Novel approaches for a highly sensitive, quantitative and three-dimensional multiplexed analysis by means of hyphenated techniques to investigate the zinc transporter pathway in the ageing mouse brain

大脑中众多的锌转运蛋白以及相关生物分子的水平在学习、记忆以及认知能力下降中起着至关重要的作用。然而，细胞和分子背景仍然知之甚少。因此，本研究的目的是开发一种新的、高灵敏的技术，用于研究锌转运蛋白途径及其在小鼠脑组织中的增龄变化。这将第一次产生关于锌和相关转运蛋白随年龄变化的作用的数据。这一方案的创新之处在于开发和应用了免疫组织化学(IHC)辅助激光消融-电感耦合等离子体质谱(LA-ICPMS)的高灵敏度、定量和多重元素和分子成像技术，并将其扩展到三维。为了实现LA-ICPMS的定量多重，抗体将与稳定的同位素富集型稀土元素偶联，可通过电感耦合等离子体质谱检测。将开发和应用基于尺寸排阻色谱(SEC)-电感耦合等离子体质谱(ICPMS)的强大分析工作流程来表征标记抗体。采用碰撞细胞技术和在线同位素稀释分析(IDA)相结合的灵敏检测方法将为标记抗体的快速现场质量控制奠定基础，并将能够确定抗体/金属化学计量比。在第二步，将开发一种使用标记抗体的可靠的IHC方法，以便利用LA-ICPMS对小鼠脑组织中选定的锌转运蛋白进行多路分析。第一部分建立的SEC-ICPMS方法将为锌转运蛋白浓度与抗体标记物中金属含量的关联奠定基础。为了获得定量测绘数据，应利用多路复用协议使用稳定的同位素富集型稀土元素这一事实，建立一种新的校准方法。为实现准确、灵敏、快速的定量，将开发一种新的在线IDA技术用于LA-ICPMS。随后将通过优化仪器以增强图像对比度和分辨率来最大化灵敏度。上述连接技术的组合最终将允许研究选定的锌转运蛋白和生物相关元素在小鼠脑组织中的表达。将对不同年龄和不同认知能力的小鼠的大脑进行研究，以了解这些关键细胞元素随年龄的基本变化，据信这些关键细胞元素通过控制细胞内锌的稳态而在记忆中发挥作用。最终将建立一种三维多路复用方法，以生成包含锌及其转运蛋白分布的小鼠脑图谱。

The levels of numerous zinc transporter proteins as well as of associated biomolecules within the brain play a crucial role in learning, memory as well as in cognitive decline. Nevertheless, the cellular and molecular background remains poorly understood. Therefore, the aim of this research project is to develop novel, highly sensitive technology for the investigation of the zinc transporter pathway and its age-related alteration in brain tissue of mice as model organism. This will produce data concerning the role of zinc and associated transporter protein alterations across age for the first time. The innovation of this proposal is based on the development and application of highly sensitive, quantitative and multiplexed elemental and molecular imaging by immunohistochemistry (IHC)-assisted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and its expansion into three dimensions. In order to perform quantitative multiplexing by LA-ICP-MS, antibodies will be conjugated with stable isotope enriched lanthanides detectable via ICP-MS. A robust analytic workflow for the characterisation of labelled antibodies based on size exclusion chromatography (SEC)-ICP-MS will be developed and applied. The combination of a sensitive detection method employing collision cell technology and on-line isotope dilution analysis (IDA) will be the basis for a rapid on-site quality control of labelled antibodies and will enable to determine the antibody/metal stoichiometry.In a second step, a robust IHC method employing the labelled antibodies will be developed in order to perform a multiplexed analysis of selected zinc transporter proteins in mouse brain tissue by LA-ICP-MS. The in the first part developed SEC-ICP-MS method will be the basis to relate the zinc transporter protein concentration with the metal content within the antibodies’ label. To obtain quantitative mapping data, a new calibration method shall be established exploiting the fact that multiplexing protocols deploy stable isotope enriched lanthanides. A new on-line IDA technique for LA-ICP-MS will be developed to perform accurate, sensitive and fast quantification. Sensitivities will subsequently be maximised by optimising the instrumentation to enhance image contrasts and resolutions.The combination of the aforementioned hyphenated techniques will finally allow to investigate the expression of selected Zn transporter proteins and biologically relevant elements in mouse brain tissue. Brains of mice across different ages and with distinct cognitive abilities will be studied in order to understand basic age-related alterations in these key cellular elements, which are believed to play a role in memory through control of the cellular homeostasis of zinc. A three-dimensional multiplexing approach will finally be established in order to generate a mouse brain atlas containing the distributions of zinc and its transporter proteins.