Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).
J Proteome Res
; 23(8): 3716-3725, 2024 Aug 02.
Article
in En
| MEDLINE
| ID: mdl-39008777
ABSTRACT
Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C160 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 µg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, 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. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 µg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Cysteine
/
Tandem Mass Spectrometry
Limits:
Animals
Language:
En
Journal:
J Proteome Res
Journal subject:
BIOQUIMICA
Year:
2024
Document type:
Article
Affiliation country:
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