title: Modification of peptide and protein cysteine thiol groups by conjugation with a degradation product of ascorbate.
abstract: Ascorbate is an important water-soluble antioxidant, which when oxidized by reactive oxygen species is converted into dehydroascorbate (DHA). If not rapidly reduced back to ascorbate, DHA decomposes to a reactive 5-carbon compound (DHA*, +130 Da) that can modify reduced cysteinyl residues in peptides and proteins in vitro. The formation of cysteine adducts by DHA* was characterized by mass spectrometry using reduced insulin B-chain, α-lactalbumin, and hemoglobin. Mass spectrometry of DHA* modified insulin B-chain revealed the presence of one and two DHA* adducts. Enzymatic cleavage and tandem mass spectrometry of modified peptides allowed unambiguous localization of DHA* to the two cysteine residues in positions 7 and 19 of the insulin B-chain. Incubations of DHA with α-lactalbumin revealed that approximately 25% of the protein population was in a reduced state and could be modified by DHA*. The adduct was assigned to the N-terminally located cysteinyl residue in position 6. Incubation of hemoglobin with DHA followed by pepsin digestion and electrospray ionization tandem mass spectrometry (ESI-MSMS) of the peptide mixture allowed for the identification of three modified peptides. Tandem mass spectrometry of the modified peptides, two from the hemoglobin A-chain with identical mass and one from the hemoglobin B-chain, gave a complete series of y-type fragment ions, which were assigned to the cysteine containing peptides (100)LLSHCL(105) (A-chain), (101)LSHCLL(106) (A-chain), and (111)VCVLAHHFGKE(121) (B-chain). Although the DHA* adduct was lost from the peptides derived from α-lactalbumin and hemoglobin before fragmentation of the peptide bond, carbamidomethylation of the proteins prior to incubation with DHA abolished the formation of DHA*-protein adducts and confirmed that the target was indeed the cysteine thiol group. Future studies are focused on the modification of proteins by DHA* in cells and in vivo.
authors.affiliation: Department of Pharmacology, ‡Department of Nuclear Medicine and Radiobiology, §Institut de pharmacologie de Sherbrooke and Department of Surgery/Urology division, Faculty of Medicine and Health Sciences, University of Sherbrooke , Sherbrooke, QC, Canada J1H 5N4.
authors.forename: Phyla
authors.initials: P
authors.lastname: Kay
authors.forename: J Richard
authors.initials: JR
authors.lastname: Wagner
authors.forename: Hugo
authors.initials: H
authors.lastname: Gagnon
authors.forename: Robert
authors.initials: R
authors.lastname: Day
authors.forename: Klaus
authors.initials: K
authors.lastname: Klarskov
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D011972:Receptor, Insulin / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006454:Hemoglobins; D013438:Sulfhydryl Compounds; D007768:Lactalbumin; D011972:Receptor, Insulin; D003545:Cysteine; D001205:Ascorbic Acid