Name: quantitative analysis of the cellular lipidome of saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry
Uploaded: 2020-03-08T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry at JoVE.com

我们感谢蒂托连科实验室的现任和前任成员进行讨论。我们感谢质谱生物学应用中心和结构和功能基因组学中心（都位于康科迪亚大学）提供出色的服务。这项研究得到了加拿大自然科学和工程研究理事会（RGPIN 2014-04482）和康科迪亚大学主席基金（CC0113）的资助。K.M.获得康科迪亚大学功绩奖的支持。

1. 培养酵母的无菌介质的准备
<ol><li>制备含有1%（v/v）酵母提取物和2%（v）百能蛋白的完整YP介质的90 mL。</li>
	<li>制备90 mL的合成最小YNB介质，含有0.67%（w/v）酵母氮碱，不含氨基酸，20毫克/L L-西丁，30毫克/L-柳氨酸，30毫克/L-赖氨酸，和20毫克/L尿素。</li>
	<li>将 90 mL 的完整 YP 介质平均分成两个 250 mL Erlenmeyer 烧瓶（即每个 45 mL）。</li>
	<li>将 90 mL 的合成最小 YNB 介质分成两个 ...

<ol>
	<li>Bou Khalil, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidomics+era:+accomplishments+and+challenges.">Lipidomics era: accomplishments and challenges.</a> Mass Spectrometry Review. 29 (6), 877-929 (2010).</li><li>Shevchenko, A., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidomics:+coming+to+grips+with+lipid+diversity.">Lipidomics: coming to grips with lipid diversity.</a> Nature Reviews Molecular Cell Biology. 11 (8), 593-598 (2010).</li><li>Br&#252;gger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidomics:+analysis+of+the+lipid+composition+of+cells+and+subcellular+organelles+by+electrospray+ionization+mass+spectrometry.">Lipidomics: analysis of the lipid composition of cells and subcellular organelles by electrospray ionization mass spectrometry.</a> Annual Review of Biochemistry. 83, 79-98 (2014).</li><li>Zechner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAT+SIGNALS+-+lipases+and+lipolysis+in+lipid+metabolism+and+signaling.">FAT SIGNALS - lipases and lipolysis in lipid metabolism and signaling.</a> Cell Metabolism. 15 (3), 279-291 (2012).</li><li>Eisenberg, T., B&#252;ttner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipids+and+cell+death+in+yeast.">Lipids and cell death in yeast.</a> FEMS Yeast Research. 14 (1), 179-197 (2014).</li><li>Richard, V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+liponecrosis,+a+distinct+mode+of+programmed+cell+death.">Mechanism of liponecrosis, a distinct mode of programmed cell death.</a> Cell Cycle. 13 (23), 3707-3726 (2014).</li><li>Arlia-Ciommo, A., Svistkova, V., Mohtashami, S., Titorenko, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+the+discovery+of+anti-tumor+pharmaceuticals:+searching+for+activators+of+liponecrosis.">A novel approach to the discovery of anti-tumor pharmaceuticals: searching for activators of liponecrosis.</a> Oncotarget. 7 (5), 5204-5225 (2016).</li><li>M&#229;rtensson, C. U., Doan, K. N., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+lipids+on+mitochondrial+functions.">Effects of lipids on mitochondrial functions.</a> Biochimica et Biophysica Acta. 1862 (1), 102-113 (2017).</li><li>Basu Ball, W., Neff, J. K., Gohil, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+non-bilayer+phospholipids+in+mitochondrial+structure+and+function.">The role of non-bilayer phospholipids in mitochondrial structure and function.</a> FEBS Letters. 592 (8), 1273-1290 (2018).</li><li>Thakur, R., Naik, A., Panda, A., Raghu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+membrane+turnover+by+phosphatidic+acid:+cellular+functions+and+disease+implications.">Regulation of membrane turnover by phosphatidic acid: cellular functions and disease implications.</a> Frontiers in Cell and Developmental Biology. 7, 83 (2019).</li><li>Goldberg, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+of+lipid+droplets+in+regulating+longevity.">A novel function of lipid droplets in regulating longevity.</a> Biochemical Society Transactions. 37 (5), 1050-1055 (2009).</li><li>Kohlwein, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obese+and+anorexic+yeasts:+experimental+models+to+understand+the+metabolic+syndrome+and+lipotoxicity.">Obese and anorexic yeasts: experimental models to understand the metabolic syndrome and lipotoxicity.</a> Biochimica et Biophysica Acta. 1801 (3), 222-229 (2010).</li><li>Titorenko, V. I., Terlecky, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+metabolism+and+cellular+aging.">Peroxisome metabolism and cellular aging.</a> Traffic. 12 (3), 252-259 (2011).</li><li>Henry, S. A., Kohlwein, S. D., Carman, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+regulation+of+glycerolipids+in+the+yeast+Saccharomyces+cerevisiae.">Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae.</a> Genetics. 190 (2), 317-349 (2012).</li><li>Kohlwein, S. D., Veenhuis, M., vander Klei, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+and+peroxisomes:+key+players+in+cellular+lipid+homeostasis+or+a+matter+of+fat--store+'em+up+or+burn+'em+down.">Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat--store 'em up or burn 'em down.</a> Genetics. 193 (1), 1-50 (2013).</li><li>Baile, M. G., Lu, Y. W., Claypool, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+topology+and+regulation+of+cardiolipin+biosynthesis+and+remodeling+in+yeast.">The topology and regulation of cardiolipin biosynthesis and remodeling in yeast.</a> Chemistry and Physics of Lipids. 179, 25-31 (2014).</li><li>Dimmer, K. S., Rapaport, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+contact+sites+as+platforms+for+phospholipid+exchange.">Mitochondrial contact sites as platforms for phospholipid exchange.</a> Biochimica et Biophysica Acta. Molecular and Cell Biology of Lipids. 1862 (1), 69-80 (2017).</li><li>Csord&#225;s, G., Weaver, D., Hajn&#243;czky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-mitochondrial+contactology:+structure+and+signaling+functions.">Endoplasmic reticulum-mitochondrial contactology: structure and signaling functions.</a> Trends in Cell Biology. 28 (7), 523-540 (2018).</li><li>Mitrofanova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+and+transport+define+longevity+of+the+yeast+Saccharomyces+cerevisiae.">Lipid metabolism and transport define longevity of the yeast Saccharomyces cerevisiae.</a> Frontiers in Bioscience (Landmark Edition). 23, 1166-1194 (2018).</li><li>Tamura, Y., Kawano, S., Endo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+contact+zones+as+sites+for+lipid+transfer.">Organelle contact zones as sites for lipid transfer.</a> Journal of Biochemistry. 165 (2), 115-123 (2019).</li><li>Weissman, J., Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to Yeast Genetics: Functional Genomics, Proteomics, and Other Systems Analyses. , (2010).</li><li>Botstein, D., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast:+an+experimental+organism+for+21st+Century+biology.">Yeast: an experimental organism for 21st Century biology.</a> Genetics. 189 (3), 695-704 (2011).</li><li>Duina, A. A., Miller, M. E., Keeney, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Budding+yeast+for+budding+geneticists:+a+primer+on+the+Saccharomyces+cerevisiae+model+system.">Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.</a> Genetics. 197 (1), 33-48 (2014).</li><li>Strynatka, K. A., Gurrola-Gal, M. C., Berman, J. N., McMaster, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+surrogate+and+chemical+genetics+in+model+organisms+can+suggest+therapies+for+human+genetic+diseases.">How surrogate and chemical genetics in model organisms can suggest therapies for human genetic diseases.</a> Genetics. 208 (3), 833-851 (2018).</li><li>Zimmermann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+as+a+tool+to+identify+anti-aging+compounds.">Yeast as a tool to identify anti-aging compounds.</a> FEMS Yeast Research. 18 (6), (2018).</li><li>Ejsing, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+the+yeast+lipidome+by+quantitative+shotgun+mass+spectrometry.">Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (7), 2136-2141 (2009).</li><li>Guan, X. L., Riezman, I., Wenk, M. R., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+lipid+analysis+and+quantification+by+mass+spectrometry.">Yeast lipid analysis and quantification by mass spectrometry.</a> Methods in Enzymology. 470, 369-391 (2010).</li><li>Guan, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+membrane+lipidomics+during+Drosophila+development.">Biochemical membrane lipidomics during Drosophila development.</a> Developmental Cell. 24 (1), 98-111 (2013).</li><li>Klose, C., Tarasov, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+yeast+lipids+by+shotgun+lipidomics.">Profiling of yeast lipids by shotgun lipidomics.</a> Methods in Molecular Biology. 1361, 309-324 (2016).</li><li>Wang, M., Wang, C., Han, R. H., Han, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+advances+in+shotgun+lipidomics+for+biology+and+medicine.">Novel advances in shotgun lipidomics for biology and medicine.</a> Progress in Lipid Research. 61, 83-108 (2016).</li><li>Sud, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LMSD:+LIPID+MAPS+structure+database.">LMSD: LIPID MAPS structure database.</a> Nucleic Acids Research. 35 (Database issue), D527-D532 (2007).</li><li>Pauling, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proposal+for+a+common+nomenclature+for+fragment+ions+in+mass+spectra+of+lipids.">Proposal for a common nomenclature for fragment ions in mass spectra of lipids.</a> PLoS One. 12 (11), e0188394 (2017).</li></ol>

以下预防措施对于成功实施此处介绍的协议非常重要：
1. 氯仿和甲醇是有毒的。它们有效地从表面提取各种物质，包括实验室塑料制品和皮肤。因此，请谨慎处理这些有机溶剂，避免在与氯仿和/或甲醇接触的步骤中使用塑料，在这些步骤中使用硼硅酸盐玻璃移液器，并在使用前用氯仿和甲醇对这些移液器进行压毛。
2. 在甲醇/氯仿（17：1）混合物提取脂?...

大量证据表明，脂质是生物分子的主要类别之一，在真核细胞的许多重要过程中起着至关重要的作用。这些过程包括组装构成细胞细胞器周围的血浆膜和膜的脂质双层物，在细胞膜之间传输小分子，对细胞外环境变化和细胞内信号转导的反应，能量的产生和储存，限制于不同细胞器的蛋白质的进出口，内膜系统和蛋白质分泌中蛋白质的静脉贩运，以及几种调节细胞死亡模式1 ，2，3，4，5，6，7，8，9，10.
萌芽酵母S.cerevisiae，一个单细胞真核生物，已经成功地揭示了一些机制，脂质在这些重要的细胞过程4，5，6，7，8，9，10，11，12，13，14，15的基本作用，16，17，18，19，20.S. cerevisiae是发现这些机制的宝贵模型有机体，因为它适用于全面的生化、遗传、细胞生物学、化学生物学、系统生物学和微流体解剖分析21、22、23、24、25。脂质代谢和细胞内迁移有助于这些重要细胞过程的相互理解机制的进一步进展，需要敏感的质谱技术，用于细胞脂质的定量表征，了解脂质分子的复杂性，并将定量脂质学整合到系统生物学1、2、3、26的多学科平台中， 27、28、29、30。
目前酵母细胞和其他真核生物细胞的质谱辅助定量脂质学方法不够通用、健壮或敏感。此外，这些目前使用的方法无法区分各种等baric或等质脂质物种。在这里，我们描述了一种多功能、健壮和灵敏的方法，它允许使用液体色谱法与串联质谱（LC-MS/MS）进行定量分析S.cerevisiae的主要细胞脂质。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL High-speed glass centrifuge tubes with Teflon lined caps</td>
			<td>PYREX</td>
			<td>05-550</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Glass sample vials with Teflon lined caps</td>
			<td>Fisher Scientific</td>
			<td>60180A-SV9-1P</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A461-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A9554</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1100 series LC system</td>
			<td>Agilent Technologies</td>
			<td>G1312A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent1100 Wellplate</td>
			<td>Agilent Technologies</td>
			<td>G1367A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Fisher Scientific</td>
			<td>A11450</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>A11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>A470-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bactopeptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiolipin</td>
			<td>Avanti Polar Lipids</td>
			<td>750332</td>
			<td></td>
		</tr>
		<tr>
			<td>Centra CL2 clinical centrifuge</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramide</td>
			<td>Avanti Polar Lipids</td>
			<td>860517</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C297-4</td>
			<td></td>
		</tr>
		<tr>
			<td>CSH C18 VanGuard</td>
			<td>Waters</td>
			<td>186006944</td>
			<td>Pre-column system</td>
		</tr>
		<tr>
			<td>Free fatty acid (19:0)</td>
			<td>Matreya</td>
			<td>1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads (acid-washed, 425-600 &#956;M)</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>L-histidine</td>
			<td>Sigma</td>
			<td>H8125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid Search software (V4.1)</td>
			<td>Fisher Scientific</td>
			<td>V4.1</td>
			<td>LC-MS/MS analysis software</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma</td>
			<td>L8912</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4564</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850340</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850704</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylglycerol</td>
			<td>Avanti Polar Lipids</td>
			<td>840446</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylinositol</td>
			<td>Avanti Polar Lipids</td>
			<td>LM1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylserine</td>
			<td>Avanti Polar Lipids</td>
			<td>840028</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse-phase column CSH C18</td>
			<td>Waters</td>
			<td>186006102</td>
			<td></td>
		</tr>
		<tr>
			<td>Sphingosine</td>
			<td>Avanti Polar Lipids</td>
			<td>860669</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Orbitrap Velos MS</td>
			<td>Fisher Scientific</td>
			<td>ETD-10600</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricylglycerol</td>
			<td>Larodan, Malmo</td>
			<td>TAG Mixed FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic sonicator</td>
			<td>Fisher Scientific</td>
			<td>15337416</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without amino acids</td>
			<td>Fisher Scientific</td>
			<td>DF0919-15-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast strain BY4742</td>
			<td>Dharmacon</td>
			<td>YSC1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative analysis of the cellular lipidome of saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry

脂质是结构上多样化的两栖分子，不溶于水。脂质是生物膜、能量储存和生产、细胞信号、蛋白质的血管传输、细胞生成和调节细胞死亡的组织和功能的基本贡献者。由于萌芽的酵母糖精，是一种单细胞真核二代，可接受彻底的分子分析，它作为模型有机体的使用有助于揭示脂质代谢和细胞内迁移与真核细胞中复杂的生物过程之间的联系。提供多种分析方法，对酵母细胞内的主要类脂质进行可靠、敏感和准确的定量评估，对于深入了解这些机制至关重要。在这里，我们提出了一个协议，使用液体色谱结合串联质谱（LC-MS/MS）定量分析S.cerevisiae的主要细胞脂质。所述 LC-MS/MS 方法用途广泛且坚固。它能够识别和定量10种脂质中每个物种（包括不同的等体或异质体形式）。该方法很灵敏，允许一些脂质物种在浓度低至0.2pmol/μL的浓度下进行识别和定量。该方法已成功应用于评估全酵母细胞及其纯化细胞体的脂质。该方法采用替代移动相添加剂进行电镀电质谱，可提高部分脂质品种的电理化效率，从而改善其识别和定量。

在LC-MS/MS的帮助下，我们定量评估酵母细胞内主要细胞脂质的方法是多功能和稳健的。它使我们能够识别和量化10个不同的脂质类在S.cerevisae细胞培养在合成最小YNB培养最初含有2%葡萄糖。这些脂质类包括自由（未酯）脂肪酸（FFA）、CL、植物酰胺（PHC）、植物蛋白酶（PHS）、PC、PE、PG、PI、PS和TAG（补充表1）。使用这种LC-MS/MS方法（补充表1），</stro...

Watch this Scientific Journal Video about Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry at JoVE.com

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

我们提出一个协议，使用液体色谱结合串联质谱法识别和量化主要细胞脂在糖体质酶。所述的酵母细胞内主要脂质类定量评估的方法用途广泛、健壮且敏感。

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

该协议提供了一种准确且可重复的方法，用于使用单个提取方法和单个LC-MS方法量化10种不同的糖精类。这种方法允许检测和定量具有不同化学和物理特性的异构血脂物种。对于糖精培养，首先在含有无菌YNB培养的两个Erlenmeyer烧瓶中加入5毫升无菌20%葡萄糖库存溶液，最终浓度为2%葡萄糖。然后，使用无菌移液器将含有五倍十至第七酵母细胞的隔夜酵母培养的体积转移到每个烧瓶中，并在30摄氏度下将烧瓶培养至少24小时，每分钟旋转200圈。对于脂质提取，确定每毫升培养酵母细胞的总数，将五倍十到第七细胞转移到15ml离心管中。加入冰冷的纳米纯水，使总体积达到15毫升，通过离心收集细胞。将酵母颗粒重新悬浮在15ml冰冷异酸铵碳酸氢盐缓冲液中，进行第二次离心，并在1.5ml新鲜冰冷碳酸氢铵溶液中重新悬浮该颗粒。在离心结束时，将颗粒储存在 80 摄氏度，直到提取。要开始脂质提取，先将冰上的细胞颗粒解冻，然后再将200微升冰纳米纯水中的细胞重新悬浮。在烟气罩中，将电池悬浮液转移到15ml高强度玻璃、螺杆顶部离心管中，带聚四氟乙烯衬里盖，并加入25微升的内脂标准混合物，以2：1氯仿甲醇制成。100微升425至600微摩尔酸洗玻璃珠和600微升17：1氯仿甲醇到管。在室温下高速旋转管5分钟，以破坏细胞。随后在室温下低速旋转一小时，以方便抽脂。在提取结束时，在冰上孵育样品15分钟，促进蛋白质沉淀和水与有机相的分离。在孵化结束时，将样品离心，将上水相与下部有机相进一步分离，并在烟气罩中，使用硅酸盐玻璃移液器将下部有机相转移到新的 15 ml 高强度玻璃螺杆顶部离心管中，带聚四氟乙烯衬里盖。将下部有机相放在氮气的流动下，并在烟气罩中，将 300 微升的 2：1 氯仿甲醇混合物加入到剩余的上水相中。为了方便磷脂酰胺、磷脂酰胺素、磷脂酰药物和心脂萃取，在室温下大力抽吸管五分钟，然后离心样品。使用硅酸盐玻璃移液器将较低的有机相转移到氮气下的下有机相中，并在烟气罩中的组合有机相中蒸发溶剂。然后，在氮气流下关闭脂膜管，将样品储存在80摄氏度。对于液相色谱，在500微升的63：35：5乙酰硝酸盐-2-丙烷醇纳米纯水混合物中分离至脂质膜管和涡流三次，每个涡流在室温下10秒。将管内内容物接受超声波声波15分钟，然后对管进行三次涡流，每个涡流10秒，正如刚才所证明的。将样品的 100 微升转移到玻璃瓶中，并采用用于 24 井板的刀片，并消除刀片内的任何气泡。将刀片放入 24 井板的一个井中，并将反向相位 C18 柱 CSH 加载到液相色谱系统中的预列中。将柱温度设置为 55 摄氏度，将流量设置为每分钟 300 微升。设置所有参数后，在加载第一个样本之前加载 10 微升的提取空白。然后，使用液相色谱梯度分离不同的脂质物种，如所示。将生成从细胞中提取的脂质的 LC-MS 数据中具有代表性的总离子色谱。对于提取的脂质的质谱分析，将样品加载到装有加热电喷电离源的质谱仪上，并设置表中概述的分析参数。使用 Fourier 变换分析仪以 60，000 的分辨率和 150-2000 道尔顿的质量范围检测 MS-1 父离子。然后，使用表中所示的设置检测 MS-2 二次离子。要识别和量化来自广泛串联LC-MS文件的不同脂质，请搜索包含完整扫描MS-1数据和数据依赖性MS-2数据的LC-MS原始文件，以获得免费的、未受附的脂肪酸， 心脂、植物素、植物素、磷脂酰胆碱、磷脂酰胺、磷脂酰胺、磷脂酰利酮醇、磷脂酰胺、磷脂酰胺和三叶草脂类，使用质量除以前体离子的电荷耐受性百万分之五和产品离子的十分之一。然后，按照软件用户手册中提供的说明，识别具有异常脂肪酸成分的内部脂质标准和脂质物种。这种敏感的串联LC-MS方法能够识别和定量浓度低至0.165皮摩尔/微升的脂质分子物种。这种定量限值不同于不同浓度范围内的不同脂质类别。该方法可用于利用电喷电离质谱法的替代移动相添加剂提高脂质电离效率。这些替代移动相添加剂可用于正常相位和反向相位 LC 柱。碰撞诱导分离法与质谱电喷电离负模结合使用醋酸铵移动相添加剂，有利于提高MS-1脂离子分离效率，形成MS-2产品。相比之下，高能量、碰撞分离法与用于质谱的电喷电离正模式的铵 formate 移动相添加剂相结合，有利于在这些条件下提高MS-1脂离子碎片对MS-2产品的效率。收集有机相时不要混合相。在氮气流下关闭管。消除刀片内的任何气泡，并将柱以正确的方向连接。如果任何峰值的标识不能由LC-MS确定，MS-NMR可用于推断该分子的结构。

Research

JoVE Journal

Biochemistry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

A Tandem Liquid ChromatographyÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

细胞外基质的Glycoproteomics：使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...