使用DESI-MSI鉴定中药空间代谢组中代谢物的可视化
1Innovative Institute of Chinese Medicine and Pharmacy,Chengdu University of Traditional Chinese Medicine, 2Tianfu Chinese Medicine Innovation Harbour,Chengdu University of Traditional Chinese Medicine, 3School of Medical Technology,Chengdu University of Traditional Chinese Medicine, 4China Resources Sanjiu (Ya’an) Pharmaceutical Co., Ltd.
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 样品制备 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。 将另一个显微镜载玻片放在样品上方，并用…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2-Propanol | Fisher | CAS:67-63-0 | HPLC grade |
| Acetonitrile | Sigma-aldrich | Number-75-05-8 | LC-MS grade |
| Adhesion Microscope slides | Citotest scientific | 80312-3161 | Microscope glass slides can adhere to the sample |
| Air cooled dry vacuum pump | EYELA | FDU-2110 | Air-vaccum equipment at -80°C |
| Formic Acid | ACS | F1089 | 64-18-6 | LC-MS grade |
| LE (Leucine Enkephalin) | Waters | 186006013-1 | LC-MS grade |
| Methanol | Sigma-aldrich | Number-67-56-1 | LC-MS grade |
| Parafilm | Bemis Company | sc-200288 | Laboratory Sealing Film |
| Paraformaldehyde | Sigma-aldrich | V900894 | Reagent grade |
| Q-Tof Mass Spectrometer with DESI source | Waters | Synapt XS |
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Cite This Article
Xu, B., Chen, L., Lv, F., Pan, Y., Fu, X., Pei, Z. Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI. J. Vis. Exp. (190), e64912, doi:10.3791/64912 (2022).