Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli.

Absolute metabolite concentrations are critical to a quantitative understanding of cellular metabolism, as concentrations impact both the free energies and rates of metabolic reactions. Here we use LC-MS/MS to quantify more than 100 metabolite concentrations in aerobic, exponentially growing Escherichia coli with glucose, glycerol or acetate as the carbon source. The total observed intracellular metabolite pool was approximately 300 mM. A small number of metabolites dominate the metabolome on a molar basis, with glutamate being the most abundant. Metabolite concentration exceeds K(m) for most substrate-enzyme pairs. An exception is lower glycolysis, where concentrations of intermediates are near the K(m) of their consuming enzymes and all reactions are near equilibrium. This may facilitate efficient flux reversibility given thermodynamic and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data.

Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach.

This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with (13)C-labeled carbon sources, with detection of (13)C-assimilation by liquid chromatography-tandem MS. It enables absolute quantitation of several dozen metabolites over approximately 1 week of work.

Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry.

A key unmet need in metabolomics is the ability to efficiently quantify a large number of known cellular metabolites. Here we present a liquid chromatography (LC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) method for reliable measurement of 141 metabolites, including components of central carbon, amino acid, and nucleotide metabolism. The selected LC approach, hydrophilic interaction chromatography with an amino column, effectively separates highly water soluble metabolites that fail to retain using standard reversed-phase chromatography. MS/MS detection is achieved by scanning through numerous selected reaction monitoring events on a triple quadrupole instrument. When applied to extracts of Escherichia coli grown in [12C]- versus [13C]glucose, the method reveals appropriate 12C- and 13C-peaks for 79 different metabolites.