Neue Methoden für ein sensitives, quantitatives und dreidimensionales Multiplexverfahren auf Basis von Kopplungstechniken zur Erforschung des Wirkmechanismus von Zinktransportern im alternden Mausgehirn
Zusammenfassung der Projektergebnisse
The implementation of immunohistochemical techniques into imaging workflows for LA-ICP-MS expanded the reach of atomic spectroscopy beyond the sole investigation of elements by employing metal-coded antibodies for the tracing of proteins. This promises new perspectives on the interface of Proteomics and Metallomics which supports more holistic investigations and interpretations of pathological disorders. However, low abundant proteins in micro-scaled anatomical structures require extremely low limits of detection and their calibration requires robust quantification strategies as well as adequate methods to characterise labelled antibodies regarding their integrity, labelling degree and affinity. This research project aimed to develop new approaches to precisely characterise metal-coded antibody probes, to develop new calibration strategies and to adapt and complement previous methodologies for the joint analysis of proteins and elements in the heterogeneous environment of biological tissues via LA-ICP-MS. The developed methods were applied to study the role of selected elements and proteins in pathological pathways associated with age-related cognitive decline in mouse models. A method based on on-line post-column isotope dilution analysis was developed for SEC-ICP- MS and was capable to fully characterise the integrity of labelled antibodies, their fragmentation, labelling degrees and their interaction with the antigen. The developed speciation method allowed rapid quality controls and enabled the quantification of protein and element levels detected via hyphenated ICP-MS. The reliable quantification of endogenous elements and metal labels required the development of new calibration strategies and therefore, a new protocol for the fabrication of gelatine-based standards was developed and validated. The application of moulds and ion extraction resins improved reproducibility, accuracy, the dynamic calibration range, manufacturing, and comparability contributing to enhanced quantification capabilities. The standardisation was further improved by developing an on-line reverse isotope dilution approach for LA-ICP-MS compensating for instrumental drift and fluctuation while allowing instant calibration. The performance of IHC-assisted LA-ICP-MS was improved by employing and adapting recently commercialised hardware and by high ion transmission bandpass mass filtering. This increased signal to noise ratios significantly, while enabling the simultaneous acquisition and correlation of major, minor and trace elements as well as metal-coded antibodies. The developed methods were subsequently employed to study the spatial distribution of proteins and elements in age-associated pathologies in murine tissues causing cognitive decline. The joint analysis of trace element and protein distributions in the murine brain enabled the convolution of metallomic and proteomic data sets, which were relevant for the prediction parkinsonian neurodegeneration in one study. In another study, the analysis of spatiotemporal metal profiles in tauopathies (including Alzheimer’s disease) revealed an age-dependent increase in brain iron levels in both wildtype mice and rTg(tauP301L)4510 mice providing valuable insight into the inter-relationship between iron and tau in ageing and neurodegeneration. The multiplexed analysis of several antigens was challenging as staining methods inferred with endogenous element levels preventing simultaneous analyses and quantitative approaches. This was solved by analysing consecutive tissue sections separately regarding the protein and elemental content. While the multiplexing was successful for the analysis of proteins including tyrosine hydroxylase, GFAP, NeuN, ZIP8 and ZIP14, several zinc transporters including ZnT3 and ZIP1-3 were not yet accessible, due to incompatibilities of antibody formulations and relatively low protein expression levels. This motivated the search for an alternative IHC protocol. Using the previously developed bandpass mass filtering method allowed the in-depth characterisation of small lanthanide-based nanoparticles which will be incorporated as new metal labels for antibodies in the future to increase the number of labelled metals per antibody and consequently sensitivity. The 3D reconstruction of elemental and proteomic levels was feasible by ablation of consecutive sections, image registration and the stacking of calibrated elemental maps. However, due to the inaccessibility to various relevant transporter proteins, the generation of the 3D atlas of the zinc transporter pathway in cognitive decline was suspended until sufficient sensitivity is ensured and adapted labelling and staining protocols are in place.
Projektbezogene Publikationen (Auswahl)
- Low background mould-prepared gelatine standards for reproducible quantification in elemental bio-imaging, Analyst (2019), 144, 6881-6888
M. T. Westerhausen, T. E. Lockwood, R. Gonzalez de Vega, A. Röhnelt, D. P. Bishop, N. Cole, P. Doble, D. Clases
(Siehe online unter https://doi.org/10.1039/c9an01580a) - On-line reverse isotope dilution analysis for spatial quantification of elemental labels used in immunohistochemical assisted imaging mass spectrometry via LA-ICP-MS, J. Anal. At. Spectrom. (2019), 34, 407-412
D. Clases, R. González de Vega, P. A. Adlard, P. A. Doble
(Siehe online unter https://doi.org/10.1039/c8ja00324f) - SEC-ICP-MS and on-line isotope dilution analysis for characterization and quantification of immunochemical assays, Anal. Bioanal. Chem. (2019), 411, 3553-3560
D. Clases, R. González de Vega, D. Bishop, P. A. Doble
(Siehe online unter https://doi.org/10.1007/s00216-019-01836-9) - Matching sensitivity to abundance: High resolution immuno-mass spectrometry imaging of lanthanide labels and endogenous elements in the murine brain, J. Anal. At. Spectrom. (2020), 35, 728-735
D. Clases, R. Gonzalez de Vega, S. Funke, T. E. Lockwood, M. T. Westerhausen, R. V. Taudte, P. A. Adlard, P. Doble
(Siehe online unter https://doi.org/10.1039/c9ja00405j)
