Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis
Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis
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DOI：
10.1002/pmic.200401300
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发表时间：
2005-07-01
期刊：
影响因子：
3.4
通讯作者：
Timms, JF
中科院分区：
文献类型：
--
作者：
Chan, HL;Gharbi, S;Timms, JF
利用标记试剂修饰选定的氨基酸进行蛋白质差异表达分析已成为定量蛋白质组学的主要方法。一种这样的方法，二维差异凝胶电泳（2-D DIGE），使用一组匹配的荧光N-羟基琥珀酰亚胺基（NHS）酯花青染料来标记不同样品中的赖氨酸残基，其可以在相同的凝胶上同时运行。在这里，我们报告了碘乙酰化花青（ICy）染料的使用（用于标记半胱氨酸硫醇，用于基于2-D DIGE的氧化还原蛋白质组学）。ICy染料标记的表征有关其化学计量，灵敏度和特异性的描述，以及比较ICy染料与NHS-Cy染料标记和几种蛋白质染色方法。我们已经优化了非还原，变性样品的标记条件，并报告了增加的灵敏度为一个子集的含巯基的蛋白质，允许准确监测氧化还原依赖的巯基修饰和表达的变化，半胱氨酸标记，然后结合赖氨酸标记的多重2-D DIGE蛋白质组学研究氧化还原依赖性和ErbB 2依赖性的变化暴露于氧化应激的上皮细胞。这项研究确定了参与细胞氧化还原调节，蛋白质折叠，增殖抑制，糖酵解和细胞骨架组织的差异修饰蛋白，揭示了氧化应激反应的复杂性和ErbB 2过表达对这种反应的影响。
Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.