Multivariable difference gel electrophoresis and mass spectrometry: A case study on transforming growth factor-β and ERBB2 signaling

David B. Friedman, Shizhen E. Wang, Corbin W. Whitwell, Richard M. Caprioli, Carlos L. Arteaga

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

Multivariable DIGE/MS was used to investigate proteins altered in expression and/or post-translational modification in response to activation of transforming growth factor (TGF)-β receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (Erbl32) oncogene. Proteome changes were monitored in response to exogenous TGF-β over time (0, 8, 24, and 40 h), and proteins were resolved using medium range (pH 4-7) and narrow range (pH 5.3-6.5) isoelectric focusing combined with up to 2 mg of protein to allow inspection of lower abundance proteins. Triplicate samples were prepared independently and analyzed together across multiple DIGE gels using a pooled sample internal standard to quantify expression changes with statistical confidence. Unsupervised principle component analysis and hierarchical clustering of the individual DIGE proteome expression maps provided independent confirmation of distinct expression patterns from the individual experiments and demonstrated high reproducibility between replicate samples. Fifty-nine proteins (including some isoforms) that exhibited significant kinetic expression changes were identified using mass spectrometry and database interrogation and were mapped to existing biological networks involved in TGF-β signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-β signaling.

Original languageEnglish (US)
Pages (from-to)150-169
Number of pages20
JournalMolecular and Cellular Proteomics
Volume6
Issue number1
DOIs
StatePublished - Jan 1 2007

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

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