Capillary Electrophoresis Time-of-flight Mass Spectrometry Research Articles

Effect of Bisphenol A on Rat Metabolic Profiling Studied by Using Capillary Electrophoresis Time-of-Flight Mass Spectrometry

Bisphenol A (BPA), a chemical widely used in the manufacture of polycarbonate plastics, has raised considerable concern in recent decades because of its hormone-like properties. Whether BPA exposure is a health risk remains controversial in many countries. A metabolomics study based on capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS) was performed to study the urine metabolic profiles of Sprague-Dawley rats fed with four dose levels of BPA (0, 1, 10, and 100 μg/kg body weight) for 45 days. Multivariate pattern recognition directly reflected the metabolic perturbations caused by BPA. On the basis of univariate analysis, 42 metabolites including amino acids, polyamines, nucleosides, organic acids, carbohydrates, pterins, polyphenols, and sugar phosphates were found as the most significantly differential metabolites. The marked perturbations were related with valine, leucine and isoleucine biosynthesis, D-glutamine and D-glutamate metabolism, etc. Significant alterations of neurotransmitters (glutamate, gamma-aminobutyric acid, and noradrenaline) and neurotransmitter-related metabolites (tyrosine, histamine, valine, and taurine) suggested that the toxic effects of small-dose BPA (below 50 mg/kg/day) may contribute to its interactions with the neuromediating system. Our study demonstrated that metabolomics may offer more specific insights into the molecular changes underlying the physiological effects of BPA.

Renal cell carcinoma with a novel germ-line mutation in isocitrate dehydrogenase 1 (IDH1) and its functional study.

e15568 Background: Recently, omics study such as metabolome analysis and whole genome sequencing has been applied to various disorders. We predicted the dysfunction of isocitrate dehydrogenase 1 (IDH1) in renal cell carcinoma (RCC) using metabolome analysis and identified a novel mutation of IDH1. In addition, we examined its function. Methods: We determined the global-scale metabolome profiling of human RCC by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), and compared the metabolite levels of tumors and paired normal tissues in 10 patients with RCC. We performed the genotyping of IDH1 and von Hippel-Lindau (VHL) gene. We also performed transfection experiments by using Lipofectamine and measured the enzymatic activity of IDH1 in RCC cell lines. All protocols were performed by the Institutional Review Board (IRB) of Hamamatsu University School of Medicine and Keio University. Informed consent was obtained from the patient in accordance with the IRB of Hamamatsu University School of Medicine. Results: The acceleration of glycolysis with low level of NADPH and citrate in one patient led to IDH1 dysfunction followed by identifying a novel missense mutation, V178I. In transfection experiment, the reductive activity of IDH1 variant (V178I) increased 2.5 times than that of IDH1 wild type while the oxidative activity of IDH1 variant oxidative activity was as well as that of IDH1 wild type. Furthermore, in the metabolome analysis using labeling glutamine, RCC cell lines deficient in the von Hippel-Lindau (VHL) protein preferentially use reductive glutamine metabolism. On the other hand, this patient with V178I mutation of IDH1 had no VHL mutation. Conclusions: Metabolome analysis led to the identification a novel IDH1 missense mutation in RCC. This mutation might be involved with acceleration of reductive reaction in tricarboxylic acid cycle. Metabolome analysis using CE-TOFMS could be a powerful tool for the global-scale exploration of disease-specific metabolism.

Metabolomic Analysis of Differential Changes in Metabolites during ATP Oscillations in Chondrogenesis

Prechondrogenic condensation is a critical step for skeletal pattern formation. Recent studies reported that ATP oscillations play an essential role in prechondrogenic condensation. However, the molecular mechanism to underlie ATP oscillations remains poorly understood. In the present study, it was investigated how changes in metabolites are implicated in ATP oscillations during chondrogenesis by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). CE-TOF-MS detected 93 cationic and 109 anionic compounds derived from known metabolic pathways. 15 cationic and 18 anionic compounds revealed significant change between peak and trough of ATP oscillations. These results implicate that glycolysis, mitochondrial respiration and uronic acid pathway oscillate in phase with ATP oscillations, while PPRP and nucleotides synthesis pathways oscillate in antiphase with ATP oscillations. This suggests that the ATP-producing glycolysis and mitochondrial respiration oscillate in antiphase with the ATP-consuming PPRP/nucleotide synthesis pathway during chondrogenesis.

Impact of 2-deoxy-D-glucose on the target metabolome profile of a human endometrial cancer cell line

2-deoxy-D-glucose (2DG) has been clinically evaluated for its potential use as an anticancer drug. Although 2DG is generally thought to inhibit the glycolytic pathway through accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), it may also interfere with various other biological processes. Here, to further understand the role of 2DG as an inhibitor of tumor progression, we assessed the metabolism of 2DG in a human endometrial cancer cell line using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). A total of 113 target metabolite peaks were identified and 90 metabolites of them were quantified. Furthermore, we present a new methodology which uses CE-TOFMS metabolome profiling following introduction of an artificial metabolite to evaluate tumor-specific metabolite traces. Aside from 2DG6P, we detected the presence of unique 2DG-derived deoxy metabolites in 2DG-treated cells. These metabolites may be responsible for the alteration of global metabolism in cells and act as various biological effectors.

A medium-chain fatty acid as an alternative energy source in mouse preimplantation development

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for ß-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-13C8] octanoate added to the culture media into intermediate metabolites of the TCA cycle via ß-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

Metabolomic profiling of lung and prostate tumor tissues by capillary electrophoresis time-of-flight mass spectrometry

Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-012-0452-2) contains supplementary material, which is available to authorized users.

Metabolomic profiling rationalized pyruvate efficacy in cybrid cells harboring MELAS mitochondrial DNA mutations

Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases.

Abstract 938: Metabolomic analysis on pharmacological responses to oxaliplatin plus 5-FU in colorectal cancer cells

Abstract Purpose: Oxaliplatin (L-OHP) in combination with 5-fluorouracil (5-FU)/leucovorin (FOLFOX regimen) is an important treatment of metastatic colorectal cancer. However, metabolic functions to determine pharmacological response to L-OHP in combination with 5-FU are still unclear. The purpose of this study is to elucidate metabolomic responses to L-OHP in combination with 5-FU and key pathways relating to chemosensitivity. Experimental Procedures: Human colorectal carcinoma, HCT116 (high-sensitive) and DLD-1 (low-sensitive) cells, were exposed to L-OHP, 5-FU or L-OHP plus 5-FU. After treatment with the anti-cancer agents, the intracellular metabolites were extracted by liquid-liquid extraction and simultaneously and quantitatively analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The mRNA expression level of spermidine/spermine N1-acetyltransferase (SSAT) was measured by real-time quantitative RT-PCR (Taqman PCR). Results: N1-acetylspermine and N8-acetylspermidine in polyamine metabolism were accumulated to a greater extent after L-OHP exposure in drug-sensitive cancer cells. The mRNA expression level of SSAT-1, a key enzyme in polyamine catabolism, increased after L-OHP exposure in drug-sensitive cancer cells but not drug-resistant cancer cells. In addition to the up-regulation of polyamine metabolism in drug-sensitive cancer cells, the intracellular levels of endogenous nucleotides remarkably increased after L-OHP exposure in drug-sensitive cancer cells. These results imply the changes in nucleotide metabolism due to the disruption of DNA synthesis by L-OHP. The intracellular level of α-alanine also accumulated after L-OHP exposure in drug-sensitive cancer cells. α-alanine is known as a pyrimidine catabolite of uracil. These results show that nucleotide metabolism and polyamine metabolism may be important metabolic pathways relating to L-OHP chemosensitivity. Furthermore, in drug-sensitive cancer cells, polyamine metabolism, glutathione metabolism, and nucleotide metabolism were up-regulated after exposure to L-OHP plus 5-FU. On the other hand, metabolites belonging to each metabolic pathway on the whole increased moderately after exposure to L-OHP plus 5-FU in drug-resistant cancer cells. Conclusions: Polyamine metabolism, glutathione metabolism, and nucleotide metabolism are important metabolic pathways relating to responsiveness to L-OHP plus 5-FU. The present CE-TOFMS metabolome analysis created a new finding on metabolomic response to L-OHP plus 5-FU in colorectal cancer cells with different chemosensitivities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 938. doi:1538-7445.AM2012-938

Abstract 838: A metabolomic analysis revealed the involvement of deoxycytidine kinase and CTP synthase in the sensitivity of human pancreatic cancer cells to gemcitabine

Abstract Gemcitabine (GEM), a pyrimidine nucleoside analogue, is used in patients with pancreatic cancer as the standard chemotherapeutic agent. However, pancreatic cancer cells often show resistance to GEM in vivo and vitro. In this study, we explored the factors involved in the sensitivity of GEM in human pancreatic cancer cells, Miapaca-2 and AsPC-1, by a metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The sensitivity of Miapaca-2 (IC50: 66 nM) to GEM was higher than that of AxPC-1 (IC50: 16 nM), whereas there were little differences in the growth rates of these cells. This result indicates that the difference in the sensitivities between Miapaca-2 and AsPC-1 is not dependent on their growth rates. At 12 hr after adding GEM, intracellular level of GEM in Miapaca-2 was higher than that in AsPC-1, whereas levels of GEM monophosphate, diphosphate and triphosphate in Miapaca-2 were lower than that in AsPC-1. In addition, expression level of deoxycytidine kinase (dCK), which catalyzes the reaction from GEM to GEM monophosphate, in Miapaca-2 was lower than that in AsPC-1. Expression levels of human equilibrium transporter (hENT1), which is known to major transporter of GEM, were similar in both cells. These results suggest that dCK is rate-limiting enzyme in GEM phosphorylation and one of the factors involved in the sensitivity. In pyrimidine metabolism, GEM was observed to inhibit CTP synthase, which converts UTP to CTP, resulting increase in UTP level, decrease in CTP level and depletion of dCTP in both cells at 12 hr after adding GEM. The degree of increase in UTP and decrease in CTP was lower in Miapaca-2 than that of AsPC-1. Indeed, the ratio of UTP to CTP in Miapaca-2 was about 2.5, whereas that of AsPC-1 was about 11. In addition, the expression level of CTP synthase in Miapaca-2 was higher than that in AsPC-1. Ribonucleotide reductase, which catalyzes the conversion of CDP to dCDP, is known to be inhibited by GEM diphosphate and be involved in the sensitivity of pancreatic cancer cells to GEM. GEM decreased CDP in AsPC-1. This may be caused by inhibiting CTP synthase. Expression level of ribonucleotide reductase in Miapaca-2 was higher than that in AxPC-1. Therefore, ribonucleotide reductase also may be involved in the sensitivity to GEM. Furthermore, we previously found that GEM mainly inhibited CTP synthase in pyrimidine metabolism, resulting in depletion of dCTP. Our current study suggests that dCK, CTP synthase and ribonucleotide reductase are involved in the sensitivity of pancreatic cancer cells to GEM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 838. doi:1538-7445.AM2012-838

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.

Metabolome analyses in human gastric cancer tissue using capillary electrophoresis time-of-flight mass spectrometry.

61 Background: In order to explore carcinogenic and prognostic biomarkers for gastric cancer, genomic, transcriptomic and proteomic approach had been extensively investigated. However, little information is possible for metabolomics profiles in gastric cancer. As cancer cells demonstrates distinguishable growth pattern from normal cells, presence of specific metabolic pathway in cancer cells is expected. Therefore, we investigated the metabolme profiles of gastric cancer tissue by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Methods: Twenty seven patients with gastric cancer underwent gastrectomy were enrolled in this study. Cancer tissue and adjacent non-cancerous tissue were obtained from surgically resected sample and immediately frozen. Frozen tissues were homogenized and then applied to CF-TOFMS.The metabolomics data by CE-TOFMS were analyzed using principal component analysis (PCA) in order to compare the metabolic profiling of the non cancerous and the cancer tissues. Correlation between metabolic profile and clinicopathologic factors were also investigated. Results: A total of 245 metabolites were detected and quantified. In PCA, the first component separated the cancer tissue data from the non-cancerous tissue data. Metabolic data from cancer tissues showed severe heterogeneity. PCA based on the difference between cancer tissue and non-cancerous tissue revealed that metabolic profile of gastric cancer was classified into two groups. One cancer group (group 1) accumulated lactate and most amino acids excessively, and another group (group 2) didn’t. In group 1, the adenylate energy charge of the cancer tissues was maintained compared to that of the non-cancerous tissues.In comparison with clinicopathologic factors, only histological type demonstrated significant correlation with metabolic classification. Undifferentiated type was significantly predominant in group 2. Conclusions: Gastric cancer was classified into two groups by quantitative metabolome profiling. As this classification significantly correlated with histological type, existence of specific metabolic pathway related to histological type is suggested.

Comprehensive Profiling of Free and Conjugated Estrogens by Capillary Electrophoresis–Time of Flight/Mass Spectrometry

The biological activity of estrogens is tightly regulated by regioselective phase I/II metabolic transformations that are critical to human health. Current methods for analysis of urinary estrogens are limited by complicated sample pretreatment and/or inadequate specificity for free estrogens and their glucuronide/sulfate conjugates that vary widely in their intrinsic polarity. In this work, direct speciation of intact estrogen conjugates and their regioisomers is demonstrated using capillary electrophoresis-time-of-flight/mass spectrometry (CE-TOF/MS) when using an alkaline buffer system with negative ion mode detection. This method allows for resolution of weakly acidic native estrogens, anionic estrogen conjugates and their positional isomers without significant matrix-induced ion suppression effects in human urine. Identification of unknown estrogen metabolites using CE-TOF/MS is supported by accurate mass together with their characteristic relative migration times, which can be predicted based on two intrinsic physicochemical properties of an ion. CE-TOF/MS offers a promising strategy for comprehensive profiling of estrogens and other classes of steroid conjugates that is needed for deeper insight into the etiology and treatment of chronic disorders associated with impaired estrogen metabolism.

Non-targeted metabolite profiling in activated macrophage secretion

Periodontal diseases are inflammatory infectious diseases that affect the periodontal tissue. Macrophages play a central role in inflammatory conditions, leading to the destruction of tissues. Identifying the signaling molecules secreted by macrophages would be valuable to the study of these diseases. Here, we present non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for the profiling of extracellular metabolites released during macrophage activation. Lipopolysaccharide (LPS)-induced activation of a mouse macrophage-like cell line RAW264.7 was used as a model system. Cells were treated without (control) or with LPS for 22 h and, after washing, were incubated for 1 h in phosphate-buffered saline. The accumulation of metabolites in the culture supernatant was monitored. LPS treatment significantly enhanced the accumulation of prostaglandins, tumor necrosis factor-α, nitric oxide and citrulline in the culture medium. RAW264.7 cells produced 46 metabolites and 66% of these showed significant changes (P < 0.05) following cell activation. In particular, the production of leucine, hypoxanthine, choline, putrecine, N 8-acetylspermidine, succinate, itaconate, and 4-methyl-2-oxopentanoate was significantly increased by cell activation (P < 0.001). Significantly elevated production of lactate and glycine was also observed. Here, we present the first catalog of the up and down-regulation of the various metabolites secreted by macrophages following inflammatory activation.

Highly sensitive profiling assay of acidic plant hormones using a novel mass probe by capillary electrophoresis-time of flight-mass spectrometry

Plant hormones play crucial roles in plant growth and development. However, up to date, identification and quantification of acidic plant hormones with trace amount in complicated plant matrix is still a challenge. In current study, we developed a high sensitive assay for the determination of acidic plant hormones in rice by combining capillary electrophoresis and electrospray ionization-time of flight-mass spectrometry (CE-ESI-TOF-MS). To improve the detection sensitivity of acidic plant hormones, 3-bromoactonyltrimethylammonium bromide (BTA) was synthesized as a new mass probe, which can react efficiently with acidic plant hormones in acetonitrile containing triethylamine (TEA). The positively charged BTA-derivatives were separated by CE using amino-coated capillary, which provided a reversed electroosmotic flow (EOF) at low pH, as well as reduced the adsorption of BTA-derivatives on the inner wall of capillary. Using the CE-ESI-TOF-MS method developed in current study, 15 acidic plant hormones, including 10 gibberellins (GAs), were identified and quantified with good linearities from 1.3 to 850 ng/mL with linear coefficient R 2 values of >0.99. The limits of detection (LODs) were in the range of 0.34–4.59 ng/mL. Recoveries of compounds from spiked beverage samples ranged from 84.6 to 112.2%. And a good reproducibility was obtained by evaluating the intra and inter-day precisions with relative standard deviations (RSDs) less than 6.7 and 9.9%, respectively.

Profiling of the charged metabolites of traditional herbal medicines using capillary electrophoresis time-of-flight mass spectrometry

The quantification of a small number of bioactive components in herbal medicines is often inadequate when attempting to elucidate a medicine’s biological effects. Despite rapid advances in analytical technologies, obtaining comprehensive metabolomic profiles of herbal medicines remains difficult, due to the complexity of natural product mixtures. Toki-Shakuyaku-San is a Chinese medicine used widely to treat gynecological and obstetric disorders, such as infertility, dysmenorrhea, toxemia during pregnancy and neural dysfunction. It consists of Angelica acutiloba Radix (Toki), Cnidium officinale Rhizoma (Senkyu), Paeonia lactiflora Radix (Shakuyaku), Atractylodes lancea Rhizoma (Sojutsu), Alisma orientale Rhizoma (Takusha) and Poria cocos Hoelen (Bukuryo). To elucidate the composition of these herbal medicines individually, we conducted non-targeted profiling analyses of extracts of these herbs using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which allows the simultaneous quantification of hundreds of charged metabolites. In total, 737 ± 183.1 (average ± SD) metabolite-derived features were observed, and of these, 119 metabolites were identified. Score plots of principal component analysis (PCA) showed a clear cluster including Shakuyaku, Bukuryo, and Sojutsu, while the other three herbs were distributed over PCA spaces. Loading plots revealed that amino acids and shikimate-derived alkaloids were the predominant metabolite constituents. Hierarchical clustering analysis revealed that few clusters overlapped in the herbal medicines tested. This report is the first demonstration of the characterization of a herbal medicine using large-scale metabolomic analysis, which is complementary to traditional quality control methods.

Metabolic profiling of human urine by CE-MS using a positively charged capillary coating and comparison with UPLC-MS

The potential of capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) using capillaries coated with a triple layer of polybrene-dextran sulfate-polybrene (PB-DS-PB) was evaluated for metabolic profiling of human urine. The method covers various metabolite classes and stable metabolic profiles of urine samples were obtained with favourable migration time repeatability (RSDs <1%). The PB-DS-PB CE-TOF-MS method was used for the analysis of human urine samples from 30 males and 30 females, which had been previously analyzed by reversed-phase UPLC-TOF-MS. Multivariate data analysis of the obtained data provided clear distinction between urine samples from males and females, emphasizing gender differences in metabolic signatures. Nearly all compounds responsible for male-female classification in CE-TOF-MS were different from the classifying compounds in UPLC-TOF-MS. Almost all compounds causing classification in the CE-TOF-MS study were highly polar and did not exhibit retention in the reversed-phase UPLC system. In addition, the CE-TOF-MS classifiers had an m/z value in the range of 50-150, whereas 95% of the classifying features found with UPLC-TOF-MS had an m/z value above 150. The CE-TOF-MS method therefore appears to be highly complementary to the UPLC-TOF-MS method providing classification based on different classes of metabolites.

Metabolomic Identification of the Target of the Filopodia Protrusion Inhibitor Glucopiericidin A

Identifying the targets of bioactive compounds is a major challenge in chemical biological research. Here, we identified the functional target of the naturalbioactive compound glucopiericidin A (GPA) through metabolomic analysis. We isolated GPA while screening microbial samples for a filopodia protrusion inhibitor. Interestingly, GPA alone did not inhibit filopodia protrusion, but synergistically inhibit protrusion with the mitochondrial respiration inhibitor, piericidin A (PA). These results suggested that GPA might inhibit glycolysis. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) provided strong evidence that GPA suppresses glycolysis by functionally targeting the glucose transporter. GPA may therefore serve asa glucose transporter chemical probe. Simultaneous inhibition of both glycolysis and mitochondrial respiration dramatically decreased intracellular ATP levels, indicating that GPA inhibits ATP-dependent filopodia protrusion with PA. Our results represent a challenge of molecular target identification using metabolomic analysis.

CE-MS for Metabolic Profiling of Volume-Limited Urine Samples: Application to Accelerated Aging TTD Mice

Metabolic profiling of biological samples is increasingly used to obtain more insight into the pathophysiology of diseases. For translational studies, biological samples from animal models are explored; however, the volume of these samples can be a limiting factor for metabolic profiling studies. For instance, only a few microliters of urine is often available from small animals like mice. Hence, there is a need for a tailor-made analytical method for metabolic profiling of volume-limited samples. In the present study, the feasibility of capillary electrophoresis time-of-flight mass spectrometry (CE-ToF-MS) for metabolic profiling of urine from mice is evaluated. Special attention is paid to the analytical workflow; that is, such aspects as sample preparation, analysis, and data treatment are discussed from the metabolomics viewpoint. We show that metabolites belonging to several chemical families can be analyzed in mouse urine with the CE-ToF-MS method using minimal sample pretreatment and an in-capillary preconcentration procedure. This exemplifies the advantages of CE-ToF-MS for metabolic profiling of volume-limited samples as loss of material is minimized. The feasibility of the CE-ToF-MS-based workflow for metabolic profiling is illustrated by the analysis of urine samples from wild-type as well as from TTD mutant mice, which are a model for the accelerated aging, with osteoporosis being one of the main hallmarks.

Abstract 2605: CE-TOFMS metabolome analysis for pharmacological responses to CPT-11 in colorectal cancer cells with different chemosensitivities

Abstract Purpose: The topoisomerase I inhibitor irinotecan (CPT-11) is widely used for the treatment of metastatic colorectal cancer. However, mechanism to determine pharmacological response to CPT-11 is still unclear. We reported the application of a novel metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) on the study of metabolic responses to anticancer agents (AACR #2563 and #4785, 2008 and #3764, 2009). The purpose of this study is to compare the metabolomic responses to SN-38, the active metabolite of CPT-11, between high-sensitive and low-sensitive colorectal cancer cells, for finding new biomarkers that can predict CPT-11 response. Experimental Procedures: To elucidate metabolomic responses to SN-38, human colorectal carcinoma, HCT116 and HT29 cells were exposed to 50 nM SN-38. At time 0, 3, 8, and 24 h after SN-38 exposure, all intracellular metabolites were extracted by liquid-liquid extraction and simultaneously analyzed by CE-TOFMS. To find out low molecular weight compounds related to the sensitivity to SN-38, the metabolomic profiles were compared across the 8 colorectal cancer cell lines and sensitivity to SN-38 was investigated. Results: The HCT116 cells were more sensitive to SN-38 than HT29. The intracellular metabolites of key pathways for cellular activity were quantitatively analyzed by CE-TOFMS, which included glycolysis, tricarboxylic acid (TCA) cycles, urea cycles, amino acid metabolism and pyrimidine metabolism. The intracellular levels of endogenous deoxyribonucleotides (dTTP and dCTP) increased after SN-38 exposure in both cells. In contrast, ribonucleotides such as CTP and UTP of RNA synthesis pathway were not affected by SN-38 exposure in either cell. These results imply the change in deoxyribonucleotide pool due to the disruption of DNA synthesis by topoisomerase I inhibition, the main anti-cancer mechanism of SN-38. It was also found that lactate and glycerol-3-phosphate were accumulated to a greater extent in HCT116 than in HT29. Furthermore, correlation analysis using 8 colorectal cancer cell lines revealed that the intracellular level of γ-aminobutyric acid (GABA) strongly correlated with cellular sensitivity to SN-38. Conclusions: The present CE-TOFMS metabolome analysis created a new finding on metabolomic mechanisms for response to SN-38 by colorectal cancer cells with different chemosensitivities. GABA was found to be a potential low molecular weight biomarker that may be used for predicting CPT-11 response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2605.

Quantitative metabolome profiling of Illicium anisatum by capillary electrophoresis time-of-flight mass spectrometry

We used capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) to characterize the metabolic profiles of the seed, pulp, stem and leaf of Illicium anisatum. CE-TOFMS detected more than 1000 polar metabolites within 40 min, of which 58 were annotated and quantified. Seed had higher levels of glycolytic metabolites than pulp, stem and leaf, while leaf had higher levels of TCA cycle and nucleoside metabolites. Among amino acid metabolites, levels of Gln, Glu, and Asp were higher in almost every organ. Levels of shikimic acid, the source compound for Tamiflu, were high in all organs, ranging from 6.84% to 28.82%. These results indicate that CETOFMS-based metabolomics offers an efficient, convenient method for comprehensive metabolite profiling, and may be a powerful tool for the screening of drug discovery.