Name: sample preparation for rapid lipid analysis in drosophila brain using matrix-assisted laser desorption/ionization mass spectrometry imaging
Uploaded: 2022-07-14T14:00:00
Description: Watch this Scientific Journal Video about Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging at JoVE.com

陈由纪、凯莉·维拉萨米和玛雅·海因得到了斯隆基金会纽约市立大学暑期研究计划（CSURP）的支持。尹俊仁得到了美国国立卫生研究院项目编号1ZIANS003137的校内研究项目的支持。该项目由PSC-CUNY奖提供给叶赫和里纳特·阿布扎利莫夫，由专业人员大会和纽约市立大学共同资助。

1. 飞头嵌入
注意：整个过程大约需要45-60分钟。
<ol><li>准备具有平坦表面的最佳切削温度化合物（OCT化合物）载物台。<ol><li>将OCT加入塑料冷冻模具（15 mm x 15 mm x 5 mm）至冷冻模型深度的一半，避免形成气泡。将模具放在平坦的表面上几分钟，然后将其转移到干冰上。</li>
		<li>将冷冻模具平放在干冰上，让OCT形成平坦均匀的表面。等到OCT在模具中完全凝固。立即?...

<ol>
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正如对突变型和野生型果蝇大脑中脂质组成变化的研究所证明的那样，MALDI MSI可以成为一种有价值的无标记成像技术，用于原位分析小昆虫器官内的分子分布模式。事实上，由于脂质分布在果蝇头的脑组织和脂肪体中，因此当使用全头提取时，基于液相色谱和质谱（LC-MS）的传统脂质组学方法只能检测来自两个区域的组合信号。从广义上讲，对于LC-MS方法18，?...

脂质参与广泛的生物过程，根据其结构多样性可大致分为五类：脂肪酸，三酰基甘油（TAGs），磷脂，甾醇脂质和鞘脂1。脂质的基本功能是为生物过程（即TAG）提供能量来源并形成细胞膜（即磷脂和胆固醇）。然而，脂质在发育和疾病中的其他作用已被注意到，并且在生物医学领域已被广泛研究。例如，报告显示，不同长度的脂肪酸可能具有独特的治疗作用。短脂肪酸链可以参与针对自身免疫性疾病的防御机制，中等长度的脂肪酸链产生可以减轻癫痫发作的代谢物，长脂肪酸链产生可用于治疗代谢紊乱的代谢物2。在神经系统中，神经胶质细胞衍生的胆固醇和磷脂已被证明对突触发生至关重要3，4。其他类型的脂质在医疗应用中显示出前景，包括用于药物输送系统的鞘脂和用于支持免疫系统的糖脂5，6。脂质在生物医学领域的众多作用和潜在的治疗应用使得脂质组学-细胞脂质的通路和相互作用的研究-成为一个关键且日益重要的领域。
脂质组学利用分析化学来大规模研究脂质组。脂质组学中使用的主要实验方法基于质谱（MS）结合各种色谱和离子淌度技术7，8。MS在该领域的使用是有利的，因为它具有高特异性和敏感性，获取速度以及（1）检测即使在低水平和瞬时水平下也发生的脂质和脂质代谢物的独特能力，（2）在单个实验中检测数百种不同的脂质化合物，（3）识别以前未知的脂质，以及（4）区分脂质异构体。在MS的发展中，包括解吸电喷雾电离（DESI），MALDI和二次离子质谱（SIMS），MALDI MSI已成为一种强大的成像技术，通过提供有关组织隔室内脂质空间分布的独特信息来补充传统的基于MS的方法9，10。
脂质组学的典型工作流程包括样品制备、使用质谱技术进行数据采集和数据分析11.对样品中脂质和代谢物的研究导致了了解生物体代谢过程的生理和病理状况的技术的出现。虽然了解生物相互作用很重要，但脂质和代谢物的敏感性使它们难以在没有染料或其他修饰的情况下成像和鉴定。代谢物水平或分布的变化可能导致表型变化。用于代谢组学分析的一种工具是MALDI MSI，这是一种无标记 的原位 成像技术，能够同时检测数百个分子。MALDI成像允许样品中代谢物和脂质的可视化，同时保持其完整性和空间分布。以前的脂质分析技术涉及使用放射性化学物质单独绘制脂质图，而MALDI成像则放弃了这一点，并允许同时检测一系列脂质。
脂质代谢和体内平衡在细胞生理学中起着重要作用，例如神经系统的维持和发展。神经系统脂质代谢的一个重要方面是神经元和神经胶质细胞之间的脂质穿梭，其由分子载体脂蛋白介导，包括极低密度脂蛋白（VLDL），低密度脂蛋白（LDL）和高密度脂蛋白（HDL）12。脂蛋白含有载脂蛋白（Apo），如ApoB和ApoD，其功能是脂质货物的结构块和脂蛋白受体的配体。脂质的神经元 - 神经胶质细胞串扰涉及多个参与者，例如神经胶质细胞衍生的ApoD，ApoE和ApoJ，以及它们的神经元LDL受体（LDLRs）13，14。在 果蝇中，ApoB家族的成员阿波利波弗林是主要的血红素脂质载体15。载脂蛋白具有两个密切相关的脂蛋白受体（LpR），LpR1和LpR2，它们是哺乳动物LDLR15，16的同源物。在以前的研究中，发现星形胶质细胞分泌的脂质细胞分泌的胶质拉扎里洛（GLaz）是人ApoD的 果蝇 同源物，其神经元受体LpR1可以合作介导神经元 - 胶质细胞脂质穿梭，从而调节树突形态发生17。因此，据推测，LpR1的丢失会导致 果蝇 大脑中整体脂质含量的降低。MALDI MSI将是分析 LpR1−/− 突变型果蝇脑和野生型 果蝇 脑小组织中脂质含量的合适工具，如本研究所示。
尽管MALDI MSI越来越受欢迎，但该仪器的高成本和实验复杂性经常阻碍其在单个实验室中的实施。因此，大多数MALDI MSI研究都是使用共享的核心设施进行的。与 MALDI MSI 的其他应用一样，仔细的脂质组学样品制备过程对于获得可靠的结果至关重要。然而，由于样品载玻片制备通常在单个研究实验室进行，因此MALDI MSI采集可能存在差异。为了解决这个问题，本文旨在为MALDI MSI测量之前的小生物样品的样品制备提供详细的方案，例如11，17，17，使用对一大群成年果蝇脑进行脂质分析。然而，一些磷脂类和大多数小代谢物在负离子模式下通过MALDI成像被有利地检测到，这在前面11中已经描述过。因此，通过这两个示例研究，我们希望提供各种组合的详细样品制备方案：独立式大组织与包埋的小组织，解冻安装与暖玻片安装，以及正离子模式与负离子模式。

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	<tbody>
		<tr>
			<td>2,5-Dihydroxybenzoic acid (DHB)</td>
			<td>Millipore Sigma Aldrich</td>
			<td>85707-1G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Andwin Scientific&#160;CRYOMOLD 15X15X5</td>
			<td>Fisher Scientific</td>
			<td>NC9464347</td>
			<td></td>
		</tr>
		<tr>
			<td>Andwin Scientific&#160;Tissue-Tek CRYO-OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Artist brush MSC #5 1/8 X 9/16 TRIM RED SABLE</td>
			<td>Fisher Scientific</td>
			<td>50-111-2302</td>
			<td></td>
		</tr>
		<tr>
			<td>autoflex speed MALDI-TOF MS system</td>
			<td>Bruker Daltonics Inc</td>
			<td></td>
			<td>MALDI-TOF MS instrument</td>
		</tr>
		<tr>
			<td>BD&#160;Syringe with Luer-Lok Tips</td>
			<td>Fisher Scientific</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Vacutainer General Use Syringe Needles</td>
			<td>Fisher Scientific</td>
			<td>23-021-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Daltonics&#160;GLASS SLIDES MALDI IMAGNG</td>
			<td>Fisher Scientific</td>
			<td>NC0380464</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, with indicator, 8 mesh, ACROS Organics</td>
			<td></td>
			<td>AC219095000</td>
			<td></td>
		</tr>
		<tr>
			<td>Epson Perfection V600 Photo Scanner</td>
			<td>Amazon</td>
			<td>Perfection V600</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;5-Place Slide Mailer</td>
			<td>Fisher Scientific</td>
			<td>HS15986</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Digital Auto-Range Multimeter</td>
			<td>Fisher Scientific</td>
			<td>01-241-1</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexImaging v3.0</td>
			<td>Bruker Daltonics Inc</td>
			<td></td>
			<td>Bruker MS imaging analysis software</td>
		</tr>
		<tr>
			<td>HPLC Grade Methanol</td>
			<td>Fisher Scientific</td>
			<td>MMX04751</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade Water</td>
			<td>Fisher Scientific</td>
			<td>W5-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HTX M5 Sprayer</td>
			<td>HTX Technologies, LLC</td>
			<td></td>
			<td>Automatic heated matrix sprayer</td>
		</tr>
		<tr>
			<td>Kimberly-Clark Professional&#160;Kimtech Science Kimwipes Delicate Task Wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>MSC&#160;Ziploc Freezer Bag</td>
			<td>Fisher Scientific</td>
			<td>50-111-3769</td>
			<td></td>
		</tr>
		<tr>
			<td>SCiLS Lab (2015b)</td>
			<td>SCiLS Lab</td>
			<td></td>
			<td>Advanced MALDI MSI data analysis software</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;CryoStar NX50 Cryostat</td>
			<td>Fisher Thermo Scientific</td>
			<td>95-713-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Nalgene Transparent Polycarbonate Classic Design Desiccator</td>
			<td>Fisher Scientific</td>
			<td>08-642-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation for rapid lipid analysis in drosophila brain using matrix-assisted laser desorption/ionization mass spectrometry imaging

脂质分析或脂质组学是一种成熟的技术，用于研究细胞或组织的整个脂质含量。从脂质组学获得的信息对于研究参与发育，疾病和细胞代谢的途径很有价值。许多工具和仪器都有助于脂质组学项目，最值得注意的是质谱和液相色谱技术的各种组合。基质辅助激光解吸/电离质谱成像（MALDI MSI）最近已成为一种强大的成像技术，可以补充传统方法。这种新技术提供了关于组织区室内脂质空间分布的独特信息，这在以前是没有使用过度修饰的情况下无法实现的。MALDI MSI方法的样品制备至关重要，因此是本文的重点。本文对嵌入在最佳切割温度化合物（OCT）中的大量 果蝇 脑进行快速脂质分析，为通过MALDI MSI制备脂质分析或代谢物和小分子分析的小组织提供了详细的方案。

假设星形胶质细胞分泌的脂质细胞分泌的脂质体Lazarillo（GLaz）的神经元受体LpR1的丢失是人类ApoD的 果蝇 同源物，能够导致 果蝇 大脑中总脂质含量的降低。为了测试这一点，MALDI MSI用于分析 LpR1−/− 突变型 果蝇 大脑中的脂质，这将在下面详细阐述。
实验根据 图1所示的工作流程进行。如上所述收获了成年苍蝇的大脑...

Watch this Scientific Journal Video about Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging at JoVE.com

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

该协议的目的是使用基质辅助激光解吸/电离（MALDI）质谱成像，为小组织（如 果蝇 脑）中的脂质和代谢物分析提供适当的样品制备的详细指导。

使用基质辅助激光解吸/电离质谱成像在 果蝇 脑中快速脂质分析的样品制备

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired ...

Maldi MS光谱成像是一种成熟且有用的工具，无需过度的样品修饰即可识别和可视化脂质丰度和分布。Maldi成像的主要优点是能够在不标记的情况下原位检测脂质，并同时分析大量样品。熟能生巧。在处理真正的实验样品之前，请确保您练习得很好。最大限度地减少样品制备时间，并避免在整个过程中受到污染。首先，将最佳切割温度或OCT添加到塑料冷冻机中，使其达到冷冻模具深度的一半，同时避免形成气泡。将模具放在平坦的表面上几分钟，然后将其转移到干冰上。将冷冻机平放在干冰上，让OCT形成平坦而平坦的表面。等到OCT在模具中完全凝固。立即使用冷冻的OCT载物台，或将其存放在零下80摄氏度。使用二氧化碳麻醉成年苍蝇后，准备含有一块实验室湿巾的培养皿。用水润湿部分湿巾，减少静电。保持湿巾半湿半干。在解剖范围一下切割苍蝇头后，收集四到五个头并将它们放在实验室擦拭布的干燥区域。将OCT载物台从干冰到显微镜二。立即将磁头转移到OCT阶段并快速排列，这大约需要30到60秒以避免OCT熔化。在每个苍蝇大脑周围留出大约一毫米的空白空间，以确保OCT有足够的支撑，并从块的边缘留出四到五毫米的空白空间，以提供足够的空间来处理该部分。将OCT载物台放回干冰上，让它保持约三分钟，以确保OCT载物台保持冻结和固体。将所有飞头对齐后，让 OCT 载物台在干冰上再静置 5 到 10 分钟。将OCT载物台从干冰转移到平坦的表面上，然后快速加入大量OCT化合物以覆盖所有样品并填充整个冷冻模具，这大约需要三秒钟。立即将冷冻机转移回干冰中，并冷冻包含嵌入组织的整个OCT块。让 OCT 载物台在干冰上再静置 5 到 10 分钟。在冷冻机的边缘标记样品，并将冷冻样品储存在零下80摄氏度，直到准备好切片。通过使用设置为电阻的电压表测试 ITO 载玻片的电导率来确认 ITO 编码侧。用电阻测量标记一面作为粘附组织的一面。贴上标签，并始终在载玻片底部设置实验室擦拭布，以避免载玻片污染。然后，让组织在低温恒温器室中平衡 30 到 45 分钟。清洁低温恒温器，最好用70%乙醇。在舞台上擦拭辊板并取出用过的刀片。在切片开始之前，使用额外的清洁湿巾确保乙醇已蒸发并且所有表面都干燥。接下来，根据组织的类型调节低温恒温器室砂标头的温度。使用 OCT 将组织安装在标本支架上。注意使用足够的 OCT 来覆盖 OCT 块的底部，并将块安装得尽可能平坦。将干净的刀片放在载物台上并锁定。根据需要将试样的头部朝向载物台，以达到所需的切割角度。然后开始厚切片切割，直到找到感兴趣的区域。用预冷的艺术家刷不断刷掉多余的作品，以保持舞台清洁。根据需要稍微调整腔室温度，并在达到所需区域后将切片的厚度更改为 10 至 12 微米。仔细收集所需的部分。取一张室温ITO载玻片并将其放置在切片上。轻轻接近该部分并将其粘附在载玻片上，而不会在低温恒温器载物台上留下痕迹。将载玻片放在架子上，或在低温恒温器外的实验室擦拭中，在多个部分的集合之间。如果需要在同一队列的不同样品之间进行比较，请将多个样品的切片放在一张载玻片上，以便可以同时分析它们以最大程度地减少差异。如有必要，可分离为两个载玻片，因为 Maldi 靶支架可以在一次运行中容纳两个载玻片。将真空盒中的载玻片运送到以干燥剂为底层的干燥剂中。在真空下驱动载玻片30至60分钟，然后进行基质沉积。使用二五二羟基苯甲酸或DHB在甲醇水中作为基质。将载玻片放入载玻片运输器中，并用蜡膜牢固地密封开口。用拉链袋密封。将其放入另一个装有干燥剂的拉链袋中。在外部贴上标签，并将样品运送到具有足够干眼症的Maldi核心设施。对于基质沉积，使用每毫升40毫克的DHB和甲醇水作为基质，并使用自动HTX M5基质喷雾器喷洒。使用 10 PSI 的氮气压力。使用Maldi飞行时间，MS仪器和正离子模式来获取质谱。通过将 0.5 微升红磷乳液在 aceto Nitro 中点样到 ITO 载玻片上来校准仪器。使用它的光谱通过应用二次校准曲线在 100 至 1000 MZ 质量范围内校准仪器。将激光光斑直径设置为中等，因为它是 40 微米光栅宽度的调制光束轮廓，并通过以每个阵列位置 1000 赫兹的激光重复率对 500 次射击求和来收集成像数据。然后记录光谱数据。使用均方根归一化执行成像数据分析，以正负0.10道尔顿的带宽生成离子图像。使用该软件对齐来自同一实验的OCT和脑组织的光谱，以评估OCT离子抑制干扰中的重叠峰。最后，通过苏木精和伊红或H和E染色处理含有组织样品的MALDI载玻片。来自Maldi MS分析的图像显示LPR1敲除突变体脑中的脂质含量普遍降低。此处显示了具有代表性的H和E染色成年苍蝇脑切片。图中还显示了脂质种类、它们各自的 MZ 值以及热图的刻度。与至少四个生物学重复的对照相比，LPR1敲除突变体的平均减少倍数显示在箭头旁边。对照果蝇和突变果蝇脑组织区域中选定的空白OCT区域的质谱图显示在MZ1至1000和MZ520至900的范围内。OCT的干扰与离子抑制现象和重叠信号问题有关。为了保持脂质组学数据的保真度，适当的样品制备、解剖和冷冻切片至关重要。进行Maldi实验后，可以通过LCMS（也称为液相色谱，基于质谱的脂肪组学）验证脂质的特性。该技术通过使用强大的软模型系统获得了有关脑脂质稳态的分子见解，该系统为理解与人类疾病相关的大脑代谢变化铺平了道路。

Research

JoVE Journal

Biology

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

MALDI Sample Preparation: the Ultra Thin Layer Method

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法：从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Sample Preparation for Mass Cytometry Analysis

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

使用基质辅助激光解吸/电离质谱成像在 果蝇 脑中快速脂质分析的样品制备

使用基质辅助激光解吸/电离质谱成像在果蝇脑中快速脂质分析的样品制备