Role of N-methylated lysines (K) and arginines (R) was underappreciated for a long time before the turn of the century. With the help of new emerging technologies, their crucial role in chromatin regulation was established and now their mention when discussing gene regulation is almost a given. Despite this, much about how they contribute to the cellular chemistry is still to be discovered. There is a major gap in current knowledge base due to an incomplete list of possible lysine and arginine methylation sites. This is because of their low copy number inside the cell which makes it difficult to detect them. New methylation sites are being added every day.
This thesis aims to provide a solution to this problem by establishing methods that can help detect N-methylated lysines and argnines that are present in really low quantity inside the cell. The work is influenced by a previously established fact that p-sulfonatocalix[4]arene binds methylated lysine over unmethylated ones. We have first attempted to improve this native affinity by decorating the calix[4]arene skeleton with different substituents. To this effect, we have developed methods for regioselective functionalization of calix[4]arene scaffold and then studied their effect on its binding profile against a set of test peptides derived from proteins found in vivo.
We then demonstrate a proof-of-concept enrichment method using selected molecules from our inventory. We use these calix[4]arene based molecules as a stationary phase modifier in a chromatography setup and then show that it can separate peptides based on presence of N-methylated lysines and arginines. We propose that introduction of such a method would improve the visibility of low level N-methylated peptides by removing the bulk of back ground unmethylated analytes and thus improving their signal strength. Finally, we establish the utility of this method by showing that more N-methylated lysines are detected from a real-world proteomics sample prepared using our enrichment method.
This work opens new avenues for use of supramolecular chemistry in proteomics studies. We believe that this thesis is a confident demonstration that host-guest chemistry can help expand the existing knowledge about bimolecular processes found in vivo and must be explored further. / Graduate / 2021-12-23
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/12526 |
| Date | 06 January 2021 |
| Creators | Shaurya, Alok |
| Contributors | Hof, Fraser Alan |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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