多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流
1Mass Spectrometry Core Facility,University of Sydney, 2Proteomics Core Facility,University of Technology Sydney, 3Neuropathology Group, Discipline of Pathology, School of Medical Sciences,University of Sydney, 4Redox Biology Group, Discipline of Pathology, School of Medical Sciences,University of Sydney
Summary
本协议描述了一种可重现且可靠的方法, 用于 sublimation-based 制备用于成像质谱的福尔马林固定组织。
Abstract
基质辅助激光解吸/电离, 质谱成像 (MALDI MSI) 的使用迅速扩大, 因为这项技术分析了大量的生物分子从药物和血脂到 n-糖。尽管存在各种样品制备技术, 但从甲醛保存的组织中检测多肽仍然是这类质谱分析中最困难的挑战之一。基于这个原因, 我们创建并优化了一个健壮的方法, 它保留了样本中包含的空间信息, 同时引出了最大数量的电离肽。我们还旨在以成本效益和简单的方式实现这一目标, 从而消除在使用自动化检测时可能出现的潜在偏差或准备错误。最终结果是一个可重现且价格低廉的协议。
Introduction
基质辅助激光解吸/电离质谱成像 (MALDI MSI) 已被用作图像技术的二十年1,2, 分析的生物分子范围包括: 脂类3, 多肽2 ,4,蛋白质2,5, 代谢物6,7, n-糖8, 以及合成分子, 如治疗药物 9,10。显示此技术实用工具的出版物数量在过去十年中显著增长6、11、12、13。某些分子, 如血脂, 是相对容易通过 MALDI MSI 分析, 因为它们电离容易由于其化学性质, 因此需要很少的前期准备3。然而, 对于更难的目标, 如肽, 有效电离这些分子所需的步骤是广泛的, 一般复杂的14。目前很少有出版物旨在处理或证明在使用这种独特的视觉技术的组织的方法15的重现性。出于这个原因, 我们已经将观察和实施的优化编译成一个单一的, 易于实施的方法, 不应该需要修改, 从甲醛交联组织来源的多肽的分析14。
在这份手稿中, 我们描述了一个验证的, 低成本的可重现性的方法, 用于检测和空间映射的多肽, 由福尔马林固定冷冻 (FFF) 和福尔马林固定石蜡嵌入 (FFPE) 组织切片。此方法不需要或依赖于任何专用的工具3。具体来说, 我们处理的许多方面的专业样品准备必要的分析多肽;步骤, 如抗原检索16和基体涂层。我们的协议还利用了廉价的设备和试剂, 从而使这种方法可供更广泛的社区使用, 否则将无法负担替代的机器人设备17。
开发人工样品制备方法的背后的原因是两个方面: 第一, 使用升华创建一个一致的和均匀的矩阵晶体的涂层, 是〜1µm 长度为18, 这是无法做到的, 更常见的喷涂技术.其次, 相对较小的设置成本: 自定义设备的总成本为 < 1500 澳元。我们注意到, 在成本效益方面, 当没有机器机器参与的时候, 每个样品的价格就会便宜得多。以前已经报告过升华的使用, 然而, 据我们所知, 在文献中没有报告也没有描述这一过程和样品制备的 step-by 步骤方法。
本协议旨在帮助研究人员获得 MALDI 质谱仪, 并意图生成与感兴趣的生物分子相关的空间信息19。在本质上, MALDI MSI 是一种组织学检查的形式, 不依赖于抗体或污点2。
Protocol
警告: 执行此过程时应遵循所有适用的安全措施, 包括使用适当的个人防护设备 (PPE) (例如,实验室大衣、腈手套、安全眼镜、等) 1. 试剂和设备的制备 解决方案的准备 准备500毫升的 Carnoy 的液体, 混合100% 乙醇 (乙醇), 氯仿和冰醋酸在一个 6:3: 1 伏/v/v 的比例在一个干净的玻璃肖特瓶。 使液体硝化纤维 (nc), 溶解在整齐的丙酮的 nc …
Representative Results
如果正确遵循, 该协议将生成清晰地表示组织的大体形态的图像, 而不需要任何划痕或其他变形 (图 1)。正确执行样本准备的理想验证是通过改变被查看的分子 (图 2) 区分不同物理结构的能力。 一个很好的指南, 确定样本是否已不正确的准备是检查是否存在域或矩阵聚合;延伸到物理?…
Discussion
该协议旨在最大限度地电离分子物种的生成, 同时消除域的分析。在应用矩阵、消化样本或升华24后, 使用相同的覆盖原则是关键因素; 结晶。也就是说, 需要建立和维护一个均匀的蒸气、基体或其他的沉积物。移溶剂洗涤为再结晶和消化, 均匀地在样品之下, 防止任何单独区域接受更多溶剂蒸气比其他任何地方。这是重要的防止形成可见的冷凝和整体抑制域的表面分子, 以及确保样…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢悉尼医学院基金会和布鲁斯和基金会的资助, 这项工作的一部分, 通过他们的博士奖学金计划的阿尔茨海默病研究和 ARC 发现补助金 (DP160102063) 授予 PKW。
Materials
| Cryo Microtome | Leica | CM3050 | For preparation and section of tissue. |
| Indium Tin Oxide Microscope slides | Bruker | 8237001 | For preparation and section of tissue. |
| Coplin Jars | Sigma Aldrich | S5516 | For preparation and section of tissue. |
| Pressure Cooker | Kambrook | KPR620BSS | For preparation and section of tissue. |
| Sublimator | Chem Glass | NA | For sublimation procedure. Similar in design to the CG-3038 however it was custom made |
| Sand bath | NA | NA | For sublimation procedure. Fine grade river sand held in folded aluminium foil sourced from outside not from any specific company |
| Glass Petri Dish | Sigma Aldrich | CLS70165100 | For sublimation procedure. |
| Vacuum Pump | NA | NA | For sublimation procedure. Sourced as a spare part from an old mass spectrometer |
| Cold trap | Chem Glass | CG-4510-02 | For sublimation procedure. |
| Hot Plate | John Morris | EW-15956-32. | For sublimation procedure. |
| Plastic petri dish | Sigma Aldrich | Z717223 | For sublimation procedure. |
| 37 °C incubator | NA | NA | For sublimation procedure. Not applicable, incubator is non sterile and over 30 years old |
| Blotting paper | Sigma Aldrich | P7796 | For sublimation procedure. |
| Nitrocellulose | Sigma Aldrich | N8395 | For washing of slides. |
| Acetone | Sigma Aldrich | 650501 | For washing of slides. |
| Xylene | Sigma Aldrich | 214736 | For washing of slides. |
| 100% EtOH | Sigma Aldrich | 1.02428 | For washing of slides. |
| 70% EtOH | Sigma Aldrich | NA | For washing of slides. Made in lab from 95% stock ethanol |
| Chloroform | Sigma Aldrich | C2432 | For washing of slides. |
| Glacial Acetic Acid | Sigma Aldrich | ARK2183 | For washing of slides. |
| Tris HCL pH 8.8 | Sigma Aldrich | TRIS-RO | For proteolytic cleavage. Powder made to 1M followed by equilibration with 32% HCl to PH 8.8 |
| Milli Q Ultra-Pure Water | Sigma Aldrich | NA | For proteolytic cleavage. Purification performed in house by sartorious water purification system |
| Ammonium Bircarbonate | Sigma Aldrich | A6141 | For proteolytic cleavage. |
| Trypsin | Sigma Aldrich | T0303 | For proteolytic cleavage. |
| CHCA Matrix | Sigma Aldrich | C2020 | For recrystallisation. |
| Acetonitrile | Sigma Aldrich | 1.00029 | For recrystallisation. |
| Trifluoroacetic Acid (TFA) | Sigma Aldrich | 302031 | For recrystallisation. |
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Cite This Article
O’Rourke, M. B., Padula, M. P., Smith, C., Youssef, P., Cordwell, S., Witting, P., Sutherland, G., Crossett, B. Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows. J. Vis. Exp. (131), e56778, doi:10.3791/56778 (2018).