Global profiling of dynamic protein palmitoylation

The reversible thioester linkage of palmitic acid on cysteines is known as protein S-palmitoylation, which facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analogue 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling of cells (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that show rapid turnover. Treatment with a serine lipase-selective inhibitor identified a special pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncogenes and other proteins linked to aberrant cell growth, migration, and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.