Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Protein cysteine thiols undergo reversible S -acylation via a thioester linkage in vivo. S -palmitoylation, modification by C16:0 fatty acid, is a common S -acylation that mediates protein-membrane and protein-protein interactions critical to an array of biological processes, from homeostatic lung surfactant function to cellular transformation. The most widely used S -acylation assays, including acyl-biotin exchange (ABE) and acyl resin-assisted capture (Acyl-RAC), utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. ABE and Acyl-RAC have enabled both the proteome-wide identification of S -palmitoylation sites and basic biochemical studies. Yet, these assays generally utilize hundreds of micrograms to milligrams of input material and require numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome this, we devised “Acyl-Trap”, a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S -acyl enrichment from 20-50 micrograms of input protein. The method is compatible with protein-level detection of S -acylated proteins as well as S -acyl site-based identification and quantification using on-quartz isobaric (tandem mass tag) labeling and LC-MS/MS. Also described are conditions for long-term hydroxylamine storage, which further expedites the assay and minimizes waste. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional intact protein detection and chemoproteomic workflows.