Mass Spectrometry Analysis of Non-Protein Amino Acid Misincorporation and Proteomic Changes in Neurotoxicity-Related Cellular Pathways

Abstract

Neurodegenerative diseases cause significant morbidity and mortality globally, with the prevalence continuing to rise due to prolonged life expectancy. Many neurodegenerative disorders share a common pathology that involves protein misfolding, aggregation and deposition in the brain. Dietary intake of non-protein amino acids has previously been linked to such proteinopathies, with indirect evidence indicating potential misincorporation of non-protein amino acids into growing protein chains. Phenotypic and proteomic investigations could provide more direct evidence of misincorporation and further elucidate the role that non-protein amino acids may play in neurodegenerative disease. The aim of this work was to determine if non-protein amino acids incorporate into the human proteome at a level detectable by mass spectrometry, with a focus on the amino acids L-DOPA, BMAA, and azetidine 2-carboxylic acid. An enzymatic method for the conversion of tyrosine residues to L-DOPA was successfully developed, providing a basis for studying the incorporation of L-DOPA into proteins. L-DOPA incorporation into proteins was also detected following treatment of human neuronal cells in vitro, with quantitative proteomics revealing activation of the unfolded protein response, evidence of oxidative stress, and changes in pathways involved in neurodegenerative diseases. Meta-analysis of proteomics datasets revealed a significant effect of sample preparation on the oxidation of samples, which could potentially mask true in vivo oxidation. Labelling techniques and mass spectrometer resolution were also found to be important for the identification of unique peptides and modifications, including misincorporated amino acids. The treatment of human neuronal cells with BMAA in vitro induced proteomic changes indicating a profile of toxicity like that previously reported for glutamate-mediated excitotoxicity, but the incorporation of BMAA into proteins was not detected. Conversely, the incorporation of azetidine 2-carboxylic acid into proteins was readily detectable following in vitro treatment of cells, importantly in proteins involved in cell proteostasis. Azetidine 2-carboxylic acid also resulted in quantitative proteomic changes, including an increased abundance of protein folding machinery and a decreased abundance of translational machinery. The significant proteomic changes in neuronal cells following exposure to all three non-protein amino acids investigated indicated changes in pathways potentially related to neurodegeneration and neurotoxicity, indicating a potential role in such pathologies that should be further explored. This thesis also provided direct evidence that certain non-protein amino acids can be incorporated into human proteins at a level detectable by mass spectrometry, paving the way for future studies to further investigate the role of such amino acids in human disease.