Multivariable Difference Gel Electrophoresis and Mass Spectrometry

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 (ErbB2) 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. 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 (ErbB2) 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. 2D 1The abbreviations used are: 2D, two-dimensional; TGF, transforming growth factor; PCA, principle component analysis; HC, hierarchical clustering; P-, phospho-; MAPK, mitogen-activated protein kinase; ANOVA, analysis of variance; MOWSE, Molecular Weight Search; PPARA, peroxisome proliferative-activated receptor-α; PCNA, proliferating cell nuclear antigen; EF-Tu, elongation factor Tu. gel-based approaches are often used to survey the proteome on a global scale, typically resolving thousands of intact proteins based on charge (using isoelectric focusing) and apparent molecular mass (using SDS-PAGE). Despite its popularity for differential display proteomics, 2D gel-based strategies have until recently lacked the ability to directly quantify abundance changes in the same fashion as in stable isotope strategies using liquid chromatography coupled with tandem mass spectrometry (1Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags..Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4362) Google Scholar, 2Mason D.E. L