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Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Published on: November 17, 2011
Quantitative assays for esterified oxylipins generated by immune cells.
Alwena H Morgan1, Victoria J Hammond, Lloyd Morgan
1Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK.
Nature Protocols
|December 4, 2010
Summary
This study introduces a new mass spectrometry method for precisely measuring esterified oxylipins, which are important bioactive lipids. This assay advances the quantitation of these complex molecules in immune cells and tissues.
Area of Science:
- Lipidomics
- Mass Spectrometry
- Immunology
Background:
- Phospholipid-esterified oxylipins are bioactive lipids produced by lipoxygenases in immune cells.
- Existing methods for quantifying these lipids are underdeveloped and not widely accessible.
- Accurate measurement is crucial for understanding their role in biological processes.
Purpose of the Study:
- To develop and validate a robust mass spectrometric protocol for the accurate quantitation of phospholipid-esterified oxylipins.
- To enable simultaneous measurement of multiple oxylipin species.
- To provide a reliable assay for researchers studying immune cell signaling and lipid metabolism.
Main Methods:
- Lipids were extracted from cells or tissues using liquid-phase organic extraction.
- Analysis was performed using High-Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS/MS) in multiple reaction-monitoring mode.
- The protocol allows for simultaneous monitoring of up to 23 esterified oxylipin species.
Main Results:
- A mass spectrometric protocol was established for the accurate measurement of key esterified oxylipins, including hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids, and keto-eicosatetraenoic acids.
- The method quantifies oxylipins esterified to phosphatidylethanolamine and phosphatidylcholine.
- The protocol demonstrates efficiency with standard generation in ~2 days, sample extraction in ~3 hours for 30 samples, and LC-MS/MS run time of 50 minutes per sample.
Conclusions:
- The described HPLC-MS/MS protocol provides a well-developed and accessible method for quantifying diverse phospholipid-esterified oxylipins.
- This assay facilitates further research into the biological functions of these lipids in various physiological and pathological conditions.
- The method enhances the field of lipidomics by offering precise quantification of previously challenging-to-measure bioactive molecules.
