Name: a strategy for sensitive, large scale quantitative metabolomics
Uploaded: 2014-05-27T13:45:00
Description: Watch this Scientific Journal Video about A Strategy for Sensitive, Large Scale Quantitative Metabolomics at JoVE.com

作者要感谢德特勒夫·舒曼，詹妮弗·萨顿（赛默飞世尔科技）和纳撒尼尔·斯奈德（宾夕法尼亚大学）为质量校准和数据处理有价值的讨论。研究报告本出版物中得到了卫生部下奖号码R00CA168997国家研究院​​国立癌症研究所的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1，准备LC-MS试剂，建立一个色谱法，并建立仪器操作规程<ol><li>液相色谱溶剂的制备<ol><li>制备500mL的流动相。 A为20mM的醋酸铵和15mM的氢氧化铵的3％乙腈/水，最终pH为9.0;和B为100％乙腈。 </li><li>松散帽的瓶子，把它放在水浴超声仪，超声10分钟，无需额外加热。 （此步骤是为了确保所有的铵盐的完全溶解和不存在残留气泡。） </li><li>转让250毫升溶剂的250毫升玻璃瓶中用于LC-MS的使用，并保持剩?...

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A., White, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+reaction+monitoring+for+robust+quantitative+proteomic+analysis+of+cellular+signaling+networks.">Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (14), 5860-5865 (2007).</li><li>Yuan, M., Breitkopf, S. B., Yang, X., Asara, J. 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在使用这种协议的细胞成功代谢物图谱中最关键的步骤是：1）控制所述生长培养基中并小心萃取的细胞; 2）调整液相色谱方法基于MS方法设置，以确保有足够的（通常至少为10）跨数据进行定量峰点; 3）做一个低质量运行校准样品前; 4）注入不超过5毫升到避免保留时间偏移和峰展宽引起的水; 5）准备和运行样品在同一批次，以减少批效果比较。 标准（ 表1）选择?...

代谢组学，定义为同时测量多个代谢物的试验，一直是激烈感兴趣的领域。代谢组学提供了分子生理学的一个直接读出，并提供了见解发育和疾病如癌症1-4。核磁共振（NMR）和气相色谱-质谱（GC-MS）是最常用的工具5-9之间。 NMR，尤其是已自重同位素标记的化合物，如13 C标记的代谢物用于通量的实验中，都NMR-活性10,11。然而，这种策略要求比较高的样品纯度和大样本量，这限制了它的应用代谢组学。同时，从核磁共振收集的数据密集型需求分析和复杂的NMR谱复合赋值是困难的。 GC-MS已广泛用于极性代谢物和脂质的研究，但它需要的挥发性化合物 -ds和因此往往衍生的代谢物，这有时会涉及复杂的化学反应，可以是耗时的，并引入实验噪音。 液相色谱（LC），耦合到三重四极杆质谱法使用第一个四极杆选择的完整母离子，然后将其分段中的第二四极，而第三个四极杆被用来选择特征的片段或子离子。该方法中，其中记录了从母体离子，以特定的子离子的过渡，被称为多反应监测（MRM）。 MRM是为小分子和蛋白质定量12-15,21非常敏感，特异，可靠的方法。然而，MRM也有其局限性。为了实现高特异性的MRM方法需要为每个代谢物兴建。该方法包括确定一个特定的片段和相应的最佳碰撞能量，这就要求PROPE的预先知识感兴趣的代谢物，如化学结构信息的rties。因此，与同一片段的中性丢失一些例外，它是不可能的，以确定未知代谢物与该方法。 在最近几年，高分辨率质谱（HRMS）的仪器已被释放，如LTQ-轨道阱和Exactive系列，QuanTof技术，和的TripleTOF 5600 16-18,22。 HRMS可以提供​​质量到几个ppm的误差内充完好离子的比（m / z）表示。因此，通过检测所有的前体离子（ 即全扫描模式）操作的HRMS的仪器可以从精确质量和分析物所产生的元素组成直接获得的结构信息，并且该信息可用于确定潜在的代谢物。确实，可以用一个精确质量来获得关于一种化合物的所有信息，至多结构异构体的水平。此外，全扫描方法不需要的代谢产物以前的知识，也不需要方法的优化。此外，由于m / z为落入扫描范围的所有离子进行分析，HRMS具有几乎无限的容量可量化在单次运行相比，MRM方法代谢物的数量而言。人力资源管理系统也相当于定量容量的三重四极杆MRM由于短的工作周期造成的，可以在一个完整的MS将扫描获得的数据点的数量相媲美。因此，人力资源管理系统提供了定量的代谢组学的另一种方法。近日，人力资源管理系统的改进版本称为Q-Exactive质谱（QE-MS）可以足够快的周期正，负模式在一个单一的方法，扩大了检测范围19之间的切换下操作。在这里，我们将介绍使用的QE-MS代谢组学我们的策略。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Positive calibration mix</td>
			<td style="width:117px;">Thermo Scientific</td>
			<td style="width:120px;">#88323</td>
			<td style="width:207px;">It is light sensitive. Store at 4 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Negative calibration mix</td>
			<td>Thermo Scientific</td>
			<td>#88324</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Diazinon</td>
			<td style="width:117px;">Sant Cruz Biotechnology</td>
			<td>#C0413</td>
			<td style="width:207px;">It causes eyes irritation, so work in hood. Store at 4 &#176;C</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">H-ESI needle insert</td>
			<td>Fisher Scientific</td>
			<td>#1303200</td>
			<td style="width:207px;">This could be replaced or cleaned with 5 % Formic acid/water (remove rubber ring) if clogged.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xbridge amide column</td>
			<td>Waters</td>
			<td>#186004860</td>
			<td style="width:207px;">Guard column is recommend to increase column lifetime.</td>
		</tr>
	</tbody>
</table>

a strategy for sensitive, large scale quantitative metabolomics

代谢产物分析一直是宝贵的资产在代谢在健康和疾病的研究。然而，当前的平台上有不同的限制因素，如劳动密集型样品制剂，低检测限，扫描速度很慢，对于每种代谢物密集的方法优化，以及无法积极地同时测量和在单一实验中带负电荷的离子。因此，一种新的代谢协议可以促进代谢组学研究。基于酰胺的亲水色谱法使极性代谢物的分析，而无需任何化学衍生。高分辨质谱使用Q-Exactive（QE-MS）有所改善离子光学系统，提高了扫描速度（256毫秒的分辨率70,000），并具有进行正/负切换的能力。使用冷甲醇提取策略，并与耦合QE-MS酰胺色谱柱能够稳健检测168有针对性的极性代谢物的附加功能和成千上万的同时进行。 DAT的处理是用市售的软件在一个高度有效的方法，并从质谱图中提取未知功能可以在数据库中进行查询。

代谢组数据的精度在很大程度上取决于对LC-QE-MS仪器的性能。评估状况良好仪器是否正在运行，并且应用该方法是否是正确的，几个已知代谢物的LC峰被从总离子色谱图（TIC）萃取， 如图1。极性代谢物，包括氨基酸，糖酵解中间体，三氯乙酸中间体，核苷酸，维生素，ATP，NADP +等有很好的保留在目前的LC条件下的酰胺列的列和良好的峰形。同时，大容量误差测试低质量校准后2...

Watch this Scientific Journal Video about A Strategy for Sensitive, Large Scale Quantitative Metabolomics at JoVE.com

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

代谢产物分析一直是宝贵的资产在代谢在健康和疾病的研究。利用正常阶段的液相色谱和高分辨率质谱与极性切换和快速的工作周期，我们描述了一个协议分析生物材料，具有灵敏度高，准确度和分辨率的极性代谢成分。

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