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Development of specimen processing workflows for mass spectrometric detection and characterization of TDP-43

University of British Columbia

Transactive response DNA-binding protein 43 kDa (TDP-43) is a highly conserved protein that regulates nucleic acid processing. In humans, TDP-43 is widely expressed across different tissues in the body. In frontotemporal dementia and amyotrophic lateral sclerosis, two progressive neurodegenerative diseases, TDP-43 forms insoluble aggregates in central nervous tissues. Unfortunately, there is no cure for these diseases and a definitive diagnosis can only be made upon autopsy. As such, there is great interest in detecting, characterizing and quantifying TDP-43 and its disease-related post-translational modifications to investigate pathogenesis and as a potential biomarker. Characteristic TDP-43 post-translational modifications of TDP-43 deposits in frontotemporal dementia and amyotrophic lateral sclerosis include ubiquitination, hyper-phosphorylation, and proteolytic fragmentation. These pathological deposits have been primarily characterized by immunometric methods, namely western blot analysis, and thus methods with greater structural resolution are needed to aid in our understanding of the pathological processes associated with TDP-43 misfolding and aggregation. Detailed analysis of TDP-43 in human tissues and biofluids is hindered by sample complexity and the relatively low abundance of TDP-43. The aims of this thesis were thus to (1) develop a selective and multiplex method for the detection and characterization of TDP-43 using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (2) develop protocols for enrichment of TDP-43 from human fluids, tissues and cells to improve analytical sensitivity. Application of the LC-MS/MS method enabled detection and characterization of TDP-43 in biological matrices including human cell lines and human brain tissue. In addition, aptamer enrichment of endogenous TDP-43 from these biological matrices led to improved signal-to-noise ratios and increased sequence coverage, when coupled to the LC-MS/MS method. This targeted multiplex mass spectrometric provides the opportunity for characterization of pathological forms of TDP-43 at higher resolution compared to ligand binding methods.

Text

2021-01-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/72981

Pathology and Laboratory Medicine

University of British Columbia

UBCV

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