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

大脑中许多锌转运蛋白以及相关生物分子的水平在学习、记忆和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，本研究项目的目的是开发新的，高灵敏度的技术，用于研究锌转运蛋白途径及其在小鼠脑组织中的年龄相关性变化作为模式生物。这将首次产生关于锌的作用和相关转运蛋白改变的数据。该提案的创新是基于免疫组织化学（IHC）辅助激光消融-电感耦合等离子体质谱（LA-ICP-MS）及其扩展到三维的高灵敏度，定量和多路复用元素和分子成像的开发和应用。为了通过LA-ICP-MS进行定量多路复用，抗体将与通过ICP-MS可检测的稳定同位素富集的镧系元素缀合。将开发并应用基于尺寸排阻色谱（SEC）-ICP-MS的标记抗体表征的稳健分析工作流程。采用碰撞池技术和在线同位素稀释分析（IDA）的灵敏检测方法的组合将是标记抗体的快速现场质量控制的基础，并且将能够确定抗体/金属化学计量。为了对小鼠脑中选定的锌转运蛋白进行多重分析，将开发使用标记抗体的稳健的IHC方法在第一部分中开发的SEC-ICP-MS方法将是将锌转运蛋白浓度与抗体标记物内的金属含量相关联的基础。为了获得定量绘图数据，应建立一种新的校准方法，利用多路复用协议部署稳定同位素富集的镧系元素的事实。为实现准确、灵敏、快速的定量，我们将开发一种新的在线IDA技术。灵敏度将随后通过优化仪器，以提高图像的对比度和resolutions.The组合的上述hyphenated技术将最终允许调查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。将研究不同年龄和具有不同认知能力的小鼠的大脑，以了解这些关键细胞元素中与年龄相关的基本变化，这些细胞元素被认为通过控制锌的细胞稳态在记忆中发挥作用。一个三维多路复用的方法将最终建立，以产生一个小鼠脑图谱包含锌及其转运蛋白的分布。

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.