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Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of ¹⁸ O-Phosphoramidites.

ABSTRACT: Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for ¹⁸ O-labelled phosphates is presented, based on a family of modified ¹⁸ O₂ -phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. ¹⁸ O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the ¹⁸ O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry.

SUBMITTER: Haas TM

PROVIDER: S-EPMC9298905 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledMicroorganisms grow under a remarkable range of extreme conditions. Environmental transcriptomic and proteomic studies have highlighted metabolic pathways active in extremophilic communities. However, metabolites directly linked to their physiology are less well defined because metabolomics methods lag behind other omics technologies due to a wide range of experimental complexities often associated with the environmental matrix. We identified key metabolites associated with acidophilic and metal-tolerant microorganisms using stable isotope labeling coupled with untargeted, high-resolution mass spectrometry. We observed >3,500 metabolic features in biofilms growing in pH ~0.9 acid mine drainage solutions containing millimolar concentrations of iron, sulfate, zinc, copper, and arsenic. Stable isotope labeling improved chemical formula prediction by >50% for larger metabolites (>250 atomic mass units), many of which were unrepresented in metabolic databases and may represent novel compounds. Taurine and hydroxyectoine were identified and likely provide protection from osmotic stress in the biofilms. Community genomic, transcriptomic, and proteomic data implicate fungi in taurine metabolism. Leptospirillum group II bacteria decrease production of ectoine and hydroxyectoine as biofilms mature, suggesting that biofilm structure provides some resistance to high metal and proton concentrations. The combination of taurine, ectoine, and hydroxyectoine may also constitute a sulfur, nitrogen, and carbon currency in the communities.ImportanceMicrobial communities are central to many critical global processes and yet remain enigmatic largely due to their complex and distributed metabolic interactions. Metabolomics has the possibility of providing mechanistic insights into the function and ecology of microbial communities. However, our limited knowledge of microbial metabolites, the difficulty of identifying metabolites from complex samples, and the inability to link metabolites directly to community members have proven to be major limitations in developing advances in systems interactions. Here, we show that combining stable-isotope-enabled metabolomics with genomics, transcriptomics, and proteomics can illuminate the ecology of microorganisms at the community scale.

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Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18 O-Phosphoramidites.

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Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of ¹⁸ O-Phosphoramidites.