Name: a quantitative assessment of the yeast lipidome using electrospray ionization mass spectrometry
Uploaded: 2009-08-21T12:45:00
Description: Watch this Scientific Journal Video about A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry at JoVE.com

We are grateful to Alain Tessier for valuable advise, discussions, and technical support. We acknowledge the Centre for Biological Applications of Mass Spectrometry at Concordia University for outstanding services. This work was supported by grants from the CIHR and the NSERC of Canada. V.I.T. is a CIHR New Investigator and Concordia University Research Chair in Genomics, Cell Biology and Aging.

Materials and methods
<ol>
	<li>Yeast strains and growth conditions 
		The wild-type strain BY4742 (MAT&alpha; his3&Delta;1 leu2&Delta;0 lys2&Delta;0 ura3&Delta;0) was grown in rich YEPD medium (1% yeast extract, 2% peptone, 2% glucose). Cells were cultured at 30&deg;C with rotational shaking at 200 rpm in Erlenmeyer flasks at a "flask volume/medium volume" ratio of 5:1.</li>
	<li>Storage of yeast cells for lipid extraction 
		<ol>
 	<li>A 50 ml culture of cells was harvested by centrifugat...

<ol>
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This method enables a rapid quantitative assessment of the yeast lipidome using readily available, inexpensive materials. The method allows the identification of most of the lipid species found in yeast cells with the exception of diacylglycerols, ergosterols and ergosteryl esters, although these lipids can be identified by replacing ammonium hydroxide with lithium hydroxide. The described method enables lipid identification and quantification at the concentrations as low as &mu;g/ml, with the concentration linearity spr...

a quantitative assessment of the yeast lipidome using electrospray ionization mass spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

Watch this Scientific Journal Video about A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry at JoVE.com

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

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