Meza Jose E, Scott Gary K, Benz Christopher C, Baldwin Michael A
Buck Institute for Age Research (Program of Cancer and Developmental Therapeutics), Novato, CA 94945, USA.
Anal Biochem. 2003 Sep 1;320(1):21-31. doi: 10.1016/s0003-2697(03)00296-3.
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.
在人乳腺癌发展过程中，过表达的雌激素受体（ER）富含半胱氨酸的DNA结合结构域（DBD）内由氧化剂诱导的结构修饰可能导致其DNA结合功能丧失及转录活性改变。以重组ER蛋白为模型，正在开发检测从乳腺癌细胞中提取的ER的DBD内两个Cys(4)型锌指中此类内源性产生的结构变化的方法。不幸的是，在常规ER纯化和制备过程中，这些ER-DBD半胱氨酸残基可能会发生体外氧化。此外，半胱氨酸残基容易发生硫醇-二硫键交换反应，这可能导致人为氧化和不正确的二硫键分配。在对双烷基化ER消化产物中的肽进行蛋白水解和高效液相色谱质谱分析之前，通过对所有游离硫醇进行初始不可逆烷基化，随后还原任何二硫键并用第二种烷基化剂处理，以区分最初游离的和形成二硫键的半胱氨酸残基，可避免这些问题。尽管使用化学性质相同但同位素不同的烷基化剂比使用化学性质不同的烷基化剂更有效，但在天然ER蛋白中特定Cys残基的烷基化不完全会带来后续问题。为克服这一限制，初始烷基化时进行变性处理，并在蛋白水解消化过程中进行第二次烷基化。这些改进的分析策略应有助于监测在氧化应激的人乳腺癌细胞内产生的结构改变的内源性ER。
