Name: visualization of metabolites identified in the spatial metabolome of traditional chinese medicine using desi-msi
Uploaded: 2022-12-16T14:50:00
Description: Watch this Scientific Journal Video about Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI at JoVE.com

这项工作得到了四川省自然科学基金（编号：2022NSFSC0171）和成都中医药大学杏林人才计划（第030058042号）的支持。

1. 样品制备
<ol><li>从2年生的 丹参 植物（图1A）收集干净的根和叶，并用手直接以约3-5毫米的横截面厚度切片。然后，使用双面胶带将样品粘附在粘附显微镜载玻片上（图1B）。 注意：确保双面胶带的尺寸大于样品。如果组织干燥，请在切片前将其浸泡在水或4%多聚甲醛中过夜。</li>
	<li>将另一个显微镜载玻片放在样品上方，并用...

<ol>
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MS技术的出现开启了近年来分子水平天然产物研究的新见解24。该质谱仪具有高灵敏度和高通量，即使在痕量浓度为25的情况下，也能对天然产物中的代谢物进行靶向和非靶向分析。因此，MS目前广泛应用于中药（TCM）化学领域。对中药化学成分进行定性和定量研究，可以提供中药成分及其相关化合物的信息，不仅为药理学研究提供了适宜的参考，而且为构建中?...

代谢物分布的可视化已成为植物科学中的一个关键课题，特别是在传统中医中，因为它揭示了植物内特定代谢物的形成过程。参考传统中药（TCM），它提供有关活性成分的信息，并指导植物部分在制药应用中的应用。通常，代谢物的可视化是通过原位杂交、荧光显微镜或免疫组织化学来实现的，但是这些实验检测到的化合物数量传达的化学信息有限。结合组织染色，质谱成像（MSI）可以通过扫描和分析微米级1的组织切片，提供大量的数据并提供化合物的空间分布信息。MSI使用分析物从样品表面解吸和电离，然后对产生的气相离子进行质量分析，并应用成像软件来整合信息并绘制记录特定离子丰度的二维图像。该技术可以通过检测靶组织和器官中药物及其诱导代谢物的特征分布来确定外源性和内源性分子2，3，4，5。
近几十年来，已经开发了各种成像MS模式;其中最突出的是解吸电喷雾电离基MSI （DESI-MSI）、基质辅助激光解吸/电离（MALDI）和二次离子质谱（SIMS）6。DESI-MSI由于其常压操作，高通量和更高的精度而通常用于生物学研究7。MALDI已被应用于鉴定转甲状腺素蛋白片段作为庆大霉素的潜在肾毒性生物标志物，并分析在小鼠大脑中管理1-甲基-4-苯基-1，2，3，6-四氢吡啶后神经毒性代谢物1-甲基-4-苯基吡啶的分布8，9。MALDI和DESI已被用于确定药物诱导的小鼠肾脏中晶体状结构的组成;这些结构主要由由于药物的去甲基化和/或氧化而形成的代谢物组成10。此外，MSI已被应用于靶器官中药物毒性代谢分布的定位。然而，植物组织中的细胞各不相同，与动物不同，需要特殊的切片程序。
在植物中，通过使用MALDI成像，到目前为止，已经分析了小麦（小麦）茎，大豆（甘氨酸max），水稻（稻（稻）种子，拟南芥花和根以及大麦（Hordeum vulgare）种子中不同化合物的分布11，12，13，14，15，16，17，18.最近的研究报告，DESI-MSI正在天然药物和产品的代谢物分析中出现，特别是在银杏叶，夫子和青蒿L19，20，21等中药中。在这些研究中，制备植物材料样品的方案不同，有些需要更复杂的设备，如冷冻切片机。DESI-MSI对检测样品的表面平整度有严格的要求。在分析动物的器官或组织时，通常通过冷冻切片22制成样品。然而，冷冻切片的程序复杂且昂贵，并且常用的粘合剂最佳切割温度（OCT）方法在成像时具有很强的信号。此外，中医的药用组织各不相同;例如，丹参的根，在中文中被称为丹参，用于药用，而在紫苏（紫苏）中，叶子被使用23，24。因此，有必要改进样品制备程序，以促进DESI在中药代谢物分析中的应用。
作为一种多年生草本植物和常用中药， 绣线菊 最初被记录在最古老的医学专著《神农本草经》（中文称为神农本草经）中。在这项研究中，我们优化了切片和DESI成像程序，并开发了一种更具成本效益的方法来鉴定S . miltiorrhiza组织中的分布和分类。这种方法还可以克服与干燥组织相关的缺点 - 它们通常在氮气吹下容易断裂 - 并促进中医的发展。本研究将促进中医/民族医学在研究相关技术方面的标准化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher</td>
			<td>CAS:67-63-0</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-aldrich</td>
			<td>Number-75-05-8</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>Adhesion Microscope slides</td>
			<td>Citotest scientific</td>
			<td>80312-3161</td>
			<td>Microscope glass slides&#160; can adhere to&#160; the sample&#160;</td>
		</tr>
		<tr>
			<td>Air cooled dry vacuum pump</td>
			<td>EYELA</td>
			<td>FDU-2110</td>
			<td>Air-vaccum equipment at -80&#176;C</td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>ACS</td>
			<td>F1089 | 64-18-6</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>LE (Leucine Enkephalin)</td>
			<td>Waters</td>
			<td>186006013-1</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-aldrich</td>
			<td>Number-67-56-1</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>Parafilm&#160;</td>
			<td>Bemis Company</td>
			<td>sc-200288</td>
			<td>Laboratory Sealing Film</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-aldrich</td>
			<td>V900894</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Q-Tof Mass Spectrometer with DESI source</td>
			<td>Waters</td>
			<td>Synapt XS</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of metabolites identified in the spatial metabolome of traditional chinese medicine using desi-msi

中药的药用主要是由于其次生代谢产物。这些代谢物分布的可视化已成为植物科学中的一个关键课题。质谱成像可以提取大量数据，并通过分析组织切片来提供有关这些数据的空间分布信息。解吸电喷雾电离质谱成像（DESI-MSI）具有高通量和高精度的优点，常用于生物学研究和中药研究。然而，本研究中使用的程序很复杂，负担不起。在这项研究中，我们优化了切片和DESI成像程序，并开发了一种更具成本效益的方法来鉴定代谢物的分布，并在植物组织中对这些化合物进行分类，特别关注中药。本研究将促进DESI在中药/民族医学代谢物分析中的应用和研究相关技术的标准化。

该协议可以导致植物样品中化合物的鉴定和分布。在特定m/z的MS图像中，每个像素的颜色代表m/z的相对强度，因此可以与整个样品中代谢物离子的自然分布和丰度相关联。收集位置的化学物质丰度越高，颜色越亮。图片中的条形图（图4A-D）显示了颜色的渐变。在这里，我们选择了两种对S. miltiorrhiza的药用有价值的化合物。如图4A-D所示，目标?...

Watch this Scientific Journal Video about Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI at JoVE.com

Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI

本研究提出了一系列从工厂制备DESI-MSI样品的方法，并详细描述了DESI组件安装、MSI数据采集和处理的过程。该协议可以应用于获取植物空间代谢组信息的几种条件。

使用DESI-MSI鉴定中药空间代谢组中代谢物的可视化

The medicinal use of traditional Chinese medicine is mainly due to its secondary metabolites. Visualization of the distribution of these metabolites has ...

本文主要介绍了一种使用DESI-MSI制备植物样品和成像的经济高效的方法，可以克服干燥组织在氮气吹入时容易断裂的缺点。根在低温恒温器切片机上冷冻切片，而叶子是通过印记制备的，这需要昂贵的机器或失去信号强度。我们的方法可以克服这些限制。该方法可用于植物的空间代谢组学，例如非生物和生物胁迫下植物中有毒物质的分布和运动。首先，从一株两年生的丹参植物中取出干净的根和叶，直接以大约三到五毫米的横截面厚度切片。然后，使用双面胶带将样品粘附在粘附显微镜载玻片上。将另一个显微镜载玻片放在样品上方，并用像三明治一样的密封膜包裹它们。接下来，将夹心样品在零下 80 摄氏度下冷冻至少四个小时，然后将其置于空气真空下两个小时，滞留温度为零下 75 至 -82 摄氏度，真空计为 2.5 至 3.7 帕斯卡。为了进行分析，从冷藏库中取出样品后，在干燥器中将它们带到室温，以避免样品表面冷凝，然后再对样品进行基质应用。在电喷雾电离或ESI模式下实施仪器检测器设置和质量校准。使用亮氨酸和头孢林在水乙腈中以1比1的比例进行检测器设置，并用甲酸钠和水异丙醇以1比1的比例进行质量校准。取出ESI源并将DESI单元安装到质谱仪上。将氮气供应连接到DESI装置，并将气体压力调节到0.5兆帕左右。在甲醇水中以 1 比 9 的比例将亮氨酸、头孢林和甲酸填充 5 毫升注射器，并将注射器连接到高性能注射泵上，为样品中化学物质的电离提供溶剂。接下来，将提供溶剂的毛细管连接到注射器和DESI喷雾器上。提供毛细管的溶剂是尺寸为75微米和外径为375微米的标准毛细管。启动注射泵并将注入速率设置为每分钟两微升，以获得恒定的流量和溶剂喷雾。关闭氮气阀，然后在大约 15 秒后打开它。一小滴溶剂吹到载物台上，如果溶剂流动处于恒定状态，则可以看到喷雾。接下来，根据喷雾角度、XYZ 轴、突起和高度调整喷雾器的位置。使用红色和黑色标记作为参考来优化质谱信号，在灵敏度模式下获得大约 1 倍 10 到 5 倍的信号强度。由于喷雾器的突出部分是影响信号强度的最重要因素，因此请使用 5 毫米扳手更换氮气防护装置来调整突起。喷涂方向会影响质量图像的质量。因此，旋转喷雾器直到喷雾笔直。完成所有这些步骤后，设置即可进行实验，并且在初始设置后通常稳定约三周的可用性。对于DESI-MS成像，不需要样品预处理。但是，如果可能，请清除载玻片上多余的介质。在载玻片上拍摄样品的图像。在不接触样品表面以避免任何杂质的情况下，将载玻片放在DESI载物台上的板位置，该载物台有两个板位置，A和B.使用标准载玻片或全载玻片以适应该位置。一张完整的载玻片最多可容纳四张载玻片，因此实验面积要大得多。打开高清海量图像处理软件。在“采集”选项卡中设置新印版，然后选择正确的印版位置、A 或 B 以及印版类型。在“图像选择”页上，选择幻灯片的四个角，然后将图像自动调整为正确的方向。要设置MS参数，请选择通常使用的DESI-MS模式，其中仅检测母离子。接下来，选择极性为正极或负极，因为仪器在实验中只能使用一个极性。然后应用灵敏度模式以获取有关少量化学品的更多信息。接下来，绘制一个矩形以定义“图案”选项卡中的扫描区域，并通过保持像素的 X 和 Y 大小相等来设置像素大小。将扫描速率设置为不超过像素大小的五倍。保存项目并导出 MS 采集软件的工作表。接下来，将工作表导入MS采集软件并将其另存为新的样品列表。按“开始运行”开始MS图像扫描，可以通过导入更多工作表将多个图像添加到实验队列中。将样品的数据文件加载到海量图像处理软件中，并设置DESI图像处理的参数。由于亮氨酸和头孢林用于内部锁定质量，并且锁定质量是识别实验极性的唯一点，因此设置正确的锁定质量至关重要。将 556.2772 设置为正模式，将 554.2620 设置为负模式。要构建目标化学品列表，请加载处理后的数据文件以可视化样品的 DESI 图像。点击归一化按钮，通过全离子色谱对数据进行归一化，得到特定化学物质与参比的相对强度，然后可以将不同的样品相互比较。绘制感兴趣区域或 ROI，并在示例图像上复制多个副本。然后选择所有ROI并导出多变量分析，以从所有ROI中提取MS信息。根部和整个叶段的质谱成像显示了所选化合物的空间分布。每个像素的颜色代表质荷比的相对强度，因此可以与整个样品中代谢物离子的自然分布和丰度相关联。目标化合物丹参酮IIA和迷迭香酸的分布在根的不同区域可见。在叶片中检测到化合物丹精醇A 作为旨在再现性的协议，最重要的是通过在多个维度上调整喷雾器的位置来优化信号强度。

Research

JoVE Journal

Medicine

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

草药的制备：洱西汤和含茜素的血清<em&gt;体内</em&gt;和<em&gt;体外</em&gt;实验

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

科研

医学

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

藏药红景天颗粒中多指标组分的 体外 溶出度测定

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...

Enema of Traditional Chinese Medicine for Patients with Severe Acute Pancreatitis

重症急性胰腺炎患者中药灌肠

Severe acute pancreatitis (SAP) is characterized by high mortality rates, numerous complications, and extreme difficulties in treatment. A traditional ...

Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

网络药理学预测及代谢组学验证 凤仙花抗高脂血症的机制

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug ...

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

使用多重光散射检查不同提取方法获得的余 甘子 L.提取物的稳定性

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact ...

Objectification of Tongue Diagnosis in Traditional Medicine, Data Analysis, and Study Application

舌诊在传统医学中的客观化、数据分析与研究应用

Tongue diagnosis is an essential technique of traditional Chinese medicine (TCM) diagnosis, and the need for objectifying tongue images through image ...

A Traditional Chinese Medicine Characteristic Therapy for Bronchial Asthma: Moxibustion

支气管哮喘的中医特色疗法：艾灸

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain (Video) | JoVE 

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Treating Knee Osteoarthritis with Tuina in a Rabbit Model

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis (Video) | JoVE

推拿干预膝骨关节炎兔模型

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...

Liujunzi Decoction: An Effective Treatment for UC-CRC Prevention

Establishment of Coloproctitis Cancer Model in Mice and Evaluation of Therapeutic Effect of Chinese Medicine

Author Spotlight: Liujunzi Decoction as a Traditional Chinese Treatment for Coloproctitis Cancer

Colorectal cancer (CRC) is a common malignancy of the digestive system and has become the third most common malignancy worldwide and the second leading ...