High-resolution lipidomics for decoding the soil biome: Improved lipid annotation, quantitation, and response to climate stress

The soil ecosystem harbors diverse biological communities, including archaea, bacteria, fungi, protists, plants, and soil fauna, that collectively drive essential belowground ecosystem processes such as nutrient cycling, soil carbon storage, and climate regulation. Current gene-based approaches offer greatest taxonomic depth but are semiquantitative and targeted by probe design. They do not cover the whole breath of the tree of life and therefore do not yet fully reflect the complexity of soil food webs. Conversely, fatty acid-based methods provide quantitative insights into microbial communities and their activity but are limited in taxonomic depth and coverage of multicellular organisms. We introduce an integrative high-resolution lipidomics workflow specifically designed to characterize the wide diversity of soil organisms. Our pipeline combines lipid extraction from complex soil matrices with liquid chromatography-high-resolution Orbitrap mass spectrometry, rigorous quality controls, and multitiered lipid annotation strategies. Additionally, we present a quantitative structure-property relationship model to predict lipid ionization efficiencies, providing a foundation for future improvements in lipid quantification without the use of chemical standards. Applying this approach, we detected similar to 17,000 lipid features of which we could annotate similar to 4800 lipid compounds, significantly expanding the coverage compared with the conventional methods. Lipid profiles effectively distinguish organisms such as bacteria, fungi, plants, and algae, underscoring the ability of this method to identify organism-specific lipid signatures. Furthermore, testing the workflow in soils subjected to simulated climate change (future climate and drought) revealed subtle but ecologically meaningful shifts in membrane and storage lipids, highlighting lipidome compositional sensitivity to environmental stress. Our integrated lipidomics approach substantially advances lipid annotation, quantification, and ecological interpretation, opening new avenues for biomarker discovery and improved understanding of soil biome responses to environmental perturbations.