Quantitative analysis of S-acylation

S-acylation is a protein post-translational modification that relies on the attachment of a hydrophobic fatty acid chain to a cysteine residue through the formation of a thioester bond. This reversible modification is controlled by the counteraction of protein acyltransferases and acyl protein thioesterases. The bonded lipid moiety can modulate the physicochemical properties of the substrate proteins, their membrane-binding affinity, subcellular localization, protein stability, or interactions with other proteins or cell components, thereby playing a key role in several cell processes such as protein trafficking, signal transduction, or cell proliferation. Moreover, increasing evidence has associated S-acylation malfunction with several pathological processes, including various types of cancers or neurodegenerative disorders, making S-acylation and its controlling enzymes an ideal target for therapeutic strategies. S-acylation has been commonly known as S-palmitoylation because palmitic acid was considered the predominant fatty acid attached to proteins. However, recent advances, especially in the mass spectrometry field, have suggested a diversity in the identity of the attached lipids greater than that previously considered. Moreover, since variations in the length and saturation degree of the fatty acyl chains may alter the intracellular localization and biological function of proteins, its potential role in the regulation of protein function is just being explored. However, this heterogeneous lipid composition could not be systematically studied, mainly due to the lack of suitable methods. In order to address these challenges, this protocol reports a method to identify and quantify the fatty acids attached to S-acylated proteins.