ZDHHC S-acyltransferases catalyse long-chain S-acylation at cysteine residues of >1000 proteins, including >140 oncoproteins. However, protein S‐acylation presents an analytical challenge with no approaches that directly identify or modulate substrates specific to a given ZDHHC. Here we present a transformational chemical genetic system for the deconvolution of ZDHHC substrates and S‐acylation sites that circumvents the problems of lability, ionisation, and solubility of native S-acylated peptides in LC-MS:. https://www.nature.com/articles/s41587-023-02030-0. This system enables direct proteome‐wide profiling of cognate ZDHHC substrates and S‐acylation sites, and identification and validation of novel substrates specific or common to sets of ZDHHCs for the first time:

Through structure guided design and homology modelling we developed and optimised a ZDHHC20 "hole" mutant and a "bumped" chemically tagged fatty acid probe. HEK293T, MDA-MB 231 and PANC1 cells transfected with WT ZDHHC20 or mutant ZDHHC20 were treated with a bumped 18-carbon fatty acid probe and proteins recovered following lysis were subjected to CuAAC ligation with azido biotin capture reagent. Non-acylated cysteines were blocked by N-ethylmaleimide treatment and labelled proteins enriched on resin. Thioester linkages were hydrolysed on-bead by hydroxylamine treatment and previously S-acylated cysteine residues capped with chloroacetamide. Followed by off-resin trypsin digestion and nanoLC-MS/MS analysis. This approach was expanded to two other ZDHHCs (7 and 15).

Mutant ZDHHCs efficiently load and transfer "bumped" probes whilst WT ZDHHCs do not. Lipidomic analysis shows that bumped probes are activated in cells without disrupting endogenous biosynthetic pathways. 99 potential S-acylation sites were identified, including ZDHHC20 auto-S-acylation and 28 sites previously reported in S-acylation studies. Known ZDHHC20 substrate IFITM3 was among the most enriched proteins, demonstrating sensitivity sufficient to identify substrates at endogenous expression levels. Versatility and adaptability were demonstrated by identification of substrates and sites of modification in MDA-MB-231 and PANC1 cell lines. ZDHHC7 and ZDHHC15 systems identified substrates across three cell lines, alongside sites of modification across multiple proteins. Among the 301 unique substrates identified, we observed common and distinct substrates between ZDHHCs with a degree of redundancy across the family. Comparisons with conventional chemical proteomics and proximity labelling approaches reveals chemical genetics as a complementary approach by circumventing redundancy within ZDHHC substrate networks, whilst enhancing specificity and sensitivity towards substrates. Our system unlocks the potential of drug development from specific ZDHHCs through deepening our understanding of the pathways regulating S‐acylation.