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

大脑内众多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发挥着至关重要的作用。然而，细胞和分子背景仍然知之甚少。因此，该研究项目的目的是开发新颖、高度敏感的技术，用于研究作为模型生物的小鼠脑组织中的锌转运途径及其与年龄相关的变化。这将首次产生有关锌和相关转运蛋白随年龄变化的作用的数据。该提案的创新之处在于通过免疫组织化学（IHC）辅助激光烧蚀电感耦合等离子体质谱（LA-ICP-MS）开发和应用高灵敏度、定量和多重元素和分子成像及其向三个维度的扩展。为了通过 LA-ICP-MS 进行定量多重分析，抗体将与可通过 ICP-MS 检测到的稳定同位素富集的镧系元素缀合。将开发和应用基于尺寸排阻色谱 (SEC)-ICP-MS 的强大分析工作流程，用于表征标记抗体。采用碰撞池技术和在线同位素稀释分析 (IDA) 的灵敏检测方法相结合，将成为标记抗体快速现场质量控制的基础，并将能够确定抗体/金属化学计量。第二步，将开发一种采用标记抗体的稳健 IHC 方法，以便通过 LA-ICP-MS 对小鼠脑组织中选定的锌转运蛋白进行多重分析。第一部分开发的 SEC-ICP-MS 方法将成为将锌转运蛋白浓度与抗体标记中的金属含量联系起来的基础。为了获得定量绘图数据，应利用多重协议部署稳定同位素富集的镧系元素这一事实，建立一种新的校准方法。将开发一种用于 LA-ICP-MS 的新在线 IDA 技术，以执行准确、灵敏和快速的定量。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。上述联用技术的结合最终将允许研究小鼠脑组织中选定的锌转运蛋白和生物学相关元素的表达。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，这些元素被认为通过控制锌的细胞稳态在记忆中发挥作用。最终将建立三维多重方法，以生成包含锌及其转运蛋白分布的小鼠大脑图谱。

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.