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

Abstract

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.

Figure 1

Overview of workflow, illustrated for the case of nonadherent cells such as E. coli.

Figure 2

Flowchart of protocol steps.

Figure 3

Schematic of potential mass spectrometry peak intensities obtained via Steps 2–6 for a 4 carbon compound after ¹³C labeling. Blue indicates signal arising from the spiked unlabeled internal standard. Yellow indicates signal arising from endogenous cellular compound after feeding the cells with uniformly ¹³C-labeled carbon source. The total amounts of spiked and endogenous compounds are shown as being equal for simplicity. (a) Case A involves complete labeling of the cellular compound, in which L = 1, Z = 0 and Steps 11–16 could be omitted. (b) Case B involves some of the cellular compounds being only partially labeled. In this case, L < 1 and Steps 11–13 provide an important quantitative correction; however, Z = 0 and Steps 14–16 could be omitted. (c) Case C involves some of the cellular compound remaining fully unlabeled. In this case, Z > 0 and all steps are required.

Figure 4

Creating filter culture from batch culture (Step 2A(vi)). Batch culture is slowly and evenly dripped onto a filter membrane, which sits on a sintered support base residing on top of a vacuum flask.

Figure 5

Triple quadrupole LC–MS/MS system.

Figure 6

Quenching and extraction of a filter culture (Step 2A(x)). Removal of the filter culture from the agarose plate (left panel) and placement of the filter culture, cell side down, into the extraction solution (right panel).

Figure 7

Rinsing the filter with extraction solution (Step 2A(xi)).

Figure 8

Representative extracted ion chromatograms of UTP. Signal is shown for unlabeled UTP arising from spiked standard (black) and for fully ¹³C-labeled UTP arising from endogenous E. coli metabolism (red). The observed signal intensities are used for the calculation of R in Step 8.