Bioorthogonal proteomics of 15-hexadecynyloxyacetic acid chemical reporter reveals preferential targeting of fatty acid modified proteins and biosynthetic enzymes

Chemical reporters are powerful tools for the detection and discovery of protein modifications following cellular labeling. The metabolism of alkyne- or azide-functionalized chemical reporters in cells can influence the efficiency and specificity of protein targeting. To evaluate the effect of degradation of chemical reporters of protein fatty acylation, we synthesized 15-hexadecynyloxyacetic acid (HDYOA), a reporter that was designed to be resistant to β-oxidation, and compared its ability to label palmitoylated proteins with an established reporter, 17-octadecynoic acid (ODYA). HDYOA was able to label known candidate S-palmitoylated proteins similarly to ODYA. Accordingly, bioorthogonal proteomic analysis demonstrated that 70% of proteins labeled with ODYA were also labeled with HDYOA. However, the proteins observed differentially in our proteomic studies suggested that a portion of ODYA protein labeling is a result of β-oxidation. In contrast, downstream enzymes involved in β-oxidation of fatty acids were not targeted by HDYOA. Since HDYOA can label S-palmitoylated proteins and is not utilized by downstream β-oxidation pathways, this fatty acid chemical reporter may be particularly useful for bioorthogonal proteomic studies in cell types metabolically skewed toward fatty acid breakdown.