Simultaneous Determination of Anionic Intermediates for Bacillus subtilis Metabolic Pathways by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Abstract

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.

Global metabolite analysis of yeast: evaluation of sample preparation methods

“…Similar conclusions were also achieved for filamentous fungi (Hajjaj et al, 1998) and bacterial cells (Maharjan and Ferenci, 2003). The recent methodologies developed to acquire accurate information on microbial metabolite levels (Buchholz et al, 2002;Soga et al, 2002;van Dam et al, 2002;Mashego et al, 2004;Villas-Bôas et al, 2005; and others) urgently need an extraction protocol that is able to trap a higher diversity of intracellular metabolites of yeasts. However, the yeast metabolome comprises a variety of compounds with a wide range of physical and chemical properties that make this task virtually impossible.…”

“…Therefore, it is important to determine the intracellular levels of metabolites accurately, which requires efficient and reliable methods for sample preparation. There have been considerable efforts to develop sensitive and accurate methods for detection of metabolites (Buchholz et al, 2002;Soga et al, 2002;van Dam et al, 2002;Mashego et al, 2004;Villas-Bôas et al, 2005;and others) and to improve the efficiency of data mining and data analysis (Goodacre et al, 2004;Kell, 2004;van der Greef et al, 2004) but relative little attention has been given to sample preparation procedures. Figure 1 summarizes the main steps in sample preparation for metabolome analysis.…”

A novel mechanism regulates H2S and SO2 production in Saccharomyces cerevisiae

“…All CE-ESI-MS experiments were performed using an Agilent Capillary Electrophoresis System, equipped with an air pressure pump, an Agilent 1100 series MSD mass spectrometer and an isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit and a G1607A Agilent CE-ESI-MS sprayer kit (Agilent Technologies) as described by Soga and Heiger (2000) and Soga et al (2002).…”

Eletroforese capilar acoplada à espectrometria de massas (CE-MS): vinte anos de desenvolvimento

“…O mesmo grupo de pesquisa comprovou a possibilidade de separar compostos aniônicos relacionados a rotas metabólicas do B. subtilis. 92 As quantidades detectadas foram da ordem de fmol, o que tornou o método desenvolvido ainda mais atraente e de grande aplicabilidade para estudos desta natureza.…”