Determination of pyridine and adenine nucleotide metabolites in Bacillus subtilis cell extract by sweeping borate complexation capillary electrophoresis
Determination of pyridine and adenine nucleotide metabolites in Bacillus subtilis cell extract by sweeping borate complexation capillary electrophoresis
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DOI：
10.1016/s0021-9673(03)00031-1
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发表时间：
2003-03-14
影响因子：
4.1
通讯作者：
Nishioka, T
中科院分区：
文献类型：
--
作者：
Markuszewski, MJ;Britz-McKibbin, P;Nishioka, T
随着人们对代谢组分析等生物分析研究新领域的兴趣日益浓厚，需要开发灵敏的毛细管电泳 (CE) 方法来分析生物样品中亚微米浓度的分析物。在本报告中，应用 CE 和硼酸盐络合扫描来分析来自细菌枯草芽孢杆菌细胞提取物的一组七种吡啶和腺嘌呤核苷酸代谢物。 CE 与 UV 光度检测对分析物的检测能力是通过使用硼酸盐络合方法有效聚焦大样品塞（类似于毛细管长度的 10%）来实现的，这通过约 2 X 10(-8) M 的检测限 (SIN = 3) 来反映。当使用葡萄糖或苹果酸作为培养基中的碳源时，观察到细胞提取物中代谢物浓度的变化。细胞提取物中吡啶和腺嘌呤核苷酸的浓度变化很大，从苹果酸中烟酰胺-腺嘌呤二核苷酸的78.6（+/-7.6）μM到葡萄糖培养基中烟酰胺-腺嘌呤二核苷酸磷酸的0.66（+/-0.12）μM。单细胞中代谢物的浓度也以毫摩尔 (mM) 水平进行估计。该方法在线性、灵敏度和重现性方面得到了验证。通过扫描硼酸盐络合应用毛细管电泳可以对复杂生物样品（如细菌细胞提取物）中的核苷酸代谢物进行灵敏且可重复的分析。 (C) 2003 Elsevier Science B.V. 保留所有权利。
With a growing interest in new areas of bioanalytical research such as metabolome analysis, the development of sensitive capillary electrophoresis (CE) methods to analyze sub-muM concentrations of analytes in biological samples is required. In this report, the application of CE with sweeping by borate complexation is used to analyze a group of seven pyridine and adenine nucleotide metabolites derived from bacteria Bacillus subtilis cell extracts. Nanomolar (nM) detectability of analytes by CE with UV photometric detection is achieved through effective focusing of large sample plug (similar to10% of capillary length) using sweeping by borate complexation method, reflected by a limit of detections (SIN = 3) of about 2 X 10(-8) M. Changes in metabolites concentrations were observed in cell extracts when using either glucose or malate as the carbon source in the culture medium. Concentration of pyridine and adenine nucleotides in cell extracts varied widely from 78.6 (+/-7.6) muM for nicotinamide-adenine dinucleotide in malate to 0.66 (+/-0.12) muM for nicotinamide-adenine dinucleotide phosphate in glucose culture medium. Concentrations of metabolites in a single cell were also estimated at milimolar (mM) level. The method was validated in terms of linearity, sensitivity and reproducibility. The application of CE by sweeping borate complexation allows for sensitive and reproducible analyses of nucleotide metabolites in complex biological samples such as bacteria cell extracts. (C) 2003 Elsevier Science B.V. All rights reserved.