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Published on: February 27, 2015
Profiling Eph receptor expression in cells and tissues: a targeted mass spectrometry approach
Roberta Noberini1, Elena Rubio de la Torre, Elena B Pasquale
1Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
Abstract:
The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.
Insights
A new mass spectrometry method efficiently identifies Eph receptors and ephrins in biological samples. This technique aids in understanding their roles in cell signaling and cancer, offering potential diagnostic and prognostic applications.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Cancer research
Background:
- Eph receptor tyrosine kinases and ephrin ligands form complex signaling networks crucial for physiological and pathological processes.
- Aberrant Eph/ephrin expression is linked to cancer pathogenesis, highlighting their potential as diagnostic and prognostic markers.
- Accurate profiling of Eph receptor and ephrin expression has been a significant challenge in biological research.
Purpose of the Study:
- To develop a novel and straightforward method for cataloging Eph receptors in cultured cells and tissues.
- To establish a reliable approach for identifying the full spectrum of Eph receptors within a biological sample.
- To validate the developed method in various biological contexts, including cancer cell lines and animal tissues.
Main Methods:
- Utilized ELISA and pull-down assays with a mixture of four ephrin Fc fusion proteins to capture EphA and EphB receptors.
- Employed mass spectrometry to identify Eph receptors from pull-down samples and estimate their relative abundance.
- Validated the approach using cultured human cancer cell lines, human tumor xenografts in mice, and mouse brain tissue.
Main Results:
- A mixture of four ephrins effectively isolated both EphA and EphB receptors in a single pull-down assay.
- Mass spectrometry successfully identified Eph receptors and their relative levels in diverse biological samples.
- The method demonstrated robustness and applicability across different tissue types and experimental models.
Conclusions:
- The developed mass spectrometry-based approach provides a valuable tool for comprehensive Eph receptor profiling.
- This method can be extended to profile ephrin expression, offering a complete view of the Eph/ephrin system.
- Accurate Eph receptor and ephrin cataloging is essential for advancing our understanding of their roles in biological processes and disease.
