The MAW Method: A Rapid Single-Phase Extraction for Bacterial Lipidomics

The increasing incidence of antibiotic resistance underscores the pressing need for studies aimed at understanding its underlying mechanisms. Bacterial membrane lipids provide an avenue for studies, as they are crucial for maintaining the integrity and functionality of bacterial cell membranes. However, in-depth studies on the involvement of bacterial membrane lipids require working with numerous bacterial samples, which necessitates the need for a high-throughput lipid extraction method. Traditional chloroform/methanol-based lipid extraction once considered the gold standard, hampers high-throughput lipidomics because of the challenge of separating the lipid-containing layer from the aqueous phase and is time-consuming.¹ Although monophasic extraction methods, such as butanol/methanol extraction (BUME), exist, they are primarily designed for mammalian cells and lack proven effectiveness in bacterial lipidomics. This presentation describes the development and optimization of a monophasic lipid extraction method that utilizes methanol, acetonitrile, and water (MAW) for Gram-positive bacteria, Staphylococcus aureus. The MAW method is compared with the biphasic Bligh and Dyer (B&D) extraction in terms of simplicity, lipid recovery, sample quantity, and high-throughput capability.² Furthermore, this monophasic method is applied to other Gram-positive and Gram-negative bacteria with different antibiotic susceptibilities. Multivariate statistical analyses of these bacteria revealed distinct clustering, emphasizing the robustness of the MAW extraction technique.

References

(1) Bligh, E. G. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 1959.

(2) Bimpeh, K.; Hines, K. M. A rapid single-phase extraction for polar staphylococcal lipids. Analytical and Bioanalytical Chemistry 2023. DOI: 10.1007/s00216-023-04758-9.