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Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient
Published on: September 3, 2014
Analysis of membrane microdomain-associated proteins in the insular cortex of post-mortem human brain
Aine T Behan1, Martina Foy, Kieran Wynne
1Department of Psychiatry, Royal College of Surgeons in Ireland, RCSI Education and Research Centre, Beaumont Hospital, Dublin, Ireland. abehan@rcsi.ie.
Abstract:
Membrane microdomains (MM) are membrane rafts within the cell membrane enriched in cholesterol and glycosphingolipids that have been implicated in the trafficking and sorting of membrane proteins, secretory and endocytotic pathways, and signal transduction. To date, MM have not been characterised in the human brain. We reason that by identifying MM in the normal human cortex, we may better understand the molecular mechanisms of human brain dysfunction. To characterize the protein composition of MM in the human brain, we have carried out a comprehensive proteomic analysis of detergent resistant membranes (DRMs) associated proteins derived from human postmortem insular cortex using 1-DE separation prior to LC coupled to MS/MS or GeLC-MS/MS. Eighty five proteins were identified including 57 unique to human brain cortex DRMs (by comparison with DRM proteins reported in other cell types). High levels of signal transduction, cell adhesion, cell transport and cell trafficking proteins were identified including synaptic proteins such as synapsin II and synaptic vesicle membrane protein, mitochondrial proteins such as ATPase subunits and metabolic enzymes such as malate dehydrogenase. This data will facilitate our understanding of protein expression changes within membranes in candidate brain regions in human brain diseases such as schizophrenia, bipolar disorder and other psychiatric and neurodegenerative disorders.
Insights
Researchers identified membrane microdomains (MM) in the human brain cortex, revealing key proteins involved in cell function. This discovery aids understanding of brain disorders like schizophrenia and neurodegenerative diseases.
Area of Science:
- Neuroscience
- Proteomics
- Cell Biology
Background:
- Membrane microdomains (MM), or lipid rafts, are crucial for cellular processes but remain uncharacterized in the human brain.
- Understanding MM composition in the human brain is vital for elucidating mechanisms of neurological dysfunction.
Purpose of the Study:
- To characterize the protein composition of membrane microdomains in the normal human cerebral cortex.
- To identify proteins unique to human brain cortex membrane microdomains for comparison with other cell types.
Main Methods:
- Proteomic analysis of detergent-resistant membranes (DRMs) from postmortem human insular cortex.
- Utilized 1-DE separation followed by LC-MS/MS or GeLC-MS/MS for comprehensive protein identification.
Main Results:
- Identified 85 proteins associated with human brain cortex DRMs, with 57 being unique.
- Found enrichment of proteins involved in signal transduction, cell adhesion, transport, and trafficking.
- Detected specific synaptic proteins (e.g., synapsin II), mitochondrial proteins (e.g., ATPase subunits), and metabolic enzymes (e.g., malate dehydrogenase).
Conclusions:
- This proteomic dataset provides a foundational understanding of membrane microdomain composition in the human brain.
- The identified proteins offer insights into molecular mechanisms underlying brain diseases such as schizophrenia and neurodegenerative disorders.
