Comprehensive profiling of folates across polyglutamylation and one-carbon states.

INTRODUCTION: One-carbon metabolism is central to carbon fixation, methylation, and biosynthesis of amino acids, lipids, and nucleotides. Folates are organic cofactors that harbor one-carbon units and shunt them across these metabolic pathways. Despite its essentiality to all life forms, the diverse nature of folate species with various polyglutamylation and one-carbon states makes their measurement challenging. OBJECTIVES: We aim to illuminate one-carbon metabolism by streamlining comprehensive profiling of folate polyglutamates. METHODS: We analyze folate standards and cellular extracts containing diverse folates species by liquid chromatography-mass spectrometry (LC-MS). RESULTS: We observe that Escherichia coli cells possess diverse folate polyglutamates with one to ten terminal glutamates. Interestingly, most folate polyglutamates form doubly charged ions as well as singly charged ions in LC-MS. Folates also undergo in-source fragmentation. The disparate fates of folates in MS make their quantitation prone to underestimation. Fragmentation by in-source collision-induced dissociation (CID) and LC separation circumvent this issue and facilitate robust and sensitive quantification of folates. In-source CID of folates generates reporter fragment ions that yield higher signals in the mass-to-charge ratio (m/z) range near the maximal mass resolution of Orbitrap MS. Our LC methods complement MS by effectively separating folates based on their polyglutamylation and one-carbon states. CONCLUSION: Our metabolomics approach tailored to folate polyglutamates reveals multiple layers of one-carbon metabolism organized by the lengths of polyglutamate tails in folates. Our analytical workflow is broadly applicable to folate profiling across various cell types to advance our knowledge of one-carbon metabolism as well as biotechnology and medicine.