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

Abstract

Protein cysteine thiols undergo reversibleS-acylation via a thioester linkage in vivo.S-palmitoylation, modification by C16:0 fatty acid, is a commonS-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 usedS-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 ofS-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 devisedAcyl-Trap, a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediatedS-acyl enrichment from 20-50 micrograms of input protein. The method is compatible with protein-level detection ofS-acylated proteins as well asS-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.

CysteineS-acetylation is a post-translational modification involved in metabolic regulation

“…Due to the presence of a reactive thiol, cysteine is an amino acid that plays a key role in enzyme catalysis. Beyond catalysis, cysteine side chains are frequently covalently modified, with succination 22 , nitrosylation 23 , and palmitoylation 24,25 being some of the more common cysteine-based PTMs. Reactive oxygen species (ROS) are also mediated through post-translational modification of the cysteine side chain.…”

“…These studies have repeatedly identified many sites of modification that are not positively identified as being palmitoylated by MS. Short-chain acyl modifications to cysteine were not known in the literature and not searched for and hence could not be observed even if present. 24,33,34 The presence of thioester bound acyl-cysteine sites that did not map to known sites of palmitoylation hinted at the existence of short-chain cysteine acylomes.…”

“…Previous studies characterizing the long-chain cysteine acylome have used a variety of chemical approaches to achieve enrichment. 24,33,34 Unfortunately, these approaches all fail to differentiate between long-chain and short-chain acylation. The ability to either enrich for specific S-acyl modifications, or match specific enriched sites to specific acyl modifications will be key to better understanding of the physiological role of short-chain acyl modifications of cysteine.…”