Separation and Quantification of Water-Soluble Cellular Metabolites in Clostridium thermocellum using Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry

Abstract

A new protocol for metabolomic studies was developed by combining liquid chromatography-tandem mass spectrometry and isotope dilution mass spectrometry with universal ¹³C labeled internal standards from Escherichia Coli. The multiple reaction monitoring mode of mass spectrometry was used for quantification. Forty-five water-soluble intracellular metabolites, including 20 amino acids, 16 organic acids (primarily from the tricarboxylic acid cycle), and 9 cofactors, were measured and 34 of them were successfully quantified using the ¹³C-labeled internal standards. The limit of detection, limit of quantification, precision, and linearity of the methods were evaluated. The methods were applied to the quantitative analysis of intracellular metabolites extracted from wild-type and ethanol-adapted strains of Clostridium thermocellum cultivated with and without ethanol stress, and all 34 metabolites including all 9 cofactors were successfully quantified. Further multivariate data analyses of the metabolic differences between wild-type and ethanol-adapted strains were performed on the quantitative data, which can help elucidate the metabolic mechanism behind ethanol adaptation in C. thermocellum.

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Keywords:

• Clostridium thermocellum
• Ethanol tolerance
• Isotope dilution mass spectrometry
• Liquid chromatography-mass spectrometry
• Water-soluble metabolites

Acknowledgments

This article contains supplementary material available online.

We thank Prof. Jian Xu for providing the wild type and ethanol tolerant strains of C. thermocellum ATCC 35609. This work was supported by National Basic Research Program of China (Grant No. 2011CB707404 and 2011BAD22B02) and the National Natural Science Foundation of China (Grant No. 30970050).

Notes

*¹³C-labeled analogs were not found for these metabolites.

^a No daughter ions were found for these metabolites regardless of the values of collision energy used in this experiment.

ND: Not detected.