Description:
<jats:p>Protein S‐acylation is an abundant post‐translation modification required for the trafficking, compartmentalization, and membrane‐tethering of numerous key signaling proteins. Traditionally, proteins are identified as targets of palmitoylation by metabolic labeling with 3H‐palmitate, and the specific cysteine sites of palmitoylation are inferred by mutagenesis. A recent method has been developed to replace acyl‐thioesters with affinity tags for enrichment and proteomic identification in yeast, yet this relies on the fidelity of the exchange reaction and is hampered by numerous false positives. We have developed a bioorthogonal palmitoylation probe for metabolic incorporation at in vivo palmitoylation sites in cultured mammalian cells. After enrichment and trypsin digestion, peptides are analyzed using an LTQ ion‐trap mass spectrometer using multidimensional protein identification technology (MudPIT). Hundreds of palmitoylated proteins have been identified with the potential to identify the exact modified cysteines. Various cell types, stimuli and labeling times have been profiled, revealing novel sites of dynamic palmitoylation across complex proteomes. This research was funded by F32NS060559, <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="DDBJ/EMBL/GenBank" xlink:href="CA087660">CA087660</jats:ext-link>, and the Skaggs Institute for Chemical Biology.</jats:p>