title: Carboxymethylation of the human estrogen receptor ligand-binding domain-estradiol complex: HPLC/ESMS peptide mapping shows that cysteine 447 does not react with iodoacetic acid.
abstract: Experiments were carried out to determine the degree of solvent and reagent accessibility of the cysteines in the ligand-binding domain of the human estrogen receptor (hER LBD). The cysteine residues were alkylated when human ER LBD was present in its ligand (estradiol)-bound conformation. Direct electrospray ionization mass spectrometry (ESMS) as well as liquid chromatography coupled with ESMS, and matrix-assisted laser ionization desorption time-of-flight mass spectrometry were used to determine the location and the yield of the derivatized residues after proteolysis with trypsin. We observed that the cysteine 447 was protected against alkylation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.
authors.affiliation: Children's Hospital Oakland Research Institute, California 94609, USA.
authors.forename: G B
authors.initials: GB
authors.lastname: Hegy
authors.forename: C H
authors.initials: CH
authors.lastname: Shackleton
authors.forename: M
authors.initials: M
authors.lastname: Carlquist
authors.forename: T
authors.initials: T
authors.lastname: Bonn
authors.forename: O
authors.initials: O
authors.lastname: Engström
authors.forename: P
authors.initials: P
authors.lastname: Sjöholm
authors.forename: H E
authors.initials: HE
authors.lastname: Witkowska
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D007461:Iodoacetates / Q000737:chemistry*; D019807:Iodoacetic Acid; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007461:Iodoacetates; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D004958:Estradiol; D003545:Cysteine; D019807:Iodoacetic Acid