Tailored extraction and ion mobility-mass spectrometry enables isotopologue analysis of tetrahydrofolate vitamers

Abstract

Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO₂ and CO₂-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for ¹³C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using ¹³C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.

Keywords: 13C-Labelling; Ion mobility-mass spectrometry; Isotopologue ratio analysis; Tetrahydrofolate pathway; Vitamers.

Fig. 1

Schematic illustration of the most important tetrahydrofolate pathways for amino acid metabolism and DNA synthesis (methylation, purine, and pyrimidine base synthesis). The tetrahydrofolate pool is represented in the yellow area. THF, tetrahydrofolic acid; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; FGAR, N-formylglycinamide ribonucleotide; FAICAR, 5-formamido-4-imidazolecarboxamide ribonucleotide

Fig. 2

Tetrahydrofolate vitamer stability in new extraction procedure. Single stock standards of each vitamer indicated above the plots were split, one fraction stored at 4 °C (blue) while the second was subject to the entire extraction procedure (olive). The relative abundance comparison of the conversion products or impurities indicated in the box beneath the plot is normalised to the main single stock compound (indicated above) as 100%. Extractions and measurements were performed in triplicate, averages are displayed with ± 1 s error bars

Fig. 3

Key LC-IM-QTOFMS results for isotopologue distribution analysis of folate vitamers. CH₃-THF mass spectra extracted between 4.65 and 4.90 min: (a) incompletely resolved TOFMS and (b) clean QTOFMS fragments. CH.⁺ = THF mass spectra extracted between 4.68 and 4.95 min: (c) incompletely resolved TOFMS and (d) clean IM-TOFMS result achieved using (e) arrival time filtering (green box)

Fig. 4

Tetrahydrofolate pathway and isotopologue distribution analysis results from ¹³C labelling experiments. a Tetrahydrofolate pathway in K. phaffii with gene annotation. b Carbon isotopologue distribution analysis results of ¹³C-formate labelling. c Carbon isotopologue distribution analysis results of.¹³C-methanol labelling. Yellow boxes (b, c) highlight the time-resolved bioreactor labelling experiment results of one strain; grey dashed boxes (b, c) highlight shake flask labelling experiments of different strains at the same timepoint. All strain names and genotype are found in Table 1. Note: 5-CHO-THF is not shown here as it is a storage molecule not involved in the transfer of the C1 pathway (see Fig. 1)