title: Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system.
abstract: The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.
authors.affiliation: Ludwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, United Kingdom.
authors.forename: Severine
authors.initials: S
authors.lastname: Gharbi
authors.forename: Piers
authors.initials: P
authors.lastname: Gaffney
authors.forename: Alice
authors.initials: A
authors.lastname: Yang
authors.forename: Marketa J
authors.initials: MJ
authors.lastname: Zvelebil
authors.forename: Rainer
authors.initials: R
authors.lastname: Cramer
authors.forename: Michael D
authors.initials: MD
authors.lastname: Waterfield
authors.forename: John F
authors.initials: JF
authors.lastname: Timms
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D015870:Gene Expression / Q000187:drug effects; D018734:Genes, erbB-2; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification; D020890:Neuregulin-1 / Q000494:pharmacology; D020543:Proteome; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
chemical_list: D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D020890:Neuregulin-1; D020543:Proteome; C087230:heregulin beta1