权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

通过联用技术进行高灵敏度、定量和三维多重分析的新方法，以研究衰老小鼠大脑中的锌转运蛋白途径

基本信息

批准号：
417283954
负责人：
Professor Dr. David Clases
金额：
--
依托单位：
Bereich für Analytische Chemie
依托单位国家：
德国
项目类别：
Research Fellowships
财政年份：
2018
资助国家：
德国
起止时间：
2017-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/417283954?language=en
关键词：
Novel approaches highly sensitive quantitative

项目摘要

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.

大脑中大量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一种新的、高灵敏度的技术来研究作为模式生物的小鼠脑组织中锌转运体通路及其与年龄相关的变化。这将首次产生关于锌和相关转运蛋白改变在不同年龄的作用的数据。该方案的创新之处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）的高灵敏度、定量和多路元素和分子成像技术的发展和应用，并将其扩展到三维空间。为了通过LA-ICP-MS进行定量多路复用，抗体将与通过ICP-MS检测到的稳定同位素富集镧系元素偶联。将开发并应用基于大小排斥色谱(SEC)-ICP-MS的标记抗体的强大分析工作流。采用碰撞细胞技术和在线同位素稀释分析（IDA）的灵敏检测方法的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/金属化学计量学。在第二步中，将开发一种使用标记抗体的强大免疫组化方法，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。第一部分建立的SEC-ICP-MS方法将是将锌转运蛋白浓度与抗体标签中金属含量联系起来的基础。为了获得定量的测图数据，需要利用多路复用协议部署稳定同位素富集镧系元素的事实，建立新的校准方法。为了实现准确、灵敏、快速的定量，我们将开发一种新的在线IDA技术用于LA-ICP-MS。随后将通过优化仪器来提高图像对比度和分辨率，从而使灵敏度最大化。上述联用技术的结合将最终允许研究选定的锌转运蛋白和生物学相关元件在小鼠脑组织中的表达。研究人员将研究不同年龄、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。三维多路复用方法将最终建立，以产生包含锌及其转运蛋白分布的小鼠脑图谱。