Quantification of Cysteine Oxidation in Human Estrogen Receptor by Mass Spectrometry

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• 作者：Christian Atsriku ; Christopher C. Benz ; Gary K. Scott ; Bradford W. Gibson ; Michael A. Baldwin
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：April 15, 2007
• 年：2007
• 卷：79
• 期：8
• 页码：3083 - 3090
• 全文大小：348K
• 年卷期：v.79,no.8(April 15, 2007)
• ISSN：1520-6882

文摘

Redox-dependent modifications of sulfhydryl groups withinthe two Cys₄ zinc fingers of the estrogen receptor DNA-binding domain (ER-DBD) result in structural damageand loss of ER DNA-binding function, which parallels thesituation observed in many ER-positive breast cancers.Quantitation of the redox status of cysteinyl thiols withinER-DBD employed cysteine-specific oxidants to inducevarying degrees of oxidation in recombinant ER, followedby differential alkylation with the stable isotopic labelingreagents [¹²C₂]-iodoacetic acid and [¹³C₂]-bromoaceticacid. Subsequent proteolysis with LysC/Asp-N generateddiagnostic peptides of which the C-terminal peptide of thesecond zinc finger is most strongly detected by massspectrometry (MS) and serves as a suitable marker of ER-DBD redox status. Data were collected from two differentMALDI-MS instruments: a time-of-flight and a linear iontrap (vMALDI-LIT). An analogous but larger syntheticpeptide 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 bothinstruments. This was also true of tandem MS/MS datafrom the vMALDI-LIT, which should facilitate selectedreaction monitoring. Relative quantitation by MS alsoclosely matched data from immunochemical methods.