Antibody-free affinity enrichment for global methyllysine discovery




Dewar, Charlotte

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Lysine methylation is a post-translational modification that regulates a large array of functionally diverse processes that are vital for cellular function. The role of methylation is best characterized on histone proteins due to their high concentration in the cell, but alongside histone modifications, lower abundance non-histone methylation is emerging as a prevalent and functionally diverse regulator of cellular processes. The direct biological impact of non-histone lysine methylation is less well understood because they are difficult to detect. The dynamic concentration range of the proteome masks their signal during proteomic analysis which impedes the detection of these low abundance methylated proteins. Increasing the concentration of proteins bearing methylation is required for improved discovery. This requires enriching the post-translational modification with a capturing reagent prior to analysis. This thesis details an optimized method for using the supramolecular host p-sulfonatocalix[4]arene as a stationary phase methyllysine enrichment reagent for real-life cell-extracted proteins. Prior to the optimizations described in this thesis, cell-derived peptide extracts were not retained within an early generation upper-rim modified calixarene column. But with the new protocols detailed in this thesis, proteins extracted from both cultured prostate cancer cells and industrially sourced brewer’s yeast were successfully retained by a lower-rim modified calixarene column. Thousands of methylated proteins with diverse functions and cellular localization were discovered using this method. Detection of low abundance methylated proteins will aid our discovery of all cellular methylation marks, which in turn, will help delineate their biological functions.



Proteomics, Post translational modifications, Chromatography, Protein enrichment, Lysine methylation