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

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 LED). The cysteine residues were alkylated when human ER LED 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 alkyation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.