title: Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry.
abstract: Trivalent arsenicals have high affinity for thiols (such as free cysteines) in proteins. We describe here the use of this property to develop a collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) technique for the identification of reactive cysteines in proteins. A trivalent arsenic species, dimethylarsinous acid (DMA (III)), with a residue mass (103.9607) and mass defect distinct from the normal 20 amino acids, was used to selectively label reactive cysteine residues in proteins. The CID fragment ions of the arsenic-labeled sequences shifted away from the more abundant normal fragments that would otherwise overlap with the ions of interest. Along with the internal and immonium ions, the arsenic-labeled fragment ions served as MS/MS signatures for identification of the binding sites and for assessment of the relative reactivity of individual cysteine residues in a protein. Using this method, we have identified two highly reactive binding sites in rat hemoglobin (Hb): Cys-13alpha and Cys-125beta. Cys-13alpha was bound to DMA (III) in the Hb of rats fed with arsenic, and this binding was responsible for arsenic accumulation in rat blood, while Cys-125beta was found to bind to glutathione in rat blood. This study revealed the relative reactivity of the cysteines in rat Hb in the following decreasing order: Cys-13alpha >> Cys-111alpha > Cys-104alpha and Cys-13alpha >> Cys-125beta > Cys-93beta. Arsenic-labeling is easy and fast for identification of active binding sites without enzymatic digestion and acid hydrolysis, and useful for characterization and identification of metal binding sites in other proteins.
authors.affiliation: Department of Chemistry and Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Meiling
authors.initials: M
authors.lastname: Lu
authors.forename: Hailin
authors.initials: H
authors.lastname: Wang
authors.forename: Zhongwen
authors.initials: Z
authors.lastname: Wang
authors.forename: Xing-Fang
authors.initials: XF
authors.lastname: Li
authors.forename: X Chris
authors.initials: XC
authors.lastname: Le
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002101:Cacodylic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D003545:Cysteine / Q000032:analysis*; D004912:Erythrocytes / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D006454:Hemoglobins; C472511:dimethylarsinous acid; D002101:Cacodylic Acid; D005978:Glutathione; D003545:Cysteine