Date of Award

10-30-2020

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Mass spectrometry, MTMR2, Phosphorylation, Proteomics, Spinal muscular atrophy, Ubiquitination

Supervisor

Panayiotis O. Vacratsis

Rights

info:eu-repo/semantics/embargoedAccess

Abstract

Advances in mass spectrometry (MS) has allowed for the deep analysis of various proteomes, providing identifications of proteins and their modifications. The true power in modern-day proteomics is the application of MS techniques to address various biological questions, propelling disease research and biochemical understanding of organisms. We have utilized high-resolution mass spectrometry to investigate biological questions leading to a greater knowledge of cellular biology. The transcriptional co-activator with PDZ-binding motif (TAZ), is regulated by reversible phosphorylation. However, sequence analysis suggests many potential uncharacterized sites of TAZ phosphorylation, specifically in regions in close proximity to a critical phosphorylation site making site assignment challenging. Using both targeted and untargeted approaches, we identified novel TAZ phosphorylation sites, using a reaction monitoring scheme to resolve positional phosphoisomers, and determined the biological consequence of a novel site, serine 93, on TAZ localization. Spinal muscular atrophy (SMA) is a motor neuron disease affecting 1 in 10,000 individuals. SMA has been shown to involve the release of extracellular vesicles (EVs), which have been used as a source of biomarkers for disease. We examined the use of EVs as a source of SMA biomarkers. We isolated and quantified >650 proteins from SMA-derived vesicles finding potential biomarkers, one of which was confirmed in patients, suggesting these vesicles coupled with our methods are suitable for SMA biomarker discovery. In the model plant species Arabidopsis thaliana gene expression is heavily regulated through post-translational ubiquitination, however a gap between the number of ubiquitinated substrates identified and genes encoding the ubiquitin machinery exists, suggesting many unidentified modifications exist. The main strategy for studying ubiquitomes across species uses diglycine enrichment followed by MS analysis. We developed a DIA-based MS method coupled with novel sample preparation methods to overcome plant-specific challenges and increase the repository of the Arabidopsis ubiquitome, identifying 160 proteins with over 400 ubiquitination sites. The prevalent Charcot-Marie-Tooth disease can be caused by mutations in the lipid phosphatase MTMR2, a protein critical for regulating endosomal dynamics. MTMR2 is regulated by phosphorylation at serine 58. However, the phosphatase and the alterations in protein-protein interactions occurring with this modification have not been thoroughly investigated. To isolate MTMR2 interacting proteins, we utilized in vivo labeling fusing BirA biotin ligase to MTMR2, followed by MS analysis, identifying a putative interactor, TSSC1. We also provide evidence that MTMR2 itself may be subjected to phosphorylation-dependent degradation. This work utilizes high-resolution MS techniques to link protein regulation and function in a variety of biological and cellular contexts. The techniques presented here can be applied to address the gaps of knowledge in various proteomes and are amenable to user-specific modifications. The techniques here provide a framework for determining disease biomarkers for neurological diseases from EVs, investigating proteome-wide changes through protein modifications, and ultimately link high-resolution analytical mass spectrometry techniques and data to address critical biological events in a robust fashion.

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