Determination of intracellular metabolites concentrations in Escherichia coli under nutrition stress using liquid chromatography-tandem mass spectrometry

Publication date: 1 November 2018
Source:Talanta, Volume 189
Author(s): Fenfen Ji, Yang Shen, Leihan Tang, Zongwei Cai
Cells show a timely and appropriate physiological adjustment on all levels of cellular activities in response to nutrient stress. However, the regulations for cells under different carbon/nitrogen influxes are poorly understood. To unveil a fully metabolic regulatory profile, we applied a mass spectrometry based "bottom-up" approach to investigate the metabolic response of Escherichia coli to nutrient stress. A novel cell sample preparation procedure was developed to decrease the variation and leakage of intracellular metabolites. Volatile ion-pair reagent tributylamine was used to improve the retention and selectivity of charged metabolites on a C18 reversed-phase column. The growth rate and intracellular concentrations of 12 central carbon metabolites were measured systematically under various carbon/nitrogen influxes by manipulating titratable promoters. Fructose-1,6-biphosphate (FBP) concentration as a sensor of carbon influx was positively correlated with the growth rate, whereas α-ketoglutarate (αkg), served as a coordinator of carbon and nitrogen flux showed different dependence on growth rate between carbon limitation and nitrogen limitation. By integrating different behaviors of the metabolites with knowledge from previous reports, a scenario of feedback control under carbon and nitrogen limitations was proposed. Our findings revealed the key role of αkg in the coordination of carbon and nitrogen utilization under nutrition stress and highlighted the great potential of mass spectrometry based approach in deciphering the complex metabolic network.

Graphical abstract

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