oa Isotopologue Ratios Identify 13C-Depleted, Methanotrophic Biomarkers in Complex Environmental Samples

Our study introduces a novel isotopologue-based approach for compound-specific isotope analysis (CSIA) using ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC/HRMS), which extends d¹³C analysis to non-volatile, intact lipids typically inaccessible to conventional gas chromatography–isotope ratio mass spectrometry (GC/irMS). By leveraging the distinct imprint of ¹³C-depletion in isotopologue distributions, particularly the M1/M0 ratio, our method enables the reliable identification of ¹³C-depleted methanotrophic lipids in complex environmental systems.

Method development, based on archaeol, a lipid amenable to both GC/irMS and UHPLC/HRMS, demonstrated a strong correlation (R = 0.94) between d¹³C values derived from our isotopologue-based approach and conventional GC/irMS. Subsequent application to lipid extracts from the Guaymas Basin, characterized by the widespread occurrence of anaerobic methane oxidation (AOM), proved highly effective in differentiating biomarkers strongly associated with methane-oxidizing archaea, such as intact archaeol derivatives, from those derived from other non-methanotrophic sources. These findings underscore the potential of isotopologue-based CSIA using UHPLC/HRMS as a powerful new tool for tracing methanotrophic microbial communities and methane cycling in modern and paleo-environments.