SILAC-based assessment of S-palmitoylated proteins in mammalian cells by metabolic labeling and click-chemistry

Abstract

S-palmitoylation of cysteine residues is the only lipid-based posttranslational modification of proteins that is reversible and therefore has important implications in cellular function. S-palmitoylation has been associated with several cellular processes (e.g., cell signaling, protein transport, cell cycle, immune response, lipid metabolism, host–pathogen interaction) and human diseases, including neurological disorders, cancer, and infectious diseases. However, S-palmitoylation research has been hampered by the cumbersome experimental protocols necessary for its study. Currently, there are two main methodologies that, coupled with mass spectrometry (MS), allow the study of S-palmitoylated proteins proteome-wide. They mainly differ in the way of labeling palmitoylated proteins: one relies on “metabolic labeling” with a palmitic acid analog in living cells, while the other is based on “chemical labeling” of thiol groups derived from palmitoylated sites in extracted proteins. Although metabolic labeling is restricted to cultured cells, we will focus on this technique as it is more sensitive and specific than others. Here, we describe the protocol to measure palmitoylation in cancer cells using metabolic labeling coupled to SILAC-based mass spectrometry quantification which can be applied to other mammalian cell models. Facilitating the use of this methodology will extend the knowledge of palmitoylation signaling and unravel potential therapeutic avenues for diseases in which this unexplored modification is implicated.