Fig. 1.

Quantitative chemical proteomics reveals potential LCFA-CoA–binding proteins. (A) Structure of C14-CoA-biotin probe. (B) Workflow for SILAC experiments to profile LCFA-CoA–binding proteins. (C) Results of the SILAC experiments with HEK293T cell lysates. Scatterplot of log₂(H/L ratio) of identified proteins in forward SILAC against log₂(L/H ratio) of proteins identified in reverse SILAC. Proteins with >1.5-fold enrichment (red dashed line) in both forward and reverse SILAC were considered as possible LCFA-CoA interacting proteins. Red spots are proteins known to use LCFA-CoA as substrates, blue spots are proteins reported to be regulated by LCFA-CoA in vitro, and NME1/2 are in pink. (D) Biological process analysis of the LCFA-CoA–binding proteins. (E) Partial list of the top hits from the SILAC experiment. (F) Validation of some identified proteins by C14-CoA-biotin pull-down and Western blot in cell lysate. Nonhydrolyzable myristoyl CoA (NH-Myr-CoA) is used as a competitor. (G) Validation of some identified proteins by C14-CoA-biotin probe pull-down and Western blot with purified proteins. C16:0 CoA or CoASH was used as a competitor. LC-MSMS, liquid chromatography-tandem mass spectrometry; Strep., streptavidin; IP, immunoprecipation; FA, fatty acid; CoASH, free coenzyme A.