Abstract
A comprehensive analysis of plasma membrane proteins is essential to in-depth understanding of brain development, function, and diseases. Proteomics offers the potential to perform such a comprehensive analysis, yet it requires efficient protocols for the purification of the plasma membrane compartment. Here, we present a novel and efficient protocol for the separation and enrichment of brain plasma membrane proteins. It lasts only 4 h and is easy to perform. It highly enriches plasma membrane proteins and can be applied to small amounts of brain tissue, such as the cerebellum of a single rat, which was used in the present study. The protocol is based on affinity partitioning of microsomes in an aqueous two-phase system. Marker enzyme assays demonstrated a more than 12-fold enrichment of plasma membranes and a strong reduction of other compartments, such as mitochondria and the endoplasmic reticulum. 506 different proteins were identified when the enriched proteins underwent LC-MS/MS analysis subsequent to protein separation by SDS-PAGE. Using gene ontology, 146 proteins were assigned to a subcellular compartment. Ninety-three of those (64%) were membrane proteins, and 49 (34%) were plasma membrane proteins. A combined literature and database search for all 506 identified proteins revealed subcellular information on 472 proteins, of which 197 (42%) were plasma membrane proteins. These comprised numerous transporters, channels, and neurotransmitter receptors, e.g. the inward rectifying potassium channel Kir7.1 and the cerebellum-specific gamma-aminobutyric acid receptor GABRA6. Surface proteins involved in cell-cell contact and disease-related proteins were also identified. Six of the 146 assigned proteins were derived from mitochondrial membranes and 5 from membranes of the endoplasmic reticulum. Taken together, our protocol represents a simple, rapid, and reproducible tool for the proteomic characterization of brain plasma membranes. Because it conserves membrane structure and protein interactions, it is also suitable to enrich multimeric protein complexes from the plasma membrane for subsequent analysis.
Highlights
A comprehensive analysis of plasma membrane proteins is essential to in-depth understanding of brain development, function, and diseases
Plasma Membrane Proteomics of Brain Tissue with the top phase being enriched in polyethylene glycol (PEG) and the bottom phase enriched in dextran [7]
Microsomes were prepared by differential centrifugation and partitioned in a PEG/dextran twophase system using wheat germ agglutinin (WGA)-dextran as affinity ligand
Summary
Tissue Preparation—Sprague-Dawley rats of both genders (8 –9 weeks old) were deeply anesthetized by a peritoneal injection of 700 mg/kg chloral hydrate and sacrificed by decapitation. After mixing and phase separation, the resulting top phase 3 was removed and extracted with a fresh bottom phase of a 4-g system prepared as above. The resulting top phase 4 was removed and mixed with a fresh affinity bottom phase of an 8-g system (6.3% (w/w) PEG 3350, 6.3% (w/w) dextran T500, 800 g of WGA in the form of WGA-dextran, 2 mM Li2SO4, 15 mM Tris borate, pH 7.8) to pull PMs into the dextranenriched bottom phase. After mixing and phase separation, the resulting top phase 6 was discarded, and the bottom phase was diluted 10-fold with 100 mM N-acetyl-D-glucosamine, 250 mM sucrose, and 5 mM Tris, pH 7.8, to release the membranes from the WGA-dextran. High Salt Washing and High pH Washing—To enrich integral PM proteins prior to LC-MS/MS analysis, the membranes obtained through two-phase affinity partitioning were resuspended in an icecold solution of 1 M KCl and 15 mM Tris, pH 7.4 (high salt washing). A manual search for subcellular location was based on literature and Genecards (bioinformatics.weizmann.ac.il/cards/ index.shtml)
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