Dual stable isotope probes of deuterium oxide and 13C fatty acid were demonstrated to probe the lipid biosynthesis cycle of a Gram-positive bacterium Enterococcus faecalis. As external nutrients and carbon sources often interact with metabolic processes, the use of dual-labeled isotope pools allowed for the simultaneous investigation of both exogenous nutrient incorporation or modification and de novo biosynthesis. Deuterium was utilized to trace de novo fatty acid biosynthesis through solvent-mediated proton transfer during elongation of the carbon chain while 13C-fatty acids were utilized to trace exogenous nutrient metabolism and modification through lipid synthesis. Ultra-high-performance liquid chromatography high-resolution mass spectrometry identified 30 lipid species which incorporated deuterium and/or 13C fatty acid into the membrane. Additionally, MS2 fragments of isolated lipids identified acyl tail position confirming enzymatic activity of PlsY in the incorporation of the 13C fatty acid into membrane lipids.
Keywords: 13C nutrients; Bioanalytical methods; Deuterium; High resolution mass spectrometry; Isotope tracing; Liquid chromatography.
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