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 Ϫ75°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. (J Am Soc Mass Spectrom 2007, 18, 898 -909)

Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria

“…6A). In agreement with this notion, recent isotope ratiobased profiling studies of E. coli folate pools in trimethoprimtreated cells have revealed decreased THF levels and increased DHF (or more oxidized compound) levels in comparison to the levels in nontreated cultures (17). Consequently, when the level of FolA activity is low, the intracellular concentration of the ThyA substrate CH 2 THF declines rapidly to a very low level, resulting in a decreased rate of the ThyA reaction.…”

Analytical strategies for LC–MS-based targeted metabolomics

“…By separating compounds into a 40-min positive ion run and a 50-min negative ion run, a total of ∼140 compounds can be readily measured with a total running time of 90 min, using only one mass spectrometer. The same column chemistry can also be used to analyze additional classes of molecules, such as folates, which themselves include >50 chemical species [48]. This method is a reasonable alternative for those occasions where limited mass spectrometry resources are available.…”

The Mitochondrial Folylpolyglutamate Synthetase Gene Is Required for Nitrogen Utilization during Early Seedling Development in Arabidopsis

“…86, 2.56, 3.13, 10.61, 11.29, 11.36, and 11.49 min, respectively. Sample preparation for the folylpolyglutamates of 5-M-THF-Glu n and 5-F-THF-Glu n (n = 1-4, 5, 7, and 8) was carried out as follows (Garratt et al, 2005;Lu et al, 2007). About 0.1 g of plant material (fresh weight) after 12 d of growth on the medium was ground to a fine powder in liquid N. The powder was transferred to an ice-cold 1.5-mL tube containing 0.25 mL of extraction buffer (50:50 methanol:water, 0.1% ascorbic acid, and 0.8% 2-mercaptoethanol, pH 6.0; freshly prepared, ice cold).…”