Details

Autor(en) / Beteiligte

• Lu, Wenyun
• Kwon, Yun Kyung
• Rabinowitz, Joshua D.

Titel

Isotope Ratio-Based Profiling of Microbial Folates

Ist Teil von

• Journal of the American Society for Mass Spectrometry, 2007-05, Vol.18 (5), p.898-909

Ort / Verlag

New York, NY: Elsevier Inc

Erscheinungsjahr

2007

Link zum Volltext

Quelle

SpringerLink (Online service)

Beschreibungen/Notizen

• 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 °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 13C-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.