A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with ␥-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.An excess dose of acetaminophen (AAP), 4 the most commonly used analgesic, can lead to possibly fatal hepatitis and more than 100 such deaths occur in the United States annually. AAP is normally detoxified by sulfation or glucoronidation followed by elimination (1). In high doses, it is metabolized by P450 cytochromes to generate the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) (2), which is further inactivated by conjugation with glutathione (GSH) before excretion. This results in a sudden drop in GSH levels (3). In absence of sufficient GSH, the reactive NAPQI can cause toxic and covalent protein modifications that lead to cell death and tissue injury (4 -7).Recent transcriptomic and proteomic studies showed that AAP can cause numerous changes in gene and protein expression levels in pathways related to cellular stress response, mitochondrial function, and metabolism, as well as in cell cycle, structural, signaling, and apoptotic proteins (8, 9). However, little is known about global changes in metabolites. Global information about when and where metabolite levels increase or decrease can reveal connections in biological networks and provide a system level understanding of the cell (10 -14). However, unlike other functional genomic approaches, metabolome analysis methods are still under development. Current large scale metabolite analysis methods are based on gas chromatography mass spectrometry (15), liquid chromatography mass spectrometry (LC-MS) (16), NMR (17), Fourier transform ion cyclotron resonance mass spectrometry) (18), and capillary electrophoresis mass spectrometry (CE-MS) (19). Whereas these analytical tools allow global metabolite profiling, the exploration and identification of changes in compounds am...

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.