Global Lipidome Workflow for <i>Mycobacterium tuberculosis</i> Using High‐Resolution Mass Spectrometry

The lipid‐rich envelope of Mycobacterium tuberculosis ( Mtb ) is a defining feature that underpins its virulence, persistence and response to antibiotics. An understanding of this envelope would allow us to link its lipid composition to the bacterium's physiology and pathogenicity. However, the chemical diversity and structural complexity of the lipid components present a major challenge in obtaining a complete profile of the lipid classes within the envelope. Herein, we report our work in the development of a high‐resolution mass spectrometry pipeline to profile the Mtb lipidome using Mtb H37Rv (mc 2 6206) as a model. Lipids were separated using a reversed phase chromatography method and analysed in dual polarity with adduct awareness in tandem mass spectrometry. This workflow enables both the relative quantification and identification of the lipids present in the sample. We found that a brief, ice‐cold cell homogenisation step before lipid extraction improved coverage and abundance of both the inner‐membrane phospholipids, neutral glycolipids and envelope glycoconjugates. These indicate that mechanical disruption of the mycomembrane enhances solvent access ensuring a higher abundance of extracted lipids. Hence, this ‘homogenisation‐first’ step serves as a practical optimisation to improve lipid yields in Mtb lipidomic analyses. Overall, this workflow establishes a robust foundation for investigating the lipid composition of Mtb .