Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods

Hurst, C. H., Turnbull, D., Plain, F., Fuller, W. and Hemsley, P. A. (2017) Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods. BioTechniques, 62(2), pp. 69-75. (doi: 10.2144/000114516) (PMID:28193150) (PMCID:PMC5400063)

Abstract

S-palmitoylation (S-acylation) is an emerging dynamic post-translational modification of cysteine residues within proteins.Current assays for protein S-palmitoylation involve either in vivo labelling or chemical cleavage of S-palmitoyl groupsto reveal a free cysteine sulfhydryl that can be subsequently labelled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acylexchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using Nethylmaleimide, 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 and also requires a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of N-ethylmaleimide 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 hands-on and overall time required for assays.

• Item Type: Articles
• Additional Information: This work was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/M024911/1 to P.A.H. and British Heart Foundation (B.H.F.) PhD studentship FS/14/68/30988 to W.F.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Fuller, Professor Will
• Authors: Hurst, C. H., Turnbull, D., Plain, F., Fuller, W., and Hemsley, P. A.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: BioTechniques
• Publisher: Future Medicine
• ISSN: 0736-6205
• ISSN (Online): 1940-9818
• Copyright Holders: Copyright © 2017 Future Medicine
• First Published: First published in BioTechniques 62(2):69-75
• Publisher Policy: Reproduced in accordance with the copyright policy of the publisher