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Related Concept Videos

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Isotope ratio-based profiling of microbial folates.

Wenyun Lu1, Yun Kyung Kwon, Joshua D Rabinowitz

  • 1Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA.

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Analyzing intracellular folates is complex. This new LC-MS/MS method reveals how the antibiotic trimethoprim alters folate metabolism, unexpectedly shortening polyglutamate chains.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Analytical Chemistry

Background:

  • Folate metabolism is crucial for cellular processes and a key target for drugs.
  • Intracellular folate analysis is challenging due to modifications like oxidation, methylation, and polyglutamylation.

Purpose of the Study:

  • To develop and validate a comprehensive method for quantifying diverse intracellular folates.
  • To investigate the impact of trimethoprim on folate pools in Escherichia coli.

Main Methods:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with hydrophilic interaction chromatography.
  • Folate extraction at -75°C with protective agents, positive mode electrospray ionization, and multiple reaction monitoring.
  • Isotope-ratio-based quantification using (13)C-labeled glucose and unlabeled standards for absolute measurements.

Main Results:

  • The method successfully profiled intracellular folate diversity in Escherichia coli and Saccharomyces cerevisiae.
  • Trimethoprim treatment led to increased oxidized and decreased reduced folates.
  • A significant, previously unrecognized shift towards shorter polyglutamate chain lengths was observed upon trimethoprim exposure.

Conclusions:

  • The developed LC-MS/MS method provides a powerful tool for comprehensive intracellular folate analysis.
  • Trimethoprim's effect on folate polyglutamylation reveals novel biological insights.
  • This approach can uncover new biological phenomena by analyzing the full spectrum of cellular folates.