Isotope ratio-based profiling of microbial folates

Abstract

Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using -75 degrees C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly (13)C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.

Fig. 1

Representative compound stability traces. The top panel shows PteGlu (folic acid) and the bottom 5-CH₃-H₄PteGlu (MTHF), whose stabilities were measured in the indicated solvent systems. Each solid square is the average of two independent experiments, with the error bar being ± one standard error of the mean. The data is fit to single-exponential decay function, except when there is no obvious decay (in which case the line is for illustration purposes only).

Fig. 2

Representative chromatography traces for 5-CH₃-H₄PteGlu_n (MTHF-Glu_n, n from 1 to 7) from (a) E. coli and (b) yeast extract.

Fig. 3

Comparing the ¹²C-signal from ¹²C-glucose-grown E. coli extract with the ¹³C-signal from ¹³C-glucose-grown E. coli extract for 17 folate compounds. No ¹²C-signals were seen in the ¹³C-glucose grown cells, or vice versa. The line of unity is drawn as a reference. When the data were fit by linear regression, the slope was 1.00 ± 0.09, R² = 0.90. Specific folate compounds were those showing quantitative signal in Table 4: pAB-Glu₃, H₂PteGlu_3-5, H₄PteGlu₃, 5,10-CH₂-H₄PteGlu_3-5, 5-CH₃-H₄PteGlu_1-6, 5/10-CHO-H₄PteGlu_2-4.

Fig. 4

Absolute quantitation of 5-CH₃-H₄PteGlu_n and 5/10-CHO-H₄PteGlu_n from E. coli. Experiments were performed in duplicate, with each sample run three times. Error bars show the biological (inter-sample) standard deviation.

Fig. 5

Isotope ratio-based experimental scheme for studying the effect of the antibiotic drug trimethoprim on the folate pools in E. coli.