MSACL 2023 Abstract

The MAW Method: An Efficient Monophasic Extraction Method for Bacterial Lipidomics Kingsley Bimpeh, Kelly Hines University of Georgia, Athens, GA Kingsley Bimpeh, B.S. Chemistry (Presenter) University of Georgia

Presenter Bio: I completed my B.S. in Chemistry with a minor in Biology from Jackson State University in 2019. During my undergraduate education, I was honored to intern at the Indiana University School of Medicine and Icahn school of medicine at Mount Sinai. These internships opened my eyes to the intersection between chemistry and human health. Hence, I was intrigued and decided to pursue a doctoral degree in Analytical Chemistry. In the Fall of 2019, I enrolled as a graduate student in the lab of Dr. Kelly M. Hines at the Department of Chemistry at the University of Georgia. My research in the Hines lab focuses on developing platforms for high-throughput lipidomics in bacteria and understanding the mechanism of infection/host-pathogen relationships of different viruses.

Introduction:
Usually overlooked, a good lipid extraction method is essential for any lipid analysis. Different extraction methods are available, but each enriches different types of lipids due to their diversity in structure and polarity. Generally, these methods rely on a methanol/chloroform/water solvent system that causes phase separation and were developed with mammalian lipids from serum, plasma, or tissue in mind. While they are suitable for finding the major lipids in bacteria, there are downsides in utilizing these methods such as poor reproducibility, inability to support high-throughput experiments, and the use of a widely known carcinogen, chloroform.

Objectives:
The main goal of this work was to develop a chloroform-free single-phase lipid extraction method based on methanol, acetonitrile, and water (MAW method) for the recovery of lipids from bacteria that circumvents the issues affecting the reproducibility and throughput of biphasic liquid-liquid extractions.

Methods:
Different combinations of acetonitrile/methanol ratios ranging from 1:4 to 4:1 were used to extract the major species of endogenous lipids (i.e., PGs, DGDGs, and LysylPGs) from the S. aureus model system. All analyses were performed with an online HILIC column and a Waters Synapt XS traveling-wave ion mobility-mass spectrometer (TWIM-MS). Subsequent mass spectrometry data were submitted to Progenesis QI (Waters) for alignment, peak picking, and multivariate statistical analysis. Given the amphipathic nature of polar phospholipids, the impact of acidification on extraction yield was examined. The optimized conditions for the single-phase extraction, consisting of 1:4 ACN/MeOH with 0.5% (v/v) acetic acid, were tested against the biphasic Bligh & Dyer (B&D) extraction method for the recovery of LysylPGs, DGDGs, and PGs in S. aureus strain JE2-Dap2. For further analytical quantitation, we determined the absolute lipid recovery using nonendogenous internal standard lipids, the limit of detection (LOD), and quantitation (LOQ) following the MAW and B&D extractions. We evaluated whether the MAW method could be scaled down into volumes compatible with 96 well plates and, to a greater extent, simplified by eliminating the drying and reconstitution steps prior to LC-MS analysis. To demonstrate the suitability of the high-throughput implementation of the MAW method for profiling, we performed untargeted lipidomics on daptomycin-resistant and susceptible strains that were extracted in 96-well microplates using the MAW method.

Results
The optimum solvent conditions for identifying all the essential lipids in S. aureus were 1:4 (%v/v) acetonitrile/methanol. Acidification enhanced the yield of LysylPGs and DGDGs, confirming our initial hypothesis that acidification of anionic and cationic phosphatides disrupts the ionic interaction present between these lipids and the ionized surrounding environment. Comparison of the MAW method against the Bligh & Dyer extraction showed minimal differences in the extracted amounts of LysylPGs, DGDGs, and PGs, but a 3-fold improvement in reproducibility. The extraction recovery of isotope-labeled internal standards confirmed that the MAW method provided comparable recoveries (ca. 90%) to the Bligh & Dyer method. Calibration curves generated for the limit of detection and the limit of quantification of PGs further reveal that the MAW method is sensitive and requires less bacteria for lipid analysis. Furthermore, the lipid peak areas of the small-scale extraction approach were statistically indifferent and strongly correlated with those of the 3.5 mL large-scale extraction when using a total volume of 175 µL (R2 > 0.99; Pearson P < 0.0001). Further simplification by eliminating the drying and reconstitution steps before LC-MS analysis of lipid extracts resulted in higher-intensity signals. Ultimately, the results from the untargeted lipidomics of the daptomycin-resistant and susceptible strains were the same as those of previous lipidomic measurements.

Conclusion:
This work presents a single-phase extraction method, the MAW method, for the recovery of lipids in bacteria. The MAW method demonstrates significant advantages, including ease of use, increased sensitivity coupled with greater reproducibility when compared to the Bligh & Dyer method.