger, R. D. Pers. Med. 2008, 5, 495– 504. (4) Brindle, J. T.; Antti, H.; Holmes, E.; Tranter, G. E.; Nicholson, J. K.; Bethell, H. W.; Clarke, S.; Schofield, P. M.; McKilligan, E.; Mosedale, D. E.; Grainger, D. J. Nat. Med. 2002, 8, 1439–1445. (5) Gavaghan, C. L.; Holmes, E.; Lenz, E. M.; Wilson, I. D.; Nicholson, J. K. FEBS Lett. 2000, 484, 169–174. (6) Soga, T.; Ueno, Y.; Naraoka, H.; Matsuda, K.; Tomita, M.; Nishioka, T. Anal. Chem. 2002, 74, 6224–6229. (7) Harada, K.; Ohyama, Y.; Tabushi, T.; Kobayashi, A.; Fukusaki, E. J. Biosci. Bioeng. 2008, 105, 249–260. (8) Pasikanti, K. K.; Ho, P. C.; Chan, E. C. Y. Rapid Commun. Mass Spectrom. 2008, 22, 2984–2992. (9) Fiehn, O. Trends Anal. Chem. 2008, 27, 261–269. (10) Edwards, J. L.; Edwards, R. L.; Reid, K. R.; Kennedy, R. T. J. Chromatogr., A 2007, 1172, 127–134. (11) Wilson, I. D.; Plumb, R.; Granger, J.; Major, H.; Williams, R.; Lenz, E. M. J. Chromatogr., B 2005, 817, 67–76. (12) Abian, J.; Oosterkamp, A. J.; Gelpı´, E. J. Mass Spectrom. 1999, 34, 244– 254. (13) Domon, B.; Aebersold, R. Science 2006, 312, 212–217. (14) Myint, K. T.; Aoshima, K.; Tanaka, S.; Nakamura, T.; Oda, Y. Anal. Chem. 2009, 81, 1121–1129. 10.1021/ac901269h CCC: $40.75  2009 American Chemical Society Published on Web 08/24/2009

metabolome with the use of nano-LC/MS in the presence of ethylenediaminetetraacetic acid (EDTA). EXPERIMENTAL SECTION Reagents and Standard Metabolites. Adenosine-5′-monophosphate (AMP), adenosine-5′-triphosphate (ADP), adenosine3′,5′-cyclic monophosphate (cAMP), glyceraldehyde-3-phosphate (Gly-3-P), O-phospho-L-serine, oxalacetic acid, thymidine-5′-monophosphate (TMP), uridine-5′-diphosphate (UDP), urocanic acid, and fumaric acid were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). D-Fructose 6-phosphate (F6P), uridine 5′-monophosphate (UMP), L-cysteine S-sulfate, D-glucose 3-sulfate, estrone 3-sulfate, L-serine O-sulfate, and taurolithocholic acid 3-sulfate were purchased from Sigma-Aldrich (St. Louis, MO). D-Fructose-1,6diphosphate (F-1,6-D) was from Fluka (Seelze, Germany), while 13 C-labeled lactic acid and 13C,15N-labeled AMP and ATP were purchased from Cambridge Isotope Laboratory (Andover, MA). Citric acid, creatine phosphate, cytidine 5′-monophosphate (CMP), glucose-6-phosphate(G6P), guanosine 5′-triphosphate(GTP), S-(carboxymethyl)-L-cysteine, UDP-glucuronic acid, uridine 5′-triphosphate (UTP), NADPH, N-acetylglycine, benzoic acid, and other analytical grade reagents used in the experiments were purchased from Wako Pure Chemicals Co. (Osaka, Japan). Liquid Chromatography/Mass Spectrometry. A capillary column (20 cm length, 8 µm opening) was filled with apHera NH2 gel (5 µm, Supelco, PA), as described previously.14 Briefly, 150 µm internal diameter fused silica capillaries of approximately 20 cm length were tapered with a laser capillary puller to a 0.008 mm opening, and stationary-phase particles in methanol slurry were introduced under nitrogen gas-pressure (7 MPa). LC/MS profiling was carried out on a nanoflow Agilent 1100 solvent delivery system coupled with an Applied Biosystems QSTAR Pulsar i mass spectrometer. The mobile phase A was 5 mM ammonium acetate in 80% methanol (MeOH), and mobile phase B was 50 mM ammonium carbonate in 20% acetonitrile (ACN) adjusted to pH 10.0 with 25% ammonia solution. The flow rate was 1000 nL/min. Before sample loading, the capillary column was equilibrated with solvent A for 50 min. After loading of the sample solution, the column was conditioned with mobile phase A for another 5 min before the gradient with solvent B was applied. The gradient elution profile was from 10 to 100% B for 50 min and then 100% B for 15 min. A sample volume of 1 µL was injected onto the column for each experiment. Sample Extraction. A standard compound mixture (200 ng/ 10 µL) spiked with 10 µL of labeled internal standards (IS) solution (13C-labeled lactic acid, 13C,15N-labeled AMP, and ATP; 50, 10, and 20 ng/mL, respectively) was used for recovery evaluation. Oasis WAX Extraction. After addition of 800 µL of 10 mM sodium acetate buffer (pH 5.0), the mixed solution was passed through an Oasis WAX 96-well plate cartridge (Waters Co. Tokyo) preconditioned with 1 mL of MeOH and 2 mL of deionized water. The cartridge was washed successively with 2 mL of water and 1 mL of MeOH. Anionic metabolites were eluted with 0.8 mL of alkaline solution (25% ammonia solution/MeOH (2:3)). apHera NH2 Extraction. Anionic metabolites were extracted using apHera NH2 materials in a pipet tip. Specifically, 500 µL of 100 mg/mL apHera NH2 slurry in MeOH was loaded onto a 1 mL blue pipet tip packed with a Fluoropore membrane filter (pore

size 3 µm, Millipore Yonezawa, Japan). The tip was washed with 2 mL of water and then conditioned with 2 mL of MeOH before loading the sample solution. The sample solution was adjusted to 80% MeOH. The tip was washed twice with 1 mL of 80% MeOH followed by 1 mL of 50% MeOH, and anionic metabolites were eluted with 1 mL of 10% NH4OH in 20% MeOH. Then, 4 µL of 100 µg/mL sodium-free EDTA dissolved in ammonia solution was added to the extracted sample. After evaporation, the residue was reconstituted with 20 µL of buffer A and 1 µL of the sample was injected for nano-LC-ESI/MS profiling of anionic metabolites in the negative ionization mode. In order to remove solid particles, samples were filtered through a 0.2 µm PTFE filter unit before nano-LC/MS analysis. Biological Samples. Hela cells were grown to a density of 2 × 106 cells/well in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). The cells to be used for metabolome profiling were treated with methotrexate (MTX) for 4 h at a final concentration of 100 nM, rinsed with 10 mL of ice-cold 155 mM ammonium acetate solution twice, and harvested. The cells were quenched with 5 mL of 80% MeOH and stored at -80 °C until extraction of the metabolites. Cell lysate (200 µL) was sonicated in an ice bath for 15 min and then centrifuged at 12 000 rpm for 20 min to precipitate proteins. The filtrate was used for analysis of polar metabolites. Frozen mouse brain tissues (n ) 4), stored at -80 °C, were homogenized with a mortar and pestle in 1 mL of ice-cold water. Homogenized brain tissue lysate (30 µL) was immediately treated with 300 µL of MeOH to stop enzymatic reactions, and then the tissue-solvent mixture was sonicated for 10 min. For the extraction of polar metabolites, 150 µL of chloroform and 100 µL of water were added to the tissue-solvent mixture. The samples were then centrifuged at 12 000 rpm for 20 min. The upper phase (MeOH and water) was used for analysis of polar anionic metabolites. Human plasma (50 µL) and cerebrospinal fluid (CSF) (100 µL), purchased from Precision Med Inc. (San Diego, CA), were filtered with an Ultrafree-MC 5 000 NMWL centrifuge filter unit (Millipore Corporation, Bedford, MA) after addition of an internal standard mixture. The filtrate was used for extraction of anionic metabolites. Data Analysis. LC/MS data were processed using Mass++ software, available from http://groups.google.com/group/massplusplus. LC/MS ion chromatograms (XIC) were taken every 0.1 m/z in the mass region of 70-900. For quantitation, the area under the original XIC was integrated at 50% resolution. Features were extracted using retention time alignment and matching by the Mass++ algorithm. RESULTS AND DISCUSSION Chromatography of Metabolite Standards. Our initial focus was to establish a highly sensitive profiling strategy for anionic metabolites (i.e., organic acids and phosphorylated and sulfated compounds). Comprehensive and simultaneous analysis of highly polar anionic metabolites in biological samples is very difficult in the separation from matrixes like inorganic salts due to very poor retention on HPLC columns. Thus, various attempts have been made by many authors. Nucleotides and phosphorus metabolites from cultured mammalian cells were separated by means of Analytical Chemistry, Vol. 81, No. 18, September 15, 2009

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Figure 1. Nano-LC/MS chromatograms of GTP, ATP, UTP citric acid, and EDTA on a apHera NH2 column, before EDTA flushing (a), fifth analysis after EDTA flushing (b), sixth analysis after EDTA flushing (c), and coinjection of 85 pmol of EDTA (d).

reversed-phase LC with an ion-pairing agent DMHA.15 Moritz et al. developed a reversed-phase separation method with the use of precolumn derivatization of polar compounds to nonpolar

propionylated or benzoylated analytes.16 Recently, Kennedy et al. developed a capillary LC/MS for metabolome analysis, in which just 15 nL of sample was injected into an offline column (not

(15) Cordell, R. L.; Hill, S. J.; Ortori, C. A.; Barrett, D. A. J. Chromatogr., B Anal. Technol. Biomed. Life Sci. 2008, 871, 115–124.

(16) Nordstro ¨m, A.; Tarkowski, P.; Tarkowska, D.; Dolezal, K.; Astot, C.; Sandberg, G.; Moritz, T. Anal. Chem. 2004, 76, 2869–2877.

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Table 1. Comparison of the Recoveries of Standards by Oasis WAX and apHera NH2 Extraction

lactic acid N-acetyl-L-glutamic acid NAD dCMP AICAR AMP ADP UDP ATP

Oasis WAX recovery (%) 42 69 25 112 95 118 123 107 99

apHera NH2 recovery (%) 84 108 79 110 82 110 93 127 101

connected to the pump during injection of the sample).17 In this study, we examined the chromatographic behavior of a group of polar anionic metabolites (Supplementary Table 1 in the Supporting Information) in a nano-LC/MS system. Polar anionic metabolites, including phosphates, organic acids, and sulfur metabolites, were well retained on a HILIC/WAX mixed mode column with ammonium as the paired ion in a MeOH-rich mobile phase. Recently, our group reported a method for phosphorus metabolite analysis using the same column in combination with multiple reaction monitoring (MRM).18 In that study, the gradient curve of the mobile phases was very steep, and the retention window of AMP, ADM, and ATP was within 5 min. When the gradient was made more gentle to expand the retention window, very poor peak shapes were observed. Moreover, work with this stationary phase revealed that manipulation of the mobile phase pH was critical for separation of the different types of anionic analytes. Conventional columns based on silica gels do not have a long life due to hydrolysis of the silica particles under alkaline conditions. In order to improve the stability and reproducibility of analysis, a polymeric apHera NH2 column packed with polyamine-bonded polymer gel, which can withstand a wide range of pH from 2 to 13, was selected for this study. During examination of the separation conditions, including gradient conditions and concentration of ammonium carbonate or organic solvent, highly polar compounds, such as citric acid, ATP, UTP, or GTP, showed very poor peak shapes as in Figure 1a, or sometimes disappeared from the chromatograms due to binding to the column. Although we could not detect metal adduct ions in the mass spectra, it is well-known that a Fe(III)-immobilized metal affinity chromatography (IMAC) column is used for the separation of phosphorylated compounds, such as nucleotides and phosphopeptides.19 Thus, we considered that our nano-LC system might contain metals derived from the injection needle, the electrode for ESI, the mobile phases, the nano-LC pump, and the column, including the stationary phase. Recently, Asakawa et al. reported that phosphorylated nucleotides interact with the stainless steel of HPLC hardware, but this undesirable binding was reduced with the use of ammonium carbonate buffer in the mobile phase.20 Although we were using ammonium carbonate buffer over the whole optimizing period, this irreversible binding to the (17) Ni, Q.; Reid, K. R.; Burant, C. F.; Kennedy, R. T. Anal. Chem. 2008, 80, 3539–3546. (18) Uehara, T.; Yokoi, A.; Aoshima, K; Tanaka, S.; Kadowaki, T.; Tanaka, M.; Oda, Y. Anal. Chem. 2009, 81, 3836–3842. (19) Cao, P.; Stults, J. T. J. Chromatogr., A 1999, 853, 225–35.

LC/MS system or serious peak tailing of strongly acidic metabolites seemed unavoidable. Thus, LC separation of multiply phosphorylated or multiple carboxylate-containing compounds is still problematic, and such anionic compounds generally show low recovery, poor peak shape, and inadequate separation. It has been demonstrated that the interaction of phosphorylated compounds with metal ions in LC/MS systems is suppressed at very low acid pH, but the sensitivity and separation of anionic compounds become worse. Although a low concentration of ammonium phosphate buffer21 or coinjection of phosphoric acid22 was also used to improve the peak shapes of phosphorus compounds, phosphate buffer is nonvolatile and not suitable for LC/MS analysis in general. Engelhardt and Lobert reported that they used a metal-free LC system, but nevertheless, metal accumulation was observed after elution of 40 L of mobile phase containing MeOH.23 Thus, Slingsby et al. introduced an in-line column packed with iminodiacetate chelation resin to capture metal impurities arising from the pump and mobile phases.24 A chelating reagent, EDTA, is frequently used for removing metal ions which are absorbed on the silica, tubes, and elsewhere in HPLC systems. Therefore, we flushed the column, connecting tubes and injector with 30 µL of 50 mM EDTA followed by injection of 100 µL of buffer B to remove the EDTA, and then restored the original condition by injecting 100 µL of buffer A. Flushing the LC system with EDTA before analysis yielded better peak shapes for all compounds, but this effect lasted only for four or five analyses. Even if the system is cleaned, metallic impurities are still introduced from the organic solvent and ammonium salt in the mobile phase, where they exist in the parts per billion or parts per million range.25,26 An EDTA peak was detected on LC/MS analysis after flushing the system until the fifth analysis (Figure 1b), but when it was no longer detectable (Figure 1c), strong anionic metabolites again showed poor peak shapes. Based on these results, EDTA seems to be required for every analysis to remove metallic impurities from the LC/MS system. As shown in Figure 1d, coinjection of 85 pmol of sodium-free EDTA with samples in every analysis resulted in no further peak tailing or disappearance of peaks after repeated analysis. We encountered no difficulty in coinjecting EDTA with samples during many repeated analyses. Sample Cleanup and Extraction. Sample cleanup and extraction are critical for low-abundance metabolites from complex biological matrixes. Extraction can provide a preconcentration step to improve the detection limits of the employed analytical technique, as well as removing matrix components that would interfere with the analysis. For cellular and tissue metabolomes, organic solvent extraction worked well for protein precipitation.27,28 Tiziani et al. compared different extraction protocols, including (20) Asakawa, Y.; Tokida, N.; Ozawa, C.; Ishiba, M.; Tagaya, O.; Asakawa, N. J. Chromatogr., A 2008, 1198-1199, 80-86. (21) Tuytten, R.; Lemie`re, F.; Van Dongen, W.; Esmans, E. L.; Slegers, H. Rapid Commun. Mass Spectrom. 2002, 16, 1205–1215. (22) Kim, J.; Camp, D.G., II; Smith, R. D. J. Mass Spectrom. 2004, 39, 208–215. (23) Engelhardt, H.; Lobert, T. Anal. Chem. 1999, 71, 1885–1892. (24) Slingsby, R. W.; Bordunov, A.; Grimes, M. J. Chromatogr., A 2001, 913, 159–63. (25) http://www.fishersci.com/wps/downloads/segment/Scientific/pdf/Literature/ LCMS_OptimizeMobilePhase.pdf. (26) http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/General_Information/lcms_chromasolv_flyer.Par.0001.File.tmp/lcms_chromasolv_flyer.pdf. (27) Want, E. J.; O’Maille, G.; Smith, C. A.; Brandon, T. R.; Uritboonthai, W.; Qin, C.; Trauger, S. A.; Siuzdak, G. Anal. Chem. 2006, 78, 743–752.

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Table 2. Composition of Metabolites in Various Biological Samples compounds

retention time (min)

lactic acid fumaric acid oxoproline N-acetylaspartate citric acid

18.11 22.33 16.16 22.23 36.32

creatine phosphate D-glucose-1-phosphate D-fructose-6-phosphate D-glucose-6-phosphate cytidine-5′-monophosphate uridine monophosphate adenosine-5′-monophosphate uridine-5′-diphosphate adenosine-5′-diphosphate uridine-5′-triphosphate adenosine-5′-triphosphate guanosine-5′-triphosphate NAD NADP+

21.88 25.50 26.40 27.00 25.45 28.10 26.00 39.22 38.77 48.53 47.73 53.70 17.92 31.26

human plasma (ng/µL)

human CSF (ng/µL)

Carbonylated Compounds 56.25 141.65 0.31 3.01 24.58 0.10 17.48 15.26

mouse brain (ng/mg (wet wt))

Hela cell (ng/1 × 104 cells)

137.81 27.83 5.03 281.51 36.89

69.07 15.18 25.28 34.52 20.98

Phosphorylated Compounds

0.04 0.04

0.04 0.05 0.09 0.04 0.05

0.04

0.61

1.19 1.29 4.12 4.17 4.19 83.78 53.24 0.80 26.99 6.70 10.77 1.00 0.22

0.29 0.42 0.59 7.59 0.23 2.46 8.00 34.52 173.67 21.47 14.62 1.64

perchloric acid, acetone, ACN, MeOH-chloroform extraction, and ultrafiltration to remove proteins from serum samples and found that ultrafiltration gave the best reproducibility.29 Nevertheless, there seems to be no universally applicable extraction method, and a compromise must be made between the number of distinct molecules to be extracted and the efficiency/reproducibility of extraction. Neither a simple one-step extraction nor a combination of organic solvent extraction followed by ultrafiltration was effective for analysis of very polar anionic metabolites. Inorganic salts and cationic metabolites would interfere with our separation method, so solid-phase extraction (SPE) after protein removal might be a good choice for our target anionic metabolites. However, extraction procedures applied to aqueous samples resulted in poor recoveries of highly polar compounds. First, we used a commercially available mixed-mode Oasis WAX cartridge for cleanup of the extracts. The WAX adsorbent contains amine groups on the surface of the hydrophobic polymeric resin, providing selectivity for the adsorption of acidic compounds. However, in practice, only moderate anionic compounds gave good reproducibility, while weak anionic compounds were not retained on this sorbent. In addition, this anion exchange sorbent with pKa 6.5 requires aqueous acidic buffers with little or no organic solvent, while typical metabolites from cells or tissue are best extracted with organic solvent, such as MeOH or acetonitrile, to obtain good recoveries. Evaporation of crude biological extracts often encounters difficulty with resolubilization of the residue. As we have found that apHera NH2 aminopropyl resin can achieve good separation with high resolution, we made a pipet tip extraction cartridge with apHera NH2 resin after packing a Fluoropore membrane filter for global extraction of anionic metabolites. As shown in Table 1, the desired metabolites gave better recoveries with apHera NH2 resin than with Oasis WAX sorbent extraction. Because apHera NH2 resin has both

anionic and hydrophilic properties, polar but weak anionic metabolites were well retained on it. The peak area normalizations were done by the ISs labeled with stable isotopes which corresponded to original structures except the diphosphates, ADP, and UDP, which were normalized by the 13C,15N-ATP (the retention time is also different from the target metabolites); thus, the recovery rates of ADP and UDP were higher than 120% in some cases. Sample Analysis. In the subsequent optimization for the nanoLC/MS and sample extraction process, we verified the applicability of our methodology by analyzing various kinds of biological samples, i.e., human cerebrospinal fluid (CSF), human plasma, mouse brain tissue, and cultured cell lysate, to profile the anionic metabolites. The extracted ion chromatograms of some of the identified anionic metabolites in cell lysate are shown in Supplementary Figure 1 in the Supporting Information; the metabolites were well separated with no interference. Individual peak area was calculated by the use of Mass ++ software after alignment of retention time at 0.1 Da intervals of m/z. The identified peaks by the commercially available standard compounds in our labora-

(28) Le Belle, J. E.; Harris, N. G.; Williams, S. R.; Bhakoo, K. K. NMR Biomed. 2002, 15, 37–44. (29) Tiziani, S.; Emwas, A. H.; Lodi, A.; Ludwig, C.; Bunce, C. M.; Viant, M. R.; Gu ¨ nther, U. L. Anal. Biochem. 2008, 377, 16–23.

Figure 2. Extracted ion chromatograms of GAR, dUMP, AICAR, SAICAR, and an unknown compound detected in nano-LC/MS analysis of a MTX-treated Hela cell sample (blue chromatogram, MTX-treated cells; red chromatogram, control cells).

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Figure 3. Chemical structure and product ion mass spectrum of UTP (a) and the unknown compound (b) detected in a MTX-treated Hela cell sample.

tory among the clearly detected peaks from human cerebrospinal fluid, human plasma, mouse brain tissue, and cultured Hela cell lysate were 8 (169), 9 (199), 33 (358), and 31 (221), respectively. Table 2 summarizes the estimated composition of metabolites in plasma, CSF, brain tissue, and Hela cells. Phosphorylated metabolites were present at significantly higher levels in brain tissue and Hela cells, as compared with plasma and CSF, while carbonylated compounds were present at comparable levels in all the samples. It seems likely that phosphorus metabolites would be unstable in plasma and CSF during circulation in the body due to

the activity of phosphatases, while phosphorus metabolites do not readily pass through the plasma membrane from the inside of cells. Therefore, metabolite profiling of cultured cells might be preferable to understand metabolic pathways. Therefore, we applied our method to metabolome analysis of Hela cells after treatment with MTX. Our group has previously shown that 5′phosphoribosylglycinamide (GAR), 5′-phosphoribosyl-5-amino-4imidazolecarboxamide (AICAR), dUMP, and 5′-phosphoribosyl4-(N-succinocarboxamide)-5-aminoimidazole (SAICAR) are formed in MTX-treated HCT116 cells, when we focused on MRM analysis Analytical Chemistry, Vol. 81, No. 18, September 15, 2009

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of phosphorus metabolites. Here, we analyzed MTX-treated Hela cells to look for changes in other metabolites. As shown in Figure 2, the levels of GAR, AICAR, dUMP, and SAICAR were significantly increased in MTX-treated samples, as expected. We found that one unknown peak detected only in MTX-treated samples at m/z 496.99 showed the same retention time as UTP (XlogP3 ) 5.8) but with a difference of 14 mass units. The fragmentation pattern in the MS/MS spectrum obtained from the unknown peak was identical to that of UTP between m/z 50 and m/z 273 (fragmentation of the ribose ring with diphosphate), as shown in Figure 3, but there was a 14 mass unit shift above m/z 273, which corresponds to the covalent attachment of CH2 (methylene group) to the uracil ring. Although we could not find any candidates in the Kyoto Encyclopedia of Genes and Genomes (KEGG), there was a candidate, methylated UTP, in PubChem. Since the XlogP3 value of this candidate was 5.8, which is the exactly the same as that of UTP, methylated UTP should be eluted at the same retention time as UTP from the HILIC/ WAX stationary phase. MTX inhibits the folate biosynthesis pathway, which is associated with one-carbon pool metabolism, and once this pathway is inhibited, one-carbon donors are accumulated. The excess precursor metabolites might be used for other one-carbon acceptors, one of which might be UTP. This pathway might not usually be active, since we did not

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detect methylated UTP in cells not treated with MTX. However, the details remain to be clarified. CONCLUSION Our results show that addition of a trace level of EDTA to the sample solution improved peak resolution and sample preparation for nano-LC/MS-based polar anionic metabolomics, resulting in better detection limits. This method proved to be effective for profiling anionic metabolites in various biological samples and is expected to be widely useful, for example, in biomarker discovery for diseases and in mode-of-action studies for drug candidates. ACKNOWLEDGMENT This work was supported by a grant from Core Research for Evolution Science and Technology, Japan (CREST). We thank Dr. Tatsuji Nakamura for helpful support and fruitful discussions. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 11, 2009. Accepted August 13, 2009. AC901269H