A Tetrazine-Terminal Alkene System for Profiling Protein Fatty Acylation Modifications

Abstract

Protein fatty acylation is included in the class of modifications where attachment via an acyl linkage is employed including myristoylation and palmitoylation. This work focuses on applying the novel tetrazine moiety to covalently target alkene functionalized fatty acids via a highly sensitive and selective inverse electron demand Diels alder cycloaddition (iEDDA) reaction with accompanied visualization via in gel fluorescence imaging or western blot analysis. This strategy was used in this study to compare the incorporation of the terminal alkene versus alkyne fatty acids mimics, by labeling of the modified proteins via iEDDA and Copper assisted Azide-Alkyne Cycloadditon (CuAAC), respectively. The successful labeling of the incorporated terminal alkene fatty acid provides another strategy for investigating fatty acyl modifications and so assist in validating the established library of curated fatty acyl modifications. Additionally, while differences in the location and intensity of labeling was observed between the alkene and alkyne fatty acid surrogates utilized in this study it was impossible to formulate conclusions on the effectiveness of the iEDDA labeling method over the traditionally used CuAAC strategies without quantitative mass spectroscopy data.