Kevin Williams (Presenter)
University of California, Los Angeles
Bio: Kevin J. Williams is a research scientist in the laboratory of Steven J. Bensinger.
Authorship: Kevin J. Williams(1), Quan D. Zhou(2), Joseph P. Argus(2), Robert Damoiseaux(2,3) and Steven J. Bensinger(1,2)
(1)Dept of Microbiology, Immunology, and Molecular Genetics, UCLA (2)Dept of Medical and Molecular Pharmacology, David Geffen School of Medicine, UCLA (3)California NanoSystems Institute, UCLA
| Short Abstract In recent years, metabolomic and isotopic enrichment analysis has led to breakthroughs in a variety of fields of research including cancer biology, immunology, and aging/regenerative medicine. This research has necessitated the preparation and analysis of ever increasing numbers of samples. There are considerable challenges to preparing metabolomics samples, especially in the preparation of cellular lipid samples. Here we describe a high-throughput procedure for the preparation of cellular fatty acids and sterols that reduces costs, labor and preparation time while increasing sample consistency. The resulting FAMEs and Sterols can then be analyzed by GC/MS for quantification and isotopic enrichment analysis. |
Long Abstract
Groundbreaking research in the last decade has solidified the central role of metabolism in a wide array of physiological and pathological processes. As such, metabolomic analysis is being applied to a diverse number of fields, including cancer biology, immunology, and aging/regenerative medicine. Unfortunately, there are considerable challenges in the preparation of metabolomic samples, especially lipid samples. The use of glassware and other consumables for derivatization and extraction steps add considerable cost to these experiments. The complexity of these steps makes such experiments labor intensive. Finally, the use of organic solvents like hexane, chloroform and toluene create safety hazards for research personnel.
To facilitate the preparation of large numbers of samples for analysis of whole cellular fatty acids and cholesterol, we adopted an acid methanolysis procedure capable of converting total cellular lipids into constituent FAMEs and cholesterol(1). We coupled this to a rapid imaging-based cell counting and collection procedure. Further, we miniaturized the acid methanolysis reaction using the 96-well Multi-Tier™ Micro Plate System with glass conical vials. Lastly, we used a liquid handler to automate the procedure. This automation expedites the most labor intensive steps including the addition of acid methanolysis reaction reagents and standards and the subsequent neutral lipid extraction. Extracted lipids are transferred to a second 96 well Multi-Tier plate that is compatible with numerous mass spectrometry autosampler systems.
Using this procedure, we can rapidly prepare samples from cells cultured in 6, 12, 24, or 96 well plates. Here we demonstrate that we can linearly measure total cellular fatty acid and cholesterol content across a number of cell samples sizes. Further, to demonstrate the utility of this assay, we silenced a number of genes known to regulate fatty acid and cholesterol homeostasis in H1299 cells, label these cells with U-13C-glucose and measure resulting fatty acid and cholesterol amounts and isotopic labeling. These results demonstrate the capabilities of this system to process medium to high-throughput experiments with cellular fatty acid and cholesterol measurements as the readout. Our current sample preparation pipeline allows for the running of 96 samples for fatty acid or cholesterol analysis every 24 hours, with GC/MS run time being the rate limiting step of the analysis.
References & Acknowledgements:
Reference:
(1) Ichihara K, Fukubayashi Y. Preparation of fatty acid methyl esters for gas-liquid chromatography. J Lipid Res. 2010 Mar;51(3):635-40
Acknowledgments:
This work was supported by Molecular Screening Shared Resource which is a core facility supported by the NCI Cancer Center Support Grant P3016042-35 to Judy Gasson
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