Name: maldi-mass spectrometric imaging for the investigation of metabolites in medicago truncatula root nodules
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Description: Watch this Scientific Journal Video about MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules at JoVE.com

作者要感谢让-米歇尔·ANE博士农艺学系威斯康星大学麦迪逊分校提供蒺藜苜蓿样品。这项工作是由美国国家科学基金会（NSF）提供资金支持部分补助CHE-0957784，威斯康星大学的研究生院和威斯康星校友研究基金会（WARF）和Romnes学院研究奖学金计划（以当地雇员）。 EG承认了美国国家科学基金会研究生研究奖学金（DGE-1256259）。

1。仪器仪表<ol><li> MALDI-TOF/TOF微星。使用质谱仪配备了MALDI源小分子的分析（见表材料/设备）。在正或负离子模式取决于目标分析物进行收购。指定所关注的质量范围内，并在50微米的间隔在两个跨样本生成离子图像的表面的x和y维度收集500次激光发射/斑点。激光照射的光栅宽度和数量可以调节，分别得到更高的空间分辨率和最大信号强度。使用DHB基质峰或施加于滑动或直接向组织校准质...

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如上所述，样品的准备是在MSI的工作流程中最关键的步骤。不均匀地嵌入组织将导致切片是在某些情况下难于或不可能的。截面尺寸和足够的平衡时间是至关重要的维持组织的完整性，避免折叠和泪水。选择矩阵和应用技术将在确定待检测的分析物的种类的作用，空间分辨率，其结果的再现性。使用矩阵或应用技术的组合可以提供互补的结果。 这个方法是专门为内源性代谢?...

代谢组学领域的发展具有许多重要的生物学应用，包括生物标志物的发现，破译代谢途径在植物和其他生物系统，和毒理学分析4,6-10。研究生物系统时，一个主要的技术挑战是研究代谢途径，而不破坏它们11。 MALDI-MSI允许完整的组织的直接分析，使敏感的检测分析物的单一器官12,13，甚至单个细胞14,15。 样品制备是生产重现性好，质量可靠的光谱图像的关键一步。的图像的质量极大地依赖于因素，例如组织包埋剂，切片厚度，MALDI基质和基质的应用技术。用于成像应用中，理想的部分的厚度是1小区（8-20微米取决于样品类型）的宽度。 MALDI需要有机，晶体沉积Ë基复合材料，通常是弱酸，对样本，以协助分析消融和电离，16个不同的矩阵提供不同的信号强度，干扰离子，以及不同类别的化合物的离子化效率。 矩阵应用技术也起着质谱图像和不同的技术质量的作用适用于不同类的分析物。三个矩阵应用方法载于本协议：喷枪，自动喷雾器，和升华。喷枪矩阵应用程序已被广泛应用于MALDI成像。喷枪矩阵应用的优点在于它是相对快速和容易的。然而，喷枪矩阵应用的质量在很大程度上取决于用户的技能，而且往往是较少重现性，并导致被分析物的扩散，特别是小分子17。自动喷雾系统也有类似的机制，以喷枪矩阵的应用，但甲肝Ë已经发展到去除看到与手动喷枪应用程序的多变性，使得喷雾更具有可重复性。这种方法有时会耗费更多的时间比传统喷枪矩阵的应用。手动喷枪，自动喷雾系统是溶剂型矩阵的应用方法。升华是，正成为越来越流行的代谢物和小分子的质谱成像，因为它减少分析物扩散的干基质的应用技术，但它缺乏必要的溶剂提取，并观察更高质量的化合物18。 自信鉴定代谢物通常需要精确质量测量，以获得公认的鉴定其次是串联质谱（MS / MS）的实验进行验证，与MS / MS谱图相比，目前的标准，文学，或理论光谱。在这个协议中高分辨率（质量分辨60,000 m / z为400的电源），液相色谱（LC）-MS连接到MALDI-MSI同时获得空间信息和内源性代谢物鉴定信心，用苜蓿根系和根瘤的生物系统。 MS / MS实验，可直接执行的组织与MALDI-MSI或组织提取物的LC-MS和用于代谢物的鉴定的验证。 该协议提供了一种简单的方法来映射内源性代谢物M。苜蓿 ，这可适应和应用于在各种组织类型和生物系统的小分子的MSI。

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			<td height="21" style="height:21px;width:316px;">Gelatin</td>
			<td style="width:181px;">Difco</td>
			<td style="width:163px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=214340">214340</a></td>
			<td style="width:611px;">heat to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat- HM 550</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:163px;">956564A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">indium tin oxide (ITO)-coated glass slides&#160;</td>
			<td>Delta Technologies</td>
			<td>CB-90IN-S107</td>
			<td>25-75-0.8 mm (width-length-thickness)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,5-Dihydroxybenzoic acid (DHB) matrix&#160;</td>
			<td>ICN Biomedicals</td>
			<td><a href="http://www.labsource.com/Catalog/Item.aspx?ItemID=1787195">PI90033</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Airbrush</td>
			<td>Paasche Airbrush Company</td>
			<td>TG-100D</td>
			<td>coupled with 75 ml steel container</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Automatic matrix sprayer system- TM-Sprayer</td>
			<td>HTX Technologies, LLC</td>
			<td style="width:163px;">HTX.TMSP.H021-U</td>
			<td>Specific start-up and shut-down instructions will be given when the instrument is installed</td>
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			<td height="21" style="height:21px;">Sublimation apparatus&#160;</td>
			<td>Chemglass Life Science</td>
			<td style="width:163px;"><a href="http://www.chemglass.com/product_view.asp?pnr=CG-3038">CG-3038-01</a></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vaccum pump- Alcatel 2008 A</td>
			<td>Ideal Vacuum Products</td>
			<td>P10976</td>
			<td>Ultimate Pressure = 1x10-4 Torr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultrafleXtreme MALDI-TOF/TOF</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td style="width:163px;">276601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">FlexImaging</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td>269841</td>
			<td>One example of &#34;vender specific software&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">MALDI LTQ Orbitrap</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMASZ</td>
			<td>High resolution MALDI instrument for accurate mass measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Q Exactive</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMAZR</td>
			<td>High resolution LC-MS instrument for accurate mass measurements</td>
		</tr>
	</tbody>
</table>

maldi-mass spectrometric imaging for the investigation of metabolites in medicago truncatula root nodules

用于研究小分子，如药物的药物或内源性代谢物，采用组织提取物需要所关注的组织的均匀化，可能潜在地会改变正在研究1中的代谢途径多数技术。质谱成像（MSI）是一种可以生物组织样品1-5的完整片内提供分析物的空间信息的强大的分析工具。这种技术已被广泛用于研究各种类型的化合物，包括蛋白，肽，脂质和小分子如内源性代谢物。用基质辅助激光解吸/电离（MALDI）的MSI，多个代谢物的空间分布，可以同时检测到。在此，特别为豆科植物的根和根瘤中进行非靶向代谢物组的MSI实验开发了一种方法，提出这可能揭示见解发生的生物过程。该方法，这里介绍显示了一个典型的MSI的工作流程，从样品制备到图像采集，并集中于矩阵的应用步骤，表明是用于检测小分子的有用的几种基质应用技术。一旦生成了多光谱图像，感兴趣代谢物的分析鉴定，讨论和论证。标准工作流程这里介绍可以很容易地修改为不同的组织类型，分子种类，和仪器仪表。

MSI的实验概述示于图1。在实验的开始，样品制备是关键的一步。结节是从植物根修剪和嵌入在明胶。该组织必须被压平的低温恒温器茶杯中，用无气泡，而它被冻结，这将确保组织更容易和正确对齐，而它被切开。当组织已经被切片，重要的是要切断的组织在适当的厚度;太薄部分撕裂，搞坏了组织的完整性，而过厚的部分会降低分析物提取的，并从组织检测到的数量。选择矩阵和?...

Watch this Scientific Journal Video about MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules at JoVE.com

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

质谱成像（MSI）是一种强大的工具，可用于在完整组织中发现和鉴定各化学物种，保护化合物中的其天然环境中，它可以提供新的见解的生物过程。用于本发明的小分子的分析开发出一种MSI方法进行说明。

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Research

JoVE Journal

Biology

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

复合植物代苜蓿truncatula</em用于检测结瘤

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

科研

生物学

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Purification of Transcripts and Metabolites from Drosophila Heads

Purification of Transcripts and Metabolites from Drosophila Heads

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

MRM 微可可性能校准和使用在 22 T 的 Medicago 特伦卡图拉根上演示

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

使用 MALDI 质谱成像进行代谢分析的示例准备

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...