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

我们非常感谢阿兰特西尔宝贵的建议，讨论和技术支持。我们承认在Concordia大学生物应用质谱杰出服务中心。这项工作得到了CIHR和加拿大NSERC资助。 VIT是一个新的CIHR调查和Concordia大学基因组学研究主席，细胞生物学和老化。

材料和方法<ol><li>酵母菌株和生长条件。 丰富YEPD培养基中生长的野生型菌株BY4742（MATαhis3Δ1leu2Δ0lys2Δ0ura3Δ0）（1％酵母提取物，2％蛋白胨，2％葡萄糖） 。细胞培养在30 ° C旋转摇晃锥形瓶在200 RPM“瓶体积/中等音量”5:1的比例。 </li><li>脂质提取的酵母细胞储存 <ol><li>文化是收获的细胞50毫升离心5分钟在3000 XG。 </li><li>用冷水冲洗两次。 <ol style=" list-style-type:lower-alpha;"><li>悬浮颗?...

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	<li>Cao, H., Gerhold, K., Mayers, J. R., Wiest, M. M., Watkins, S. M., Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+lipokine,+a+lipid+hormone+linking+adipose+tissue+to+systemic+metabolism.">Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.</a> Cell. 134, 933-944 (2008).</li><li>Claypool, S. M., Oktay, Y., Boontheung, P., Loo, J. A., Koehler, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+defines+the+interactome+of+the+major+ADP/ATP+carrier+protein+of+the+mitochondrial+inner+membrane.">Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane.</a> J. Cell Biol. 182, 937-950 (2008).</li><li>Guo, T., Gregg, C., Boukh-Viner, T., Kyryakov, P., Goldberg, A., Bourque, S., Banu, F., Haile, S., Milijevic, S., San, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+signal+from+inside+the+peroxisome+initiates+its+division+by+promoting+the+remodeling+of+the+peroxisomal+membrane.">A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane.</a> J. Cell Biol. 177, 289-303 (2007).</li><li>Russell, S. J., Kahn, 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=Endocrine+regulation+of+ageing.">Endocrine regulation of ageing.</a> Nat. Rev. Mol. Cell Biol. 8, 681-691 (2007).</li><li>Czabany, T., Athenstaedt, K., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+storage+and+degradation+of+neutral+lipids+in+yeast.">Synthesis, storage and degradation of neutral lipids in yeast.</a> Biochim. Biophys. Acta. 1771, 299-309 (2007).</li><li>Br&amp;uuml, g. g. e. r., Erben, B., Sandhoff, G., Wieland, R., &amp;amp, F. T., Lehmann, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+biological+membrane+lipids+at+the+low+picomole+level+by+nano-electrospray+ionization+tandem+mass+spectroscopy.">Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectroscopy.</a> Proc. Natl. Acad. Sci. USA. 94, 2339-2344 (1997).</li><li>Schneiter, R., Br&#252;gger, B., Sandhoff, R., Zellnig, G., Leber, A., Lampl, M., Athenstaedt, K., Hrastnik, C., Eder, S., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+tandem+mass+spectrometry+(ESI-MS/MS)+analysis+of+the+lipid+molecular+species+composition+of+yeast+subcellular+membranes+reveals+acyl+chain-based+sorting/remodeling+of+distinct+molecular+species+en+route+to+the+plasma+membrane.">Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.</a> J. Cell Biol. 146, 741-754 (1999).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-917 (1959).</li></ol>

这种方法使酵母lipidome使用现成的，廉价的材料的快速定量评估。该方法允许大部分diacylglycerols，ergosterols和ergosteryl酯的例外在酵母细胞中发现的脂质物种的鉴定，虽然这些脂质可取代氢氧化锂，氢氧化铵确定。所描述的方法可低至微克/毫升的浓度脂质识别和量化，与浓度的线性超过2〜3个数量级（脂质种而定）的蔓延。虽然市售磷酸肌醇的适当的标准，本磷脂不同的分子形式可以使用其他磷脂?...

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

脂质是生物分子的主要类别之一，并发挥重要作用膜动力学，能量存储，和信令<sup&gt; 1-4</sup&gt;。芽殖酵母<em&gt;酿酒酵母</em&gt;，一种基因和生物化学来操作带注释的基因组，很简单lipidome的单细胞真核生物，是一个有价值的模型，研究在多细胞真核生物中各种脂质物种的生物功能<sup&gt; 2,3,5</sup&gt;。<em&gt; S。酵母</em与链长，主要是16或18个碳原子和零或一个不饱和度&gt; 10血脂的主要类别<sup&gt; 6,7</sup&gt;。现有脂质的鉴定和定量的方法 - 如高效液相色谱法，薄层色谱法，荧光显微镜，气相色谱仪由MS - 已经非常成熟，但灵敏度低，不够独立的各种脂类分子形式，要求前脂质derivitization分析，或可相当费时。在这里，我们提出了我们的实验方法的详细说明，以解决这些固有的局限性，所使用的整个酵母细胞中的脂类补充的识别和定量调查扫描ESI / MS。所描述的方法不需要复杂的脂质混合物从酵母细胞中提取的色谱分离，从而大大加快了数据采集的过程。这种方法能够在低克/毫升的浓度脂质识别和量化，并已成功地应用于评估整个酵母细胞及其纯化细胞器lipidomes。血脂，使用这种方法血脂分析的整个酵母细胞中提取需要两到三个小时。它只有五到十分钟，上运行的Q - TOF质谱光谱仪配备了一个纳米电源每个样本提取和干燥血脂。

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

我们描述了一个新的定量确定使用调查的扫描电喷雾电离质谱（ESI / MS）酵母大量的脂质物种lipidomics方法。这种方法超过目前可用的方法脂质识别和量化的能力来解决血脂，灵敏度和速度不同的分子形式。

一个定量评估的酵母使用电喷雾质谱法Lipidome

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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