The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC ‘hole’ mutants and ‘bumped’ chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical–genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

23 个含有 Asp-His-His-Cys 基序 （ZDHHC） 的人锌指 S-酰基转移酶在由数百种对正常生理学重要或疾病失调的蛋白质组成的广泛网络中催化半胱氨酸残基的长链 S-酰化。在这里，我们提出了一种技术，可以在完整细胞的整个蛋白质组水平上直接定位特定 ZDHHC 的蛋白质底物。配对 ZDHHC“空穴”突变体和“凸块”化学标记脂肪酸探针的结构引导工程使探针能够转移到特定的蛋白质底物上，比野生型 ZDHHC 具有极好的选择性。化学遗传系统以 5 种人 ZDHHC（3、7、11、15 和 20）为例，并应用于生成从头 ZDHHC 底物谱，鉴定 >300 底物和 S-跨多个细胞系的新功能多样化蛋白质的酰化位点。我们预计该平台将阐明各种模型和生物体的 S-酰化生物学。