Chemical Proteomic Analysis of S-Fatty Acylated Proteins and Their Modification Sites

• DOI: 10.1007/978-1-4939-9532-5_4
• Publication Date: 2019-05-31T09:02:33Z
• Authors: Emmanuelle Thinon; Howard C. Hang
• Keywords: Thioester; Palmitoylation; Lipid-anchored protein; Hydroxylamine; Native chemical ligation; Chemical modification

Protein S-fatty-acylation, the covalent addition of a long-chain fatty acid, predominantly palmitate (S-palmitoylation), to cysteine, is a highly dynamic and regulated process that controls protein function and localization of membrane-associated proteins in eukaryotes. The analysis of S-fatty acylated peptides by mass spectrometry remains challenging due to the hydrophobic and potentially labile thioester linkage of the S-fatty acylated peptides.Here we describe an optimized protocol for the global analysis of S-palmitoylated proteins based on the combination of an alkyne-tagged chemical reporter of palmitoylation, alk-16 with hydroxylamine-selective hydrolysis of thioester bonds. This protocol decreased the number of false positive proteins and was applied to identify S-fatty acylation sites, providing modification sites for 44 proteins out of the 106 S-fatty acylated proteins identified.