Quantitative assays for esterified oxylipins generated by immune cells

• Published in: Nature protocols Vol. 5; no. 12; pp. 1919 - 1931
• Main Authors: O'Donnell, Valerie B, Morgan, Alwena H, Hammond, Victoria J, Morgan, Lloyd, Thomas, Christopher P, Tallman, Keri A, Garcia-Diaz, Yoel R, McGuigan, Christopher, Serpi, Michaela, Porter, Ned A, Murphy, Robert C
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-12-2010; Nature Publishing Group
• Subjects: 631/1647/2230; 631/1647/527/296; 631/250/2504; 631/45/287/1183; Acids; Analytical Chemistry; Assaying; Bacterial infections; Biological Techniques; Biomedical and Life Sciences; Chemical Fractionation - methods; Chromatography, High Pressure Liquid - methods; Computational Biology/Bioinformatics; Immune response; Life Sciences; Lipids; Liquid chromatography; Mass spectrometry; Methods; Microarrays; Molecular Structure; Organic Chemistry; Oxylipins - isolation & purification; Phosphatidylcholines - chemistry; Phosphatidylethanolamines - chemistry; Phospholipids; Properties; Protocol; Tandem Mass Spectrometry - methods; United Kingdom; United Kingdom > UK; United States > US
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2010.162

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins—in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids—attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC–tandem mass spectrometry (LC/MS/MS) in multiple reaction–monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ∼2 d. Following this, extraction of 30 samples takes ∼3 h, with LC/MS/MS run time of 50 min per sample.