Essential cysteine-alkylation strategies to monitor structurally altered estrogen receptor as found in oxidant-stressed breast cancers.
Oxidant-induced structural modifications within the cysteine-rich DNA-binding domain (DBD) of the overexpressed estrogen receptor (ER) likely contribute to its loss of DNA-binding function and altered transcriptional activity during human breast cancer development. Using recombinant ER protein as a model, procedures to detect such endogenously produced structural changes in the two Cys(4)-type zinc fingers within the DBD of ER extracted from breast cancer cells are being developed. Unfortunately, ex vivo oxidation of these ER-DBD cysteine residues can occur during routine ER purification and preparation procedures. Also, cysteine residues readily undergo thiol-disulfide exchange reactions that can result in artificial oxidation and incorrect disulfide bond assignments. These problems can be circumvented by an initial irreversible alkylation of all free thiols followed by reduction of any disulfides and treatment with a second alkylating agent, prior to proteolysis and high-performance liquid chromatography mass spectrometry analysis of peptides in the doubly alkylated ER digest, to differentiate between the originally free and the disulfide-bonded cysteine residues. Although the use of chemically identical but isotopically different alkylating agents was more effective than the use of chemically different alkylating agents, subsequent problems were encountered with incomplete alkylation of particular Cys residues in the native ER protein. To overcome this limitation, the initial alkylation was accompanied by denaturation and the second alkylation was carried out during the proteolytic digestion. These improved analytical strategies should facilitate the monitoring of structurally altered endogenous ER produced within oxidant-stressed human breast cancer cells.
Meza
Jose E
JE
Buck Institute for Age Research (Program of Cancer and Developmental Therapeutics), Novato, CA 94945, USA.
Scott
Gary K
GK
Benz
Christopher C
CC
Baldwin
Michael A
MA
D000477:Alkylating Agents / Q000737:chemistry; D000478:Alkylation; D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000737:chemistry* / Q000175:diagnosis; D003545:Cysteine / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D011960:Receptors, Estrogen / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
D000477:Alkylating Agents; D011960:Receptors, Estrogen; D010450:Endopeptidases; D003545:Cysteine