用于 MALDI 成像质谱法检测和可视化大鼠脑组织中神经节苷脂的 DAN 基质升华法

摘要

本文介绍了一种在大鼠脑组织上升华DAN基质以通过基质辅助激光解吸电离成像质谱法检测神经节苷脂的实验方案。

摘要

在基质辅助激光解吸/电离成像质谱（MALDI成像质谱，IMS）实验中，样品制备是实现分析物最佳检测与可视化效果的关键。在整个样品制备过程中，确定合适的实验方案可能具有挑战性，因为每一步操作都必须根据目标分析物的独特性质进行优化。该过程不仅需要选择一种能够高效解吸并电离目标分子的相容性基质，还需确定合适的基质沉积技术。例如，湿法基质沉积技术需将基质溶解于溶剂中，适用于大多数蛋白质和肽类的解吸；而干法基质沉积技术则对脂质的电离尤为有效。已有文献报道，升华法作为一种干法基质沉积方法，在MALDI IMS检测组织中脂质时表现出高效率，其优势在于基质晶体沉积均匀，且与多种湿法沉积方法相比，分析物的位移程度最小^1，2。该方法通常包括：将样品与粉末状基质置于真空密封的腔室内，样品紧贴冷表面；随后将装置浸入加热浴（如沙浴或油浴）中，使粉末基质升华并沉积于冷却的组织样品表面。本文描述了一种采用1,5-二氨基萘（DAN）基质，结合MALDI成像质谱技术对大鼠脑组织中神经节苷脂进行检测与可视化的升华沉积实验方案。

引言

基质辅助激光解吸/电离（MALDI）成像质谱（IMS）正成为一种备受青睐的技术，可用于可视化完整样品表面脂质、肽和蛋白质的空间分布。MALDI IMS 以往主要用于预纯化分析物的分析，但近年来，由于其能够将质谱技术的精确性与高分辨率的视觉/解剖学参考点相结合，且无需任何外部标记，因而受到多个学科领域的广泛关注。随着使用该技术的科研人员群体不断扩大，迫切需要标准化且易于遵循的操作流程，以协助开展和优化 IMS 实验。神经节苷脂是一类在中枢神经系统中高度富集的膜脂，非常适合用于 MALDI IMS 实验，因为它们嵌入在膜结构中的位置使得某些种类无法通过传统的免疫标记方法检测到。此外，我们已通过 MALDI IMS 证明，这些脂质除了具有调控细胞信号传导等多种功能外，在健康啮齿类动物脑组织中具有独特的解剖分布模式，而在脑损伤后该分布模式会发生改变^3,4,5。与其他大多数脂质种类相比，神经节苷脂位于较高的质荷比范围，因此特别适用于 MALDI 成像平台。

图 1：MALDI 成像质谱（IMS）实验的工作流程。 使用升华法进行 MALDI 成像质谱实验的一般流程示意图。在 -80 °C 下冷冻的组织于冷冻切片机中切片，获得 10 µm 厚的切片，并将切片贴附于导电性 ITO 载玻片上。随后将载玻片置于干燥器中直至进行升华处理。将载玻片插入升华装置，在组织样本表面均匀涂布基质层。样品在 -20 °C 冰箱中冷冻过夜，然后置于干燥器中 10 分钟。在施加标准品后，将样品放入 MALDI 仪器中，激光扫描组织表面，使基质中的分子解吸并离子化。离子沿飞行管行进，根据其质量（飞行时间/TOF）分离，最终到达检测器。在预设的质荷比（m/z）范围内，分析物离子丰度的信息以分子图像和质谱图两种形式呈现。该数据可用于对成像组织中目标分析物的离子丰度进行可视化和定量分析。 请点击此处查看该图的放大版本。

基质辅助激光解吸电离成像质谱（MALDI IMS）的样品制备具有高度可变性，因为该过程的每一步都必须根据目标分析物进行定制。基于MALDI的实验的决定性特征是在分析前将基质涂层沉积到样品表面。除了在消融过程中吸收并传递激光辐射能量外，基质还起到将样品中各种分析物分离的作用，从而促进目标化合物的分析^6,7。将基质均匀地涂布于样品表面是样品制备过程中最关键的步骤。基质沉积不当可能导致形成大而不均一的基质晶体，产生伪影、离子信号弱以及重复性差等问题⁷。

由于某些基质对特定分析物具有亲和性，实验中所选用的基质类型可能显著影响实验结果。用于蛋白质和肽类成像的基质通常不同于用于脂质成像的基质，而为了成功检测组织中的信号，还需进行洗涤和再水化等额外步骤，这进一步增加了实验的复杂性。尽管存在旨在增强脂质信号的洗涤步骤⁸，但这些步骤并非检测大多数脂质种类的必要前提。在为脂质成像实验选择基质时，必须考虑目标脂质的极性，因为这将缩小合适基质的选择范围。例如，神经节苷脂含有唾液酸残基，使其整体呈现负极性。已有多种基质可有效从组织中解吸并离子化神经节苷脂；然而，还需考虑诸如质谱图中基质自身产生的峰以及基质在真空条件下的稳定性等因素。1,5-二氨基萘（DAN）基质在大多数成像应用的仪器真空条件下具有足够的稳定性，并且对脂质解吸表现出高度灵敏性，可用于正离子和负离子模式下的脂质分析²。与其他对负极性脂质有亲和性的基质（如二羟基苯甲酸（DHB）、9-氨基吖啶（9-AA）和5-氯-2-巯基苯并噻唑（CMBT））相比，DAN基质在负离子模式下能更高效地从大鼠脑组织中解吸神经节苷脂（相关论文正在准备中）。

选择合适的基质沉积方法与选择基质本身同样重要。湿法基质沉积技术是将固态基质溶解于有机溶剂中，再通过气动或自动喷雾器或点样仪进行沉积，该方法特别适用于蛋白质和肽类的解吸，因为液体可渗透样品，从而实现化合物的提取以及与基质的共结晶。尽管这些技术也可用于脂质分析，但由于脂质在溶剂中丰度高且溶解性好，尤其是在组织中，常常导致分析物的扩散和基质晶体分布不均^2,9。由于脂质在组织中易于离子化，干法基质沉积技术（如升华法）提供了一种简单且成本较低的替代喷雾法的方案，同时避免了这些湿法技术的诸多缺点。升华法在基质辅助激光解吸电离成像质谱（MALDI IMS）实验中的成功归因于其多种特性，例如微晶状的基质形态可增加基质与分析物结合的表面积、基质纯度更高，以及基质沉积更加均匀，从而相比湿法基质技术具有更高的重复性^1,10。

升华是指在真空条件下，将粉末基质置于冷却的样品表面正下方进行加热，使粉末基质发生从固相到气相的转变，随后沉积到组织样品表面。在升华过程中，可通过调节时间、温度和压力等因素来控制基质的沉积，从而获得高度可重复的结果。一次升华实验通常需要5至20分钟，具体时间取决于所选基质的类型，且该基质可在废弃前多次重复使用。该装置可商业购得，价格仅为自动喷涂仪的一小部分，且易于拆卸以进行清洁和维护。由于该基质沉积技术成本低且相对简单，因此非常适合刚开始或正在拓展MALDI成像质谱（IMS）中脂质成像应用的研究人员。尽管已有文献报道了用于IMS组织升华的技术方案¹¹，但目前仍缺乏标准化的操作流程，特别是针对在负离子模式下对高分子量脂质进行成像时所涉及的基本升华实验步骤，导致在没有大量试错的情况下难以建立该技术。以下实验方案旨在填补这一空白，提供一种将DAN基质升华沉积至大鼠脑切片上，用于高分辨率成像及神经节苷脂检测的详细操作流程。

图 2：升华装置。 升华装置的照片（A）和示意图（B）。真空泵通过橡胶管连接至装有 300 mL 乙醇的冷阱，冷阱再通过橡胶管连接至升华装置。该装置由两个独立的玻璃部件组成，通过金属U型接头密封连接。升华仪的上半部分包含冷凝器，内部填充冰浆。样品板用胶带固定在密封玻璃装置内部的冷凝器底部。升华仪的下半部分装有DAN基质，均匀铺展并正对样品板。升华过程中，将玻璃装置置于沙浴上，沙浴由正下方的加热板加热至 140 °C。温度探头通过反馈沙浴温度与实验预设温度的比较，有助于在整个升华实验过程中维持稳定的温度。请点击此处查看该图的放大版本。

方案

All animal handling procedures described below adhere to the University of Western Ontario's animal care committee (2016-014).

1. Tissue Preparation and Sectioning

1. Extract brain tissue from the rat through a process known as "Fresh Frozen Extraction" (FFE).
   1. Euthanize the rat with an overdose of pentobarbital sodium and monitor reflexes in the limbs. Once all reflexes have ceased, sever the head of the rat using a guillotine.
   2. Carefully separate the muscles and other tissues from the skull using a scalpel. Use bone-cutting forceps to break the skull to expose the brain, starting from the base of the skull (foramen magnum) to the anterior portion.
   3. Once the brain is exposed, carefully scoop it out with a surgical spatula and immediately place on crushed dry ice for flash freezing.
      NOTE: The brain will be very malleable. Therefore, take extreme care when placing the brain on dry ice. If the brain is crushed or pressed against a surface, it will alter its shape and freeze in that position.
2. Remove fresh frozen tissue (not perfused with formaldehyde or embedded in OCT) from - 80° C freezer and place on dry ice.
3. Place several drops of water on a cryostat holder and place on either dry ice or cryostat freeze bar. Press tissue lightly onto cryostat holder as it begins to freeze and hold until water freezes around the base of the tissue and anchors it in place. Add additional water to further secure the tissue on the holder if necessary.
   NOTE: Water is used instead of OCT media for mounting tissue in order to avoid contamination of the tissue with compounds which can affect the detection of the desired signal.
4. Position tissue in cryostat. Section tissue up to the desired anatomical location. Ensure that the cutting thickness is between 8 - 12 µm.
   NOTE: When sectioning tissue in communal cryostat devices, it is imperative that separate materials such as blades, brushes, anti-roll bars, and holders be used in order to avoid contamination with embedding compounds. The inside of the cryostat should also be cleaned thoroughly with ethanol before use.
5. Using the cryostat anti-roll bar, slowly section flattened tissue sections. Holes or markings on the tissue can occur if the roll bar placement is incorrect or blade is dull. Move the tissue to the center of slicing platform using clean paintbrushes.
6. Mounting
   1. Freeze conductive slides or metal plates by placing them in the cryostat while slicing. When the slide is completely frozen, carefully move the sectioned tissue onto the conductive surface of the slide using clean paintbrushes. Once all tissue sections are positioned correctly on the slide, place a finger under the slide, opposite the tissue, and press until the section thaws.
      NOTE: Sections may fold or curl during the thawing process when using this mounting method. This can be reduced by keeping the slide in the cryostat when thawing the tissue. If these issues become problematic, an alternative, warm-mount method is listed below.
   2. Alternatively, take a room temperature Indium-tin Oxide (ITO) slide (or metal plate) and lightly press down on frozen tissue section on the slicing platform surface, conductive side down. This will lead to the tissue section thawing evenly onto the surface of the slide with little curling or folding of the tissue.
      NOTE: Condensation may appear below the section when mounting using this method which may lead to loss of certain proteins and lipids.
7. Place slides with tissue in a desiccator for 5 - 10 min.

2. Sublimator Apparatus Set-up

NOTE: Perform these steps in a fume hood.

1. Place a sand bath in an aluminum container onto a hot plate, with the hot plate on a metal scissor lift of appropriate surface area (i.e. larger than the hot plate). Turn on the hot plate and set the temperature to 140 °C.
   NOTE: The melting point for DAN matrix is between 187 - 190 °C. Do not exceed this temperature on the hotplate.
   1. If the hot plate is equipped with a temperature feedback probe, use it to monitor sand temperature throughout the experiment and ensure temperature consistency. This feature can assist with experiment reproducibility across various sublimation experiments.
      NOTE: The sand bath should be contained within an aluminum container as a glass container may shatter at high temperatures.
2. Place 300 mg of DAN matrix onto the bottom surface of sublimation apparatus. Place the matrix in the center of the apparatus and spread out in an even layer in the approximate width and length of the slide being sublimated.
   CAUTION: DAN matrix is toxic. Therefore, it is important wear gloves, masks, and safety goggles at all times when handling the powdered matrix. DAN should be stored in the dark as it is light sensitive.
3. Tape a metal plate onto the inner surface of the apparatus with the plate making direct contact with the bottom of the condenser in order to ensure even distribution of temperature cooling across the entire surface of the slide during sublimation. Alternatively, sand the bottom of the condenser to ensure a flat surface.
   1. Place the tape along the outer edges of the plate and adhere to the sides of the inner glassware. If the tape is placed under the plate and adheres to the bottom of the inner glass surface, the temperature distribution may not be even and could result in uneven matrix distribution across the surface of the slide.
   2. Tape a blank (test) slide diagonally across the surface of the metal plate with the tape again placed on the outer edges of the slide.
4. Connect the top and bottom portions of the apparatus, with a rubber O-ring in the middle to ensure a complete seal.
5. Place a metal U-joint around the center of the apparatus and tighten vices until the top and bottom half of the apparatus are sealed tightly together. Place in the metal O-ring above the sand bath.
6. Take a handful of crushed ice and place it in the condenser. Fill condenser ¼ to ½ full with cold water to create ice slush. The ice slush will cool the metal plate on the inside of the apparatus and subsequently the slide adhered to it. Wait at least 5 min for the temperature to reach a steady state.
7. Pour 300 mL of ethanol in the cold trap container and place the glassware into the container. Drop 2 - 3 small pieces of dry ice into the ethanol in the bottom of the cold trap container. The cold trap input has a glass tube running to the bottom of the cylinder, while the output does not.
8. Connect the vacuum pump to the cold trap output using rubber tubing. Use another piece of rubber tubing to connect the cold trap input to the sublimation apparatus. Ensure that the tubing is tightly secured using metal clamps if available.
9. Use the vacuum pump to deliver a vacuum of 30 - 50 mT. Allow the pump to run for at least 5 min for pressure equilibration. Vices on U-ring of sublimation apparatus may have to be tightened again once the vacuum pump is turned on because of decreased pressure in the apparatus.
   NOTE: It is highly recommended that a vacuum gauge be attached to the pump to monitor the pressure of vacuum during the experiment and to test for leaks in pressure in order to achieve the highest possible reproducibility between experiments. However, most pumps are designed to maintain a constant pressure, therefore the gauge may not be essential for experienced users. Additionally, vacuum seal grease can be used to help maintain vacuum pressure.

3. Sublimation

1. Ensure that the sand bath temperature has stabilized at 140 °C and that the apparatus is secured in the metal O-ring above the sand bath with all tubing connected and the vacuum turned on.
2. Set timer for 7 min but do not start timer. Slowly raise the scissor lift and sand bath up to the sublimation apparatus until the U-joint of the sublimator is well above the metal O-ring. This extra space allows for adjustment of the apparatus on the sand.
   1. Quickly press the sublimator gently on the sand surface to ensure that the apparatus is sitting evenly on the sand, and then immediately start the timer.
3. When the timer sounds, turn off the vacuum pump and carefully lower the scissor lift until the sublimator is no longer touching the sand bath and is sitting securely in the metal O-ring.
   1. Slowly loosen the micro-vent valve to release pressure in the apparatus. Loosen the metal clamp around the rubber tubing of the sublimation apparatus and slowly begin to loosen the tube. Once the rubber tube has been loosened slightly, bend the tube to one side to allow residual pressure to escape.
   2. When ambient pressure returns, carefully remove the rubber tubing from the sublimation apparatus.
4. Loosen the vices on the U-joint of the apparatus and remove. Carefully separate the two halves of the sublimation apparatus and pull off the slide from the top of the inner glassware. Examine slide to confirm even matrix distribution.
   NOTE: If matrix is uneven, the powdered matrix in the bottom of the sublimator can be repositioned or the sublimator apparatus can be repositioned in the sand for the next slide. The sublimation process can be repeated several times using the same matrix, however, the matrix will eventually become darker from repeated heat exposure and the quantity of powder will decrease such that the sublimation time will have to be adjusted slightly to compensate. For this reason, it is important to monitor the amount of matrix being sublimated after each experiment to ensure consistency in matrix deposition between slides
5. If the distribution and amount of matrix sublimated is sufficient, tape a new slide with tissue onto the inside of the sublimator and repeat Section 3.
   NOTE: For quality control (QC) purposes, the amount of matrix sublimated can be measured after each experiment by weighing the slide before and after sublimation and dividing the weight of sublimated matrix with the surface area of the slide ², it should be noted that due to the lack of precision of most scales beyond 4 decimal points and variability between scales, these measurements should be only be used a guide for optimal matrix deposition as opposed to a fully quantifiable means of QC unless a high precision balance is used (see Figure 3).

4. Tissue Storage/Rehydration

1. After sublimation, store slides in a sealed container or small cassette in sealed plastic bag.
2. Incubate slides in -20 °C freezer for 2 h or overnight.
   NOTE: The goal of the freezing process is to act as a rehydration step for the desorption of tissue materials into the matrix. The freezing process has also been shown to prevent degradation of lipid signals for up to 1 week when stored at -80 °C ¹². For this reason, the amount of time the samples are stored in the freezer can be varied to a certain degree to suit the needs of the experimenter without significantly altering signal detection (as observed in our lab). However, we have noticed some discoloration of matrix when samples are frozen for longer than 24 h at -20 °C. Thus, we recommend imaging the sublimated tissue before that time or storing tissue at -80 °C for longer incubation periods.
3. Remove slides from freezer (and container) and place in a desiccator for 5 - 10 min.

5. Imaging and Analysis

1. Remove slides from desiccator and apply instrument standards to ensure mass accuracy of MALDI instrument during imaging. The type of standards will vary depending on instrumentation. Standards are generally applied evenly across entire slide, surrounding tissue to be imaged (Figure 3D).
2. Insert slide into MALDI instrument and follow manufacturer's instructions for imaging experiments (instrument methods should be optimized for a 1,000 - 2,000 mass range). Representative result (Figure 4) was acquired in reflectron negative mode with a 70 µm raster and 20 shots/spectrum (acquisition time ~ 2 h).

结果

升华实验完成后，应在通风橱中将玻璃装置的两半分开，并取下固定在冷凝管上的载玻片（图3A）。此时应检查载玻片上基质分布是否均匀，必要时需调整下一次实验的升华时间、温度或装置在沙浴中的位置。成功的DAN升华实验应在载玻片表面形成均匀的灰褐色基质涂层，组织的解剖结构可清晰可见，且载玻片上的基质量与组织上的基质量相近（图3B）。例如，若组织上沉积的基质过多，基质厚度将掩盖组织特征，仅能分辨其大致轮廓；而若基质沉积过少，组织区域将比载玻片其他部分颜色更深（图3C）。基质过多或过少均会导致MALDI仪器中信号质量下降。为进行质量控制，Thomas et al. 称量了沉积在载玻片上的基质量，并将重量除以载玻片表面积。他们报道了包括DAN在内的多种基质的最佳沉积量（110 µg/cm²）²。尽管大多数天平缺乏精确复现该结果所需的精度，我们仍尝试采用此质量控制方法；但由于变异性较高，我们仅能建议一个与DAN基质升华实验成功...

讨论

本研究详细阐述了一种用于基质辅助激光解吸电离成像质谱（MALDI IMS）实验中检测带负电荷脂质（如神经节苷脂）的组织基质升华标准化方案。MALDI IMS 的样品制备方法具有高度可变性，必须根据目标分析物的独特性质进行优化。将基质施加到组织样品表面是样品制备过程中影响 IMS 结果质量的关键步骤。在选择基质时应格外谨慎，确保所选基质与目标分析物相容，并且在质谱中不会产生基质来源的干扰信号。升华法是一种干法基质沉积技术，可实现高度均匀的基质晶体分布，同时几乎不会引起组织中脂质的位移。特别是 DAN 基质对一类称为神经节苷脂的带负电荷膜脂具有很高的解吸灵敏度，这已在大鼠脑切片中得到验证；此外，DAN 基质在真空条件下长时间保持稳定，因而适用于大多数成像应用。DAN 基质的另一优势在于其对带正电荷的脂质种类也具有高亲和力，从而提高了该基质在 MALDI IMS 应用中的通用性²。还应注意的是，采用此 IMS 方案可同时检测多种脂质种类，包括磷脂。

此前已发表一项关于DAN基质与其他9种常用基质特性的系统比较研究

材料

本文使用的材料清单

姓名	公司	目录编号	评论
升华器	Chemglass Life Sciences	CG3038-01
1,5-二氨基萘（DAN）基质	Sigma-Aldrich	D21200	100 G
冷冻切片机	Thermo-Fisher Scientific		CryoStar NX50
带温度反馈的加热板	Thermo-Fisher Scientific	HP88857290	Isotemp ADVD 7x7 HP 100 - 120 V
不锈钢千斤顶	Thermo-Fisher Scientific	2216479	10x10
冷阱			现场定制
真空泵	Franklin Electric	1102180403	Savant VP100 两级
氧化铟锡（ITO）载玻片	Hudson Surface Technology	PSI 1111000	II型，1.1 mm/每盒25片
MALDI TOF/TOF 5800 仪器	AB Sciex
干燥器	Sigma-Aldrich	D2797	台式干燥器