Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.
Biotechniques
; 62(2): 69-75, 2017 02 01.
Article
in En
| MEDLINE
| ID: mdl-28193150
ABSTRACT
S-palmitoylation (S-acylation) is emerging as an important dynamic post-translational modification of cysteine residues within proteins. Current assays for protein S-palmitoylation involve either in vivo labeling or chemical cleavage of S-palmitoyl groups to reveal a free cysteine sulfhydryl that can be subsequently labeled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acyl-exchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using N-ethylmaleimide (NEM), to prevent non-specific detection. This in turn necessitates multiple precipitation-based clean-up steps to remove reagents between stages, often leading to variable sample loss, reduced signal, or protein aggregation. These combine to reduce the sensitivity, reliability, and accuracy of these assays, which also require a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of NEM by 2,3-dimethyl-1,3-butadiene in an aqueous Diels-Alder 4+2 cyclo-addition reaction, it is possible to greatly improve sensitivity and accuracy while reducing the hands-on time and overall time required for the assay.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Proteins
/
Protein Processing, Post-Translational
/
Maleimides
Language:
En
Journal:
Biotechniques
Year:
2017
Document type:
Article
Affiliation country:
United kingdom