Quantitative Chemical Proteomics of Dynamic Palmitoylation

Abstract

Protein palmitoylation is an essential post‐translational modification necessary for trafficking and localization of numerous regulatory proteins that play key roles in cell growth and signaling. The commercially available fatty acid analog 17‐octadecynoic acid is readily metabolically incorporated in living cells at endogenous sites of protein palmitoylation. Labeled proteins can be chemically coupled to azide‐linked reporters for robust non‐radioactive analysis by multiple platforms, including fluorescent gel‐based detection and mass spectrometry‐based identification. This approach shows unprecedented sensitivity for profiling palmitoylated proteins in complex biological systems, leading to the identification of hundreds of palmitoylated proteins in human cell lines and mouse primary cells. In order to understand the processes regulating dynamic palmitoylation, we have developed a quantitative platform for global comparative proteomic analysis of palmitoylated proteins, including identification of exact sites of palmitoylation. This platform has been applied to interrogate the population of palmitoylated proteins regulated by both palmitoyl transferases and thioesterases by using novel small molecule inhibitors, shRNA knockdowns, and physiological stimulation. Funded by F32NS060559, CA087660 and the Skaggs Institute for Chemical Biology.