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High throughput antibody-based methods to track the architecture and operations of cell signalling systems

University of British Columbia

The antibody microarray platform has emerged as an innovative and economical tool for ultra- sensitive, semi-quantitative interrogation of protein expression, post-translational modifications such as phosphorylation, and interactions with other proteins and small molecules. While this technology possesses unique strengths such as compatibility to directly assay biofluids as well as tissue and cell specimens without enrichment, the interpretation of antibody microarray findings have been plagued by the biological complexity of protein-protein interactions, poor sample handling, and inadequately characterized antibodies. Further refinement of the antibody microarray platform can identify proteomic cell signalling events in a targeted approach that is still high throughput and relatively accurate. To this end, this thesis describes recent innovations in the design, production and characterization of over 600 phosphosite-specific antibodies used in printing antibody microarrays, as well as refinements in sample preparation and detection systems to yield significant improvements in data quality. The resulting KAM-1325 antibody microarray was then used to investigate the associated similarities and differences between epidermal growth factor and insulin-mediated metabolic signal transduction pathways, as well as the effects of inhibition of protein-tyrosine phosphatases and protein synthesis. Taken together, along with over 250 different experiments performed with the same array with samples from diversified model systems and treatments, I used correlative analysis to propose physiological protein-protein relationships with a focus on kinase-substrate connections. I have also developed maps of common cell signalling networks of known and proposed novel protein connections relating to the targets that are tracked with antibodies on the KAM-1325 antibody microarray. To validate some of these connections, in vitro kinase assays were performed. These experiments involved direct incubation of cell lysates with purified recombinant protein kinases and surveillance of enhanced phosphorylation of substrate proteins with the antibody microarray. The continued elucidation of cell signalling systems will help discern between healthy and pathological states of cells and organisms, so that future therapies that utilize these signalling pathways to restore homeostasis and wellbeing can be developed.

Text

2020-10-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/76322

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/