Multivariable difference gel electrophoresis and mass spectrometry: a case study on transforming growth factor-beta and ERBB2 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)-beta receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (ErbB2) oncogene. Proteome changes were monitored in response to exogenous TGF-beta 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-beta signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-beta signaling.
Friedman
David B
DB
Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. david.friedman@vanderbilt.edu
Wang
Shizhen E
SE
Whitwell
Corbin W
CW
Caprioli
Richard M
RM
Arteaga
Carlos L
CL
D000595:Amino Acid Sequence; D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism; D016000:Cluster Analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D016147:Genes, Tumor Suppressor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D015999:Multivariate Analysis; D025341:Principal Component Analysis; D020543:Proteome / Q000737:chemistry; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D015203:Reproducibility of Results; D015843:Serpins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
D002402:Cathepsin D; D020543:Proteome; D018719:Erb-b2 Receptor Tyrosine Kinases; D015843:Serpins; D053773:Transforming Growth Factor beta1; C085344:SERPIN-B5