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Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry
Published on: June 8, 2021
Multivariable difference gel electrophoresis and mass spectrometry: a case study on transforming growth factor-beta
David B Friedman1, Shizhen E Wang, Corbin W Whitwell
1Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. david.friedman@vanderbilt.edu
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)-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.
Insights
Transforming growth factor-beta (TGF-β) signaling in HER2-overexpressing breast cancer cells reveals novel proteins. This study identifies key molecules like maspin and cathepsin D involved in TGF-β pathways.
Area of Science:
- Proteomics
- Cell Biology
- Cancer Research
Background:
- HER2/Neu (ErbB2) oncogene overexpression is common in breast cancer.
- Transforming growth factor-beta (TGF-β) signaling plays a crucial role in mammary gland development and cancer progression.
- Understanding protein alterations in response to TGF-β signaling in HER2-positive cells is vital for targeted therapies.
Purpose of the Study:
- To investigate proteome changes in MCF10A mammary epithelial cells overexpressing HER2/Neu upon TGF-β receptor activation.
- To identify proteins with altered expression or post-translational modifications in response to TGF-β stimulation over time.
- To map identified proteins to biological networks and discover novel molecules associated with TGF-β signaling in this cellular context.
Main Methods:
- Multivariable Difference Gel Electrophoresis (DIGE) coupled with Mass Spectrometry (MS) was employed.
- Proteome analysis was performed at multiple time points (0, 8, 24, 40 h) following exogenous TGF-β stimulation.
- Isoelectric focusing (IEF) in medium (pH 4-7) and narrow (pH 5.3-6.5) ranges, along with pooled internal standards and unsupervised statistical analysis (PCA, hierarchical clustering), ensured robust quantification and reproducibility.
Main Results:
- Fifty-nine proteins, including isoforms, showed significant kinetic expression changes.
- Proteins were identified via mass spectrometry and database interrogation.
- Identified proteins were mapped to existing biological networks related to TGF-β signaling.
Conclusions:
- Novel proteins associated with TGF-β signaling were identified in HER2-overexpressing mammary epithelial cells.
- Proteins such as maspin and cathepsin D, with potential roles in breast cancer, were highlighted.
- This research provides insights into the molecular mechanisms of TGF-β signaling in HER2-positive breast cancer.
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