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Analysis of mouse brain microvascular endothelium using laser capture microdissection coupled with proteomics
Nivetha Murugesan1, Jennifer A Macdonald, Qiaozhan Lu
1Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 18, 2010
Summary
Investigating the blood-brain barrier (BBB) at the protein level is crucial for understanding its function. This study details a method using laser capture microdissection and mass spectrometry to profile BBB proteins in mice.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- The blood-brain barrier (BBB) is vital for brain function, but its molecular mechanisms are not fully understood.
- Genomic studies of the BBB transcriptome exist, but comprehensive proteomic studies of the cerebral microvasculature are limited due to technical challenges.
Purpose of the Study:
- To describe a method for comprehensive proteomic profiling of mouse brain microvascular endothelium.
- To enable a detailed understanding of signaling mediators and BBB function in normal and pathological conditions.
Main Methods:
- Utilized immunohistochemistry-guided laser capture microdissection (immuno-LCM) to isolate brain microvascular endothelial cells.
- Employed Fourier transform mass spectrometry (FTMS) for sensitive, high-resolution proteomic analysis.
- Detailed protocols for immuno-LCM, sample processing, protein digestion, and mass spectrometric analysis were provided.
Main Results:
- Successfully profiled the proteome of mouse brain microvascular endothelium in situ.
- Demonstrated the feasibility of using immuno-LCM coupled with FTMS for BBB proteomic studies.
Conclusions:
- This integrated approach overcomes previous technical limitations in studying BBB proteins.
- Provides a foundation for understanding the molecular underpinnings of BBB function and dysfunction.
