Essential cysteine-alkylation strategies to monitor structurally altered estrogen receptor as found in oxidant-stressed breast cancers Jose E. Meza, a,b,c Gary K. Scott, a Christopher C. Benz, a,d and Michael A. Baldwin b, * a Buck Institute for Age Research (Program of Cancer and Developmental Therapeutics), Novato, CA 94945, USA b Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA c Life Sciences and Chemical Analysis, Agilent Technologies, 1601 California Ave., Palo Alto, CA 94304, USA d Comprehensive Cancer Center and Division of Oncology-Hematology, University of California, San Francisco, CA 94143, USA Received 26 November 2002 Abstract 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 as- signments. 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. Ó 2003 Elsevier Science (USA). All rights reserved. The DNA-binding and transactivating functions of many transcription factors are redox sensitive and reg- ulated by oxidation of critical thiol groups within their DNA-binding domains (DBDs) 1 [1]. In particular, zinc finger transcription factors contain oxidant-sensitive and zinc-coordinating cysteine residues essential for their binding specificity within the major groove of DNA [1–3]. Tissues subjected to aging or repeated epi- sodes of hypoxia–reperfusion (such as aggressive tumors outgrowing their vascular supply) accumulate extra- and intracellular damage from excess exposure to free radi- cals and reactive oxygen species. Interestingly, normal organ aging is associated with in vivo accumulation of oxyradical protein damage and selective loss of DNA- binding activity by the Cys 2 His 2 -type zinc finger tran- scription factor Sp1; this dithiothreitol (DTT)-reversible loss of DNA-binding function occurs without any sig- nificant decline in overall Sp1 tissue content [1,2]. Likewise, an age-dependent loss of Sp1 DNA binding without loss of total Sp1 content is observed in human breast cancers [4]. The Cys 4 -type zinc finger estrogen receptor (ER), whoseoverexpressionisthoughttounderliedevelopment of most human breast cancers, appears to be at least as sensitive to oxidative stress as the Cys 2 His 2 -type zinc Analytical Biochemistry 320 (2003) 21–31 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: 415-502-1655. E-mail address: mikeab@itsa.ucsf.edu (M.A. Baldwin). 1 Abbreviations used: DBD, DNA-binding domain; DTT, dith- iothreitol; ER, estrogen receptor; EMSA, electrophoretic mobility shift assay; ICAT, isotope-coded affinity tag; IAA, iodoacetic acid; IAC, iodoacetamide; Gdn  HCl, guanidine hydrochloride; BAA**, [ 13 C 2 ]bromoacetic acid; ESIMS, electrospray ionization mass spec- trometry; oa-TOF, orthogonal acceleration time-of-flight. 0003-2697/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00296-3