Rule-Based Ion Prediction with Orthogonal Constraints Reveals Bacterial Phospholipid Remodeling Signatures

Background: Phospholipids are essential components of bacterial membranes and play central roles in membrane integrity and adaptation to antibiotic stress. However, confident annotation of phospholipid molecular species remains challenging due to the complexity of the lipidome and the limited structural constraints in conventional lipidomics workflows. Methods: Here, we present a bacterial phospholipidomic framework that integrates orthogonal structural evidence to achieve high-confidence and traceable annotation. Thin-layer chromatography (TLC) provides phospholipid headgroup assignment, gas chromatography–mass spectrometry (GC–MS) defines the acyl-chain pool, and Paternò–Büchi derivatization enables C=C localization, collectively restricting the structural search space prior to liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. A rule-based ion prediction library further standardizes diagnostic ion assignment and reduces annotation ambiguity. Results: Applying this platform, we found Escherichia coli in the stationary phase remodeled the membrane phospholipids, with cardiolipin (CL) increasing from ~5% to ~10% and cyclopropane-containing phospholipid species rising to ~75%. Similar remodeling patterns are observed under diverse antibiotic exposures at sub-inhibitory concentrations, consistent with convergence toward a tolerance-associated membrane state. Extension of the framework to Enterococcus faecium supports proof-of-concept application in an additional Gram-positive model, with vancomycin-resistant strains exhibiting pronounced phosphatidylglycerol (PG) enrichment and reduced CL. Conclusions: Our work provides a scalable and reproducible strategy for bacterial phospholipid annotation, enabling molecular-species-resolved investigation of membrane adaptation and offering a framework for future exploration of lipid homeostasis pathways as potential antimicrobial targets.