Quantification of cysteine oxidation in human estrogen receptor by mass spectrometry.
Redox-dependent modifications of sulfhydryl groups within the two Cys4 zinc fingers of the estrogen receptor DNA-binding domain (ER-DBD) result in structural damage and loss of ER DNA-binding function, which parallels the situation observed in many ER-positive breast cancers. Quantitation of the redox status of cysteinyl thiols within ER-DBD employed cysteine-specific oxidants to induce varying degrees of oxidation in recombinant ER, followed by differential alkylation with the stable isotopic labeling reagents [12C2]-iodoacetic acid and [13C2]-bromoacetic acid. Subsequent proteolysis with LysC/Asp-N generated diagnostic peptides of which the C-terminal peptide of the second zinc finger is most strongly detected by mass spectrometry (MS) and serves as a suitable marker of ER-DBD redox status. Data were collected from two different MALDI-MS instruments: a time-of-flight and a linear ion trap (vMALDI-LIT). An analogous but larger synthetic peptide treated with three isotopic variants of the alkylating reagent modeled isotopic overlaps that might complicate the relative quantitation of cysteine oxidation. Despite the isotopic overlaps, excellent relative quantitation was achieved from MS data obtained from both instruments. This was also true of tandem MS/MS data from the vMALDI-LIT, which should facilitate selected reaction monitoring. Relative quantitation by MS also closely matched data from immunochemical methods.
Atsriku
Christian
C
Buck Institute for Age Research, Novato, California 94945, USA.
Benz
Christopher C
CC
Scott
Gary K
GK
Gibson
Bradford W
BW
Baldwin
Michael A
MA
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D047628:Estrogen Receptor alpha; D003545:Cysteine