Flux Analysis of Free Amino Sugars and Amino Acids in Soils by Isotope Tracing with a Novel Liquid Chromatography/High Resolution Mass Spectrometry Platform

Soil fluxomics analysis can provide pivotal information for understanding soil biochemical pathways and their regulation, but direct measurement methods are rare. Here, we describe an approach to measure soil extracellular metabolite (amino sugar and amino acid) concentrations and fluxes based on a ¹⁵N isotope pool dilution technique via liquid chromatography and high-resolution mass spectrometry. We produced commercially unavailable ¹⁵N and ¹³C labeled amino sugars and amino acids by hydrolyzing peptidoglycan isolated from isotopically labeled bacterial biomass and used them as tracers (¹⁵N) and internal standards (¹³C). High-resolution (Orbitrap Exactive) MS with a resolution of 50 000 allowed us to separate different stable isotope labeled analogues across a large range of metabolites. The utilization of ¹³C internal standards greatly improved the accuracy and reliability of absolute quantification. We successfully applied this method to two types of soils and quantified the extracellular gross fluxes of 2 amino sugars, 18 amino acids, and 4 amino acid enantiomers. Compared to the influx and efflux rates of most amino acids, similar ones were found for glucosamine, indicating that this amino sugar is released through peptidoglycan and chitin decomposition and serves as an important nitrogen source for soil microorganisms. d-Alanine and d-glutamic acid derived from peptidoglycan decomposition exhibited similar turnover rates as their l-enantiomers. This novel approach offers new strategies to advance our understanding of the production and transformation pathways of soil organic N metabolites, including the unknown contributions of peptidoglycan and chitin decomposition to soil organic N cycling