Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).
J Proteome Res
; 23(8): 3716-3725, 2024 Aug 02.
Article
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| 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Cisteína
/
Espectrometría de Masas en Tándem
Límite:
Animals
Idioma:
En
Revista:
J Proteome Res
Asunto de la revista:
BIOQUIMICA
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos