Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Abstract

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.

Oxidative stress response: a proteomic view

“…There are only a few studies of this type, examining how mammalian cells can adapt to a constitutive, increased oxidative stress. A good example is provided for myeloid [21] and epithelial cells [22] . These papers…”

“…Besides the induction of some redox regulators (peroxiredoxins in [21] and [22], superoxide dismutase in [20]) a change in only a few enzymes involved in the glycolysis pathway was observed. In addition, only a few proteins involved in protein folding are induced.…”

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…Cys residues are also less likely to be at trypsin cleavage sites. As such, proteins labeled at cys residues can still be identified by MS following standard digestion protocols [133,146,147]. Focusing on thiols also allows the pIs of labeled proteins to be maintained since the dyes are neutrally charged.…”

“…Iodoacetylated cyanine dyes (ICy3 and ICy5) are a variation of the commercially available maleimides that were originally synthesized non-commercially [147]. The only reported LLD value was for BSA, a protein with 35 cys residues (5.8% of the total amino acid complement), at 2 ng [20].…”

Proteomics in human cancer research

“…It demonstrates some relationship triggering both kinds of breast carcinomas. 2-D DIGE technology was also successfully applied in breast cells and epithelial cells with ErbB-2 overexpression [101,102]. Li et al [103] used the SELDI-TOF in combination with bioinformatics tests to identify three serum biomarkers with high sensitivity and specificity for detection of breast cancer.…”