Lipidome isotope labelling of gut microbes (LILGM): A method of discovering endogenous microbial lipids

Lipidomics analysis of gut microbiome has become critical in recent surge of extensive human disease studies that investigate microbiome contributions. However, challenges remain in comprehending the origins of thousands of lipid species produced by the diverse microbes. Here, we proposed the development and utilization of a liquid chromatography-mass spectrometry-based approach, named lipidome isotope labelling of gut microbes (LILGM), which enables confident detection and identification of endogenous gut microbial lipidome via ¹³C/¹⁵N labeling strategy and high-resolution mass spectrometry. Our method leveraged in vitro microbial cultures and stable isotope-labeled ¹³C and ¹⁵N, allowing a reasonable degree of isotope incorporation into microbial lipids over short-term of inoculation. We then systematically detected the mass spectral patterns of 182 labeled lipid species by our in-house data analysis pipeline. Further bioinformatics analyses confidently identified biologically relevant microbial lipids from lipid classes such as diacylglycerols (DGs), fatty acids (FAs), phosphatidylglycerols (PGs), and phosphatidylethanolamines (PEs) that may have profound impacts to human physiology. Our study also demonstrated the application of LILGM by showcasing the confident detection of dysregulated microbial lipids post antibiotic perturbation. The debiased sparse partial correlation analysis provides insights into lipid metabolism intricacies. Overall, our method can provide unambiguous analyses to the endogenous microbial lipids in given biological context, and can also instantly reflect the lipidomic changes of gut microbes in response to environmental factors. We believe our LILGM approach has the potential to provide new body of knowledge by combining promising analytical approaches for sensitive and specific lipid detection to support functional microbiome studies.