Difference Gel Electrophoresis (DIGE)

Originally published in: Proteomics in Drug Research. Edited by Michael Hamacher, Katrin Marcus, Kai Stuhler, Andre van Hall, Bettina Warscheid and Helmut E. Meyer. Copyright © 2006 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-31226-9 In the last years the application of two-dimensional electrophoresis (2-DE) has often been declared outdated and a new century of gel-free proteomics was announced. Nevertheless, 2-DE is still the method of choice when analyzing complex protein mixtures. With a separation of 10 000 proteins, 2-DE gives access to high-resolution proteome analysis. Continuous development has consolidated 2-DE application in proteomics, where the introduction of difference gel electrophoresis (DIGE) is the latest improvement. DIGE is based on fluorescently tagging all proteins in each sample with one set of matched fluorescent dyes designed to minimally interfere with protein mobility during 2-DE. For a DIGE analysis, two different fluorescence dyes, CyDye® minimal dyes and CyDye saturation dyes, are available. CyDye minimal dyes react with an NHS-ester bond of lysine ɛ-amino residues and enable coelectrophoresis of up to three different samples in one approach. For special applications, e.g., samples from microdissection, the CyDye saturation dyes allow complete 2-D analysis and quantification of protein abundance changes in scarce sample amounts. The dyes react via a maleimide group with all available cysteine residues in the protein sample, giving a high labeling concentration. Here we show the application of both CyDye types for the analysis of relevant clinical samples and an approach for evaluating DIGE data statistically. We used CyDye minimal dyes to detect kinetic proteome changes resulting from ligand activation of either tyrosine kinases TrkA or TrkB in neuroblastoma cells. TrkA and TrkB are members of the family of neurotrophin receptors, which mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. In addition, we applied CyDye saturation dyes for the identification of new molecular markers of the pancreatic tumor progression. One thousand microdissected cells were analyzed from different pancreatic intraepithelial neoplasias (PanIN) grades, the precursor lesion of pancreatic ductal adenocarcinoma (PDAC). Based of the multiplexing strategy and the application of an internal standard, DIGE enables one to perform quantitative proteomics with high accuracy allowing statistical approaches for high-confidence data analysis. The sections in this article are Introduction Difference Gel Electrophoresis: Next Generation of Protein Detection in 2-DE Application of CyDye DIGE Minimal Fluors (Minimal Labeling with CyDye DIGE Minimal Fluors) General Procedure Example of Use: Identification of Kinetic Proteome Changes upon Ligand Activation of Trk-Receptors Application of Saturation Labeling with CyDye DIGE Saturation Fluors General Procedure Example of Use: Analysis of 1000 Microdissected Cells from PanIN Grades for the Identification of a New Molecular Tumor Marker Using CyDye DIGE Saturation Fluors Statistical Aspects of Applying DIGE Proteome Analysis Calibration and Normalization of Protein Expression Data Detection of Differentially Expressed Proteins Sample Size Determination Further Applications Keywords: proteomic technologies; difference gel electrophoresis (DIGE); two-dimensional gel electrophoresis for clinical research; protein detection in 2-DE; statistical aspects of applying DIGE proteome analysis