Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis.
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.
Chan
Hong-Lin
HL
Ludwig Institute for Cancer Research, Department of Biochemistry and Molecular Biology, University College London, London, UK.
Gharbi
Severine
S
Gaffney
Piers R
PR
Cramer
Rainer
R
Waterfield
Michael D
MD
Timms
John F
JF
D000818:Animals; D002232:Carbocyanines / Q000138:chemical synthesis; D002460:Cell Line; D003545:Cysteine; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D018734:Genes, erbB-2 / Q000502:physiology*; D007091:Image Processing, Computer-Assisted; D008239:Lysine; D018832:Molecular Chaperones; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis
D002232:Carbocyanines; D005456:Fluorescent Dyes; D018832:Molecular Chaperones; D011506:Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5; D008239:Lysine; D003545:Cysteine