Figure 1. Methods for the enrichment of S-acylated proteins: acyl-biotinyl exchange (ABE, left panel) and metabolic labeling with a palmitic acid analog followed by click chemistry (MLCC, right panel).

In ABE, free thiols are blocked by an alkylation reagent (shown as a red pentagon), such as methyl methanethiosulfonate or N-ethylmaleimide. S-acyl groups are specifically cleaved off by neutral hydroxylamine, and then the newly exposed cysteine residues are biotinylated by biotin-HPDP. Here, the green double pentagon shows a biotin group and the light blue square shows the linker group between the disulfide and biotin groups. The biotinylated (formerly S-acylated) proteins are captured by streptavidin affinity purification and eluted by a reducing agent. In MLCC, an alkyne- or azide-functionalized palmitate analog (e.g., 17-octadecynoic acid) is metabolically incorporated into native S-acylation sites by endogenous S-acylation machinery. After cell lysis, palmitate analog-labeled (S-acylated) proteins are conjugated to a biotin analog (e.g., azido-azo-biotin) by click chemistry, selectively enriched by streptavidin affinity purification, and eluted by sodium dithionite. In this panel, the green triple bond shows an alkyne group, the green double pentagon shows a biotin group, the green single pentagon shows a triazole group, and the orange square shows the linker group between the triazole and biotin groups.