Analysis of carboxylic acid metabolites from the tricarboxylic acid cycle in Bacillus subtilis cell extract by capillary electrophoresis using an indirect photometric detection method.
Analysis of carboxylic acid metabolites from the tricarboxylic acid cycle in Bacillus subtilis cell extract by capillary electrophoresis using an indirect photometric detection method.
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DOI：
10.1016/s0021-9673(03)01063-x
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发表时间：
2003-08
期刊：
影响因子：
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通讯作者：
M. Markuszewski;K. Otsuka;S. Terabe;K. Matsuda;T. Nishioka
中科院分区：
文献类型：
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作者：
M. Markuszewski;K. Otsuka;S. Terabe;K. Matsuda;T. Nishioka
随着对代谢组学分析的兴趣日益浓厚，需要开发可靠的方法来分析真实样品（如细菌细胞）中的细胞内代谢物谱。由于TCA循环中的三羧酸、二羧酸（柠檬酸、异柠檬酸、2-氧戊二酸、琥珀酸、富马酸、苹果酸）、糖酵解途径中的羧酸代谢物、尿素循环中的羧酸代谢物和氨基化合物（甲酸、丙酮、乳酸、乙酸、谷氨酸）的代谢物的吸光度较弱，采用间接紫外检测毛细管电泳（CE）对其进行了分析。以4 mM 2,6-吡啶二羧酸为高紫外吸收载体电解质，0.2 mM十六烷基三甲基溴化铵，10%乙二醇和10%乙腈，pH为3.5，对葡萄糖和苹果酸两种不同培养物的枯草芽孢杆菌细胞提取物中羧酸代谢产物进行了分析。在电动注射模式下，CE的检出限为13-54 ppb（1.12·10−7-5.96·10−7M）。在迁移时间小于1.3%、相关系数可接受的情况下，考察了方法的重现性和线性。采用优化后的CE法比较了以葡萄糖或苹果酸盐为碳源的两种不同培养基中提取的细胞提取物的代谢组含量。观察了不同培养基中羧酸代谢物谱的变化。羧酸浓度范围：苹果酸培养细胞提取物中乳酸和柠檬酸盐浓度范围为59 ~ 0.5 μM，葡萄糖培养细胞提取物中谷氨酸和柠檬酸盐浓度范围为133 ~ 0.5 μM。估计单个细菌细胞中羧酸的适当浓度为mM和亚mM水平。
With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell. Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection. Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures: glucose and malate. CE with an electrokinetic injection mode achieved limits of detection in the range of 13–54 ppb (1.12·10−7–5.96·10−7M). The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients. The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source. The changes in carboxylic acid metabolites profile were observed depending from used culture medium. Carboxylic acid concentrations ranged: in cell extract from malate culture from 59 to 0.5 μM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 μM for glutamate and citrate, respectively. Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.