方法文章

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

DOI：

10.3791/55254

⸱

2017年3月23日

 ,  ,  ,  , 
1Department of Anatomy and Cell Biology, The University of Western Ontario, 2Department of Chemistry and Biochemistry, The University of Western Ontario

本文内容

摘要

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提出了一种使用 MALDI 成像质谱法将 DAN 基质升华到大鼠脑组织上以检测神经节苷铛苷的方案。

摘要

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Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

引言

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Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) is becoming a highly sought after technique for visualization of the spatial distribution of lipids, peptides and proteins across intact sample surfaces. MALDI IMS was previously known as an analytical technique for pre-purified analytes, but in recent years, it has been drawing attention in many other disciplines because of the ability to combine the accuracy of mass spectrometry with high resolution visual/anatomical reference points without the need for any external labelling. As the scientific pool of researchers utilizing this technique continues to grow, there is increased need f....

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方案

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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 ....

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结果

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Upon completion of the sublimation experiment, the two halves of the glass apparatus are separated in the fume hood and the slide, taped to the condenser, can be removed (Figure 3A). At this point, the slide should be examined for uneven matrix distribution and the time, temperature, or placement of the apparatus in the sand bath may have to be adjusted for the next slide. A successful DAN sublimation experiment will result in an even coating of grey/brown matrix along th.......

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讨论

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This work details a standardized protocol of matrix sublimation on tissue for the detection of negatively charged lipids, such as gangliosides, in MALDI IMS experiments. Sample preparation for MALDI IMS is highly variable and must be customized to suit the unique properties of the analyte of interest. The application of matrix onto the tissue sample surface is a crucial aspect of the sample preparation process with regards to the quality of results in IMS. Particular care should be taken when selecting matrices, ensuring.......

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披露

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作者没有什么可透露的。

致谢

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我们要感谢西安大略大学 MALDI MS 设施的 Kristina Jurcic 的技术援助，以及美国国家科学与工程委员会 （NSERC） 为这项工作提供资金。作者还要感谢 Caprioli 小组（田纳西州范德堡大学）和 Chaurand 小组（魁北克省蒙特利尔大学）在优化本手稿中介绍的升华技术方面的建议。

....

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材料

本文使用的材料清单
姓名公司目录编号评论
升华剂Chemglass Life SciencesCG3038-01
1,5-二氨基萘 （DAN） 基质Sigma-AldrichD21200 100 克
低温恒温器Thermo-Fisher ScientificCryoStar NX50
带温度反馈的加热板Thermo-Fisher ScientificHP88857290Isotemp ADVD 7x7 HP 100 - 120 V
不锈钢插孔Thermo-Fisher Scientific221647910x10
冷阱定制现场
真空泵Franklin Electric1102180403Savant VP100 两级
铟锡氧化物 （ITO） 滑轨Hudson Surface TechnologyPSI 1111000类型 II，每个 1.1 毫米/25
个 MALDI TOF/TOF 5800 仪器AB Sciex
干燥器Sigma-AldrichD2797台式干燥器

参考文献

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  1. Hankin, J. A., Barkley, R. M., Murphy, R. C. Sublimation as a method of matrix application for mass spectrometric imaging. J. Am. Soc. Mass Spectrom. 18, 1646-1652 (2007).
  2. Thomas, A., Charbonneau, J. L., Fournaise, E., Chaurand, P.

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标签

DAN

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