From top to bottom: the methylproteome at the systems and molecular levels

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Copyright: Winter, Daniel
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Abstract
In recent years, research into the methylproteome (the subset of the proteome that is post-translationally methylated) has gained traction, with the report of novel protein methyltransferases and large-scale analyses of protein methylation sites in several organisms. However, there is little understanding on how methylation processes are regulated and how protein methylation itself is a regulator of protein activity. Moreover, notwithstanding the recent development of enrichment strategies to concentrate methylated proteins or peptides for mass spectrometry analyses, the identification of protein methylation sites remains challenging. In this thesis, network analysis and mass spectrometry strategies were employed to investigate the regulatory roles of protein methylation and the regulation of methyltransferases; and mass spectrometry and ion mobility spectrometry techniques were employed to refine methods to detect methylation events and their precise localisation. With network analysis, we demonstrate that the integration of post-translational modification (PTM) data, including methylation, with protein-protein interaction networks can reveal candidates of “interaction codes”, whereby combinations of PTMs modulate the interactions of a protein. This strategy can be extended to other types of data to be integrated with interaction networks, in a process we term “interactome annotation”. With recombinant methods and mass spectrometry, we characterise six yeast protein methyltransferases, and reveal that these are extensively post-translationally modified. Using structural data, we contextualise these novel PTMs and determine those that are most likely of functional relevance. These results suggest that methyltransferases may be subject to post-translational regulation. To refine the detection of arginine methylation in yeast, we used a combination of isotope labelling techniques and mass spectrometry to shortlist peptides of interest for further mass spectrometry analyses. Unexpectedly, this strategy led to several complications in the identification of peptides, which are discussed in detail. Finally, we demonstrate that field asymmetric ion mobility separation (FAIMS) is superior to reverse phase liquid chromatography to separate isobaric methylated peptides. In combination with mass spectrometry, FAIMS showed the potential for more precise identification of methylation sites and to reveal potential mechanisms whereby methylation affects the physicochemistry of peptides. Together, this thesis represents a significant advance in understanding the methylproteome in the cell.
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Author(s)
Winter, Daniel
Supervisor(s)
Wilkins, Marc
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Publication Year
2018
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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