Cargando…

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids(1-9). Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)(10-14). These signalling molecules are difficult to study because of their...

Descripción completa

Detalles Bibliográficos
Autores principales: Prasain, Jeevan K., Hoang, Hieu D., Edmonds, Johnathan W., Miller, Michael A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728984/
https://www.ncbi.nlm.nih.gov/pubmed/23851568
http://dx.doi.org/10.3791/50447
_version_ 1782278943662931968
author Prasain, Jeevan K.
Hoang, Hieu D.
Edmonds, Johnathan W.
Miller, Michael A.
author_facet Prasain, Jeevan K.
Hoang, Hieu D.
Edmonds, Johnathan W.
Miller, Michael A.
author_sort Prasain, Jeevan K.
collection PubMed
description Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids(1-9). Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)(10-14). These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways(10,15). C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases(6,16,17). A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF(2 α)-d(4 )as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.
format Online
Article
Text
id pubmed-3728984
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher MyJove Corporation
record_format MEDLINE/PubMed
spelling pubmed-37289842013-08-07 Prostaglandin Extraction and Analysis in Caenorhabditis elegans Prasain, Jeevan K. Hoang, Hieu D. Edmonds, Johnathan W. Miller, Michael A. J Vis Exp Developmental Biology Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids(1-9). Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)(10-14). These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways(10,15). C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases(6,16,17). A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF(2 α)-d(4 )as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways. MyJove Corporation 2013-06-25 /pmc/articles/PMC3728984/ /pubmed/23851568 http://dx.doi.org/10.3791/50447 Text en Copyright © 2013, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Developmental Biology
Prasain, Jeevan K.
Hoang, Hieu D.
Edmonds, Johnathan W.
Miller, Michael A.
Prostaglandin Extraction and Analysis in Caenorhabditis elegans
title Prostaglandin Extraction and Analysis in Caenorhabditis elegans
title_full Prostaglandin Extraction and Analysis in Caenorhabditis elegans
title_fullStr Prostaglandin Extraction and Analysis in Caenorhabditis elegans
title_full_unstemmed Prostaglandin Extraction and Analysis in Caenorhabditis elegans
title_short Prostaglandin Extraction and Analysis in Caenorhabditis elegans
title_sort prostaglandin extraction and analysis in caenorhabditis elegans
topic Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728984/
https://www.ncbi.nlm.nih.gov/pubmed/23851568
http://dx.doi.org/10.3791/50447
work_keys_str_mv AT prasainjeevank prostaglandinextractionandanalysisincaenorhabditiselegans
AT hoanghieud prostaglandinextractionandanalysisincaenorhabditiselegans
AT edmondsjohnathanw prostaglandinextractionandanalysisincaenorhabditiselegans
AT millermichaela prostaglandinextractionandanalysisincaenorhabditiselegans