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

引用本文: 牟翰, 洪铭, 刘晓云, 李敏超, 黄明志, 储炬, 庄英萍, 张嗣良. 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[J]. 分析化学, 2017, 45(9): 1264-1270. doi: 10.11895/j.issn.0253-3820.170205 shu

Citation: MOU Han, HONG Ming, LIU Xiao-Yun, LI Min-Chao, HUANG Ming-Zhi, CHU Ju, ZHUANG Ying-Ping, ZHANG Si-Liang. Accurate Detemination of Isotopic Abundance of Intracellular Metabolites of Saccharopolysporaerythraea Based on Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, 2017, 45(9): 1264-1270. doi: 10.11895/j.issn.0253-3820.170205 shu

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

华东理工大学生物反应器工程国家重点实验室, 上海 200237
基金项目:
本文系国家自然科学基金（No.21276081）和国家科技支撑项目（No.2011BAF02B05）资助

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¹³C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¹³C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¹³C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¹³C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¹³C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¹³C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。

关键词:

English

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

State Key Laboratory of Bioreactor Engnieering, East China University of Science and Technology, Shanghai 200237, China
Received Date: 30 March 2017
Revised Date: 12 June 2017
Fund Project:
This work was supported by the National Natural Science Foundation of China (No. 21276081).

Abstract: A method for measuring ¹³C 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 contain ¹³C atoms, the one-to-one method, one-to-many method and SIM method were established for measuring ¹³C isotopic abundance. Then these methods were used to measure naturally labeled intracellular metabolite standards and ¹³C 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 containing ¹³C atoms represented by sugar phosphates, one-to-many method was the best for measuring the metabolites of both parent and daughter ions containing ¹³C short carbon chains represented by carboxylic acids, SIM method could play a role in measuring the metabolites of both parent and daughter ions containing ¹³C 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.

Key words:

1. [1]
  Wiechert W. Metab. Eng.,2001,3(3):195-206
2. [2]
  Ahn W S, Antoniewicz M R. Metab. Eng., 2011,13(5):598-609
3. [3]
  Zamboni N, Fischer E, Muffler A, Wyss M, Hohmann H P, Sauer U. Biotechnol. Bioeng., 2005,89(2):219-232
4. [4]
  Ruhl M, Le Coq D, Aymerich S, Sauer U. J. Biol. Chem., 2012,287(33):27959-27970
5. [5]
  Hong M, Huang M Z, Chu J, Zhuang Y P, Zhang S L.J. Biotechnol.,2016,231:1-8
6. [6]
  Yang Y T, Aristidou A A, San K Y, Bennett G N.Metab. Eng.,1999,1(1):26-34
7. [7]
  Ahn W S, Antoniewicz M R.Metab. Eng.,2011,13(5):598-609
8. [8]
  Zamboni N, Fischer E, Muffler A, Wyss M, Hohmann H P, Sauer U.Biotechnol. Bioeng.,2005,89(2):219-232
9. [9]
  Crown S B, Antoniewicz M R. Metab. Eng., 2013,16(1):21-32
10. [10]
  GUO Meng-Lei, LIU Xiao-Yun, HUANG Ming-Zhi, LI Min-Chao, CHU Ju, ZHUANG Ying-Ping,ZHANG Si-Liang. Chinese J. Anal. Chem., 2016,44(2):232-2 郭孟磊, 刘晓云, 黄明志, 李敏超, 储炬, 庄英萍, 张嗣良.分析化学,2016,44(2):232-240
11. [11]
  Buchholz A, Takors R, Wandrey C. Anal. Biochem., 2001,295(2):129-137
12. [12]
  Lu W, Bennett B D, Rabinowitz J D. J. Chromatogr. B,2008,871(2):236-242
13. [13]
  LI Min-Chao, HUANG Min-Zhi, LIU Yu-Wei, CHU Ju, ZHUANG Ying-Ping, ZHANG Si-Liang. Chinese J. Anal. Chem.,2014,42(10):1408-1413 李敏超, 黄明志, 刘玉伟, 储炬, 庄英萍, 张嗣良.分析化学,2014,42(10):1408-1413
14. [14]
  Kiefer P, Nicolas C, Letisse F, Portais J C. Anal. Biochem.,2007,360(2):182-188
15. [15]
  Ruhl M, Rupp B, Noh K, Wiechert W, Sauer U, Zamboni N. Biotechnol. Bioeng.,2012,109(3):763-771
16. [16]
  Wahl S A, Dauner M, Wiechert W. Biotechnol. Bioeng.,2004,85(3):259-268
17. [17]
  Seifar R M, Ras C, Deshmukh A T, Bekers K M, Suarez-Mendez C A, da Cruz A L B, van Gulik W M, Heijnen J J. J. Chromatogr. A,2013,1311(18):115-120

扫一扫看文章

计量

PDF下载量: 3
文章访问数: 582
HTML全文浏览量: 31

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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

English

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

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

计量

通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

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

English

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

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

计量

文章相关

通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs