title: Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.
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.
authors.affiliation: Division of Plant Sciences, School of Life Sciences, University of Dundee, Dundee, UK; Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, UK.
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Hurst
authors.affiliation: Division of Plant Sciences, School of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Dionne
authors.initials: D
authors.lastname: Turnbull
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, School of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, UK.
authors.forename: Fiona
authors.initials: F
authors.lastname: Plain
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, School of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, UK.
authors.forename: William
authors.initials: W
authors.lastname: Fuller
authors.affiliation: Division of Plant Sciences, School of Life Sciences, University of Dundee, Dundee, UK; Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, UK.
authors.forename: Piers A
authors.initials: PA
authors.lastname: Hemsley
mesh_terms: D000215:Acylation; D000475:Alkenes / Q000737:chemistry; D017360:Arabidopsis; D002073:Butanes / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005033:Ethylmaleimide; D054878:Lipoylation; D008301:Maleimides / Q000737:chemistry* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D000475:Alkenes; D002073:Butanes; D008301:Maleimides; D011506:Proteins; C436724:2,3-dimethyl-1,3-butadiene; D003545:Cysteine; D005033:Ethylmaleimide
keywords: ABE; Diels-Alder; N-ethylmaleimide; S-acylation; S-palmitoylation; acyl biotin exchange; acyl-RAC; biotin switch; cysteine; maleimide; palmitoylation; thiol