Assessing the reproducibility of labelled antibody binding in quantitative multiplexed immuno-mass spectrometry imaging.

Autor: Mello MG; Atomic Medicine Initiative, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Ultimo, NSW, 2007, Australia., Westerhausen MT; Atomic Medicine Initiative, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Ultimo, NSW, 2007, Australia., Singh P; Atomic Medicine Initiative, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Ultimo, NSW, 2007, Australia., Doble PA; Atomic Medicine Initiative, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Ultimo, NSW, 2007, Australia., Wanagat J; Division of Geriatrics, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, 90095, USA. jwanagat@mednet.ucla.edu., Bishop DP; Atomic Medicine Initiative, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Ultimo, NSW, 2007, Australia. david.bishop@uts.edu.au.
Jazyk: angličtina
Zdroj: Analytical and bioanalytical chemistry [Anal Bioanal Chem] 2021 Sep; Vol. 413 (21), pp. 5509-5516. Date of Electronic Publication: 2021 Jul 25.
DOI: 10.1007/s00216-021-03536-9
Abstrakt: Immuno-mass spectrometry imaging (iMSI) uses laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to determine the spatial expression of biomolecules in tissue sections following immunolabelling with antibodies conjugated to a metal reporter. As with all immunolabelling techniques, the binding efficiency of multiplexed staining can be affected by a number of factors including epitope blocking and other forms of steric hindrance. To date, the effects on the binding of metal-conjugated antibodies to their epitopes in a multiplexed analysis have yet to be quantitatively explored by iMSI. Here we describe a protocol to investigate the effects of multiplexing on reproducible binding using the muscle proteins, dystrophin, sarcospan, and myosin as a model, with antibodies conjugated with Maxpar® reagents before histological application to murine quadriceps sections using standard immunolabelling protocols and imaging with LA-ICP-MS. The antibodies were each individually applied to eight sections, and multiplexed to another eight sections. The average concentrations of the lanthanide analytes were determined, before statistical analyses found there was no significant difference between the individual and multiplexed application of the antibodies. These analyses provide a framework for ensuring reproducibility of antibody binding during multiplexed iMSI, which will allow quantitative exploration of protein-protein interactions and provide a greater understanding of fundamental biological processes during healthy and diseased states.
(© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)
Databáze: MEDLINE
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