文献类型：期刊文献

中文题名：超高效液相色谱-三重四极杆质谱联用检测红色糖多孢菌胞内中间代谢物同位素丰度

英文题名：Accurate Detemination of Isotopic Abundance of Intracellular Metabolites of Saccharopolysporaerythraea Based on Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry

作者：牟翰[1];洪铭[1];刘晓云[1];李敏超[1];黄明志[1];储炬[1];庄英萍[1];张嗣良[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2017

卷号：45

期号：9

起止页码：1264

中文期刊名：分析化学

外文期刊名：Chinese Journal of Analytical Chemistry

收录：CSTPCD;;Scopus;北大核心:【北大核心2014】；CSCD:【CSCD2017_2018】；

基金：国家自然科学基金(No.21276081);国家科技支撑项目(No.2011BAF02B05)资助~~

语种：中文

中文关键词：红色糖多孢菌;胞内代谢物;13C同位素丰度;超高效液相色谱-三重四极杆串联质谱

外文关键词：Saccharopolysporaerythraea;Intracellular metabolites;13C isotopic abundance;Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry

摘要：采用超高效液相色谱-三重四极杆串联质谱联用仪(LC-MS/MS),建立了能精确检测红色糖多孢菌胞内代谢物^(13)C同位素丰度的方法。在优化的超高效液相色谱的条件及三重四极杆串联质谱的离子传输电压和碰撞池电压下,筛选出各胞内代谢物的最佳离子对。根据物质在LC-MS/MS中生成的母离子和子离子碳链长短及子离子是否含有^(13)C等特性,建立了"一对一"法、"一对多"法和单级质谱SIM法等同位素丰度检测方法。利用这些方法,检测了自然标记标准品和^(13)C标记实验样品,根据实验值与理论值的接近程度筛选出最优方法。结果表明,对于以磷酸糖类为代表的子离子不含有^(13)C的代谢物,"一对一"法最有效;对于以有机酸类为代表的母离子和子离子都含有^(13)C的短碳链代谢物,"一对多"法更有优势;对于以辅酶A类为代表的母离子和子离子都含有^(13)C且碳链较长的代谢物,单级质谱SIM法才能发挥作用。建立的同位素丰度检测方法具有较好的准确度,可应用于红色糖多孢菌胞内代谢物同位素丰度的检测,为后续研究菌体的代谢机理,实现红霉素的高效表达奠定了基础。
A method for measuring13C isotopic abundance of intracellular metabolites of Saccharopolysporaerythraea by ultra-high performance liquid chromatography（ UPLC）-triple quadrupole mass spectrometry was established. First,the chromatographic conditions of UPLC were optimized,and then the MS conditions such as unique tube lens voltage,collision energy,and ion pair were optimized. On the bases of length of the parent and daughter ions carbon chains and whether the daughter ions contain13C atoms,the oneto-one method,one-to-many method and SIM method were established for measuring13C isotopic abundance.Then these methods were used to measure naturally labeled intracellular metabolite standards and13C labeled samples,and according to the gap between the experimental value and the theoretical value,the best method was established for each metabolite of different characteristics. The results showed that one-to-one method was most effective for measuring the metabolites of daughter ions not containing13C atoms represented by sugar phosphates,one-to-many method was the best for measuring the metabolites of both parent and daughter ions containing13C short carbon chains represented by carboxylic acids,SIM method could play a role in measuring the metabolites of both parent and daughter ions containing13C long carbon chains represented by coenzyme A.This method had a good measurement precision and could be applied to the measurement of Saccharopolysporaerythraea intracellular metabolites,which contributed to the consequent study of metabolic mechanism and the efficient expression of erythromycin.