title: OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins.
abstract: In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.
authors.affiliation: a Sir William Dunn School of Pathology, University of Oxford , Oxford , UK.|c School of Biological Sciences, University of Reading , UK.
authors.forename: Lisa-Marie
authors.initials: LM
authors.lastname: Holbrook
authors.affiliation: a Sir William Dunn School of Pathology, University of Oxford , Oxford , UK.
authors.forename: Lai-Shan
authors.initials: LS
authors.lastname: Kwong
authors.affiliation: a Sir William Dunn School of Pathology, University of Oxford , Oxford , UK.
authors.forename: Clive L
authors.initials: CL
authors.lastname: Metcalfe
authors.affiliation: b CNRS, Institut des Sciences Biologiques , Marseille , France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Fenouillet
authors.affiliation: c School of Biological Sciences, University of Reading , UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Jones
authors.affiliation: a Sir William Dunn School of Pathology, University of Oxford , Oxford , UK.
authors.forename: A Neil
authors.initials: AN
authors.lastname: Barclay
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D005033:Ethylmaleimide / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational
chemical_list: D000911:Antibodies, Monoclonal; D015699:HIV Envelope Protein gp120; C514793:gp120 protein, Human immunodeficiency virus 1; C042945:N-ethylmaleimide-cysteine; D003545:Cysteine; D005033:Ethylmaleimide
keywords: Allosteric; detection; disulfide bond; labile; maleimide; reduction