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

• Applicant: Professor Dr. David Clases
• Subject Area: Analytical Chemistry
• Term: from 2018 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 417283954

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.

• DFG Programme: Research Fellowships
• International Connection: Australia
• Host: Professor Philip Doble, Ph.D.