Proteomic Analysis of Brain Plasma Membranes Isolated by Affinity Two-phase Partitioning

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 twophase 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. Ninetythree 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 ␥-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.

Extraction and Purification of Bioproducts and Nanoparticles using Aqueous Two‐Phase Systems Strategies

“…The coupling of 2-DE with other analytical techniques has been reported to overcome some of the drawbacks of SDS-PAGE, e.g., difficulties in detecting low abundance proteins, aggregation of proteins, particularly hydrophobic proteins and reproducibility issues, by adding a preliminary analytical step. Some examples include a non-denaturing anion exchange chromatography prior to 2-DE to simplify the proteome and detect functional associations between proteins [30], and a pre-fractionation/concentration step using affinity partitioning in ATPS prior to 2-DE-LC/MS for membrane proteins enrichment [31]. It has been recently reported that optimized ATPS can serve as a presorting stage in proteomic studies since it can be customized for selective extraction and/or partition of large fractions of proteins from crude extracts.…”

Neuroproteomics – the tasks lying ahead

“…This dissection can take place on the anatomical level (by analyzing defined brain regions instead of whole brain samples), on the subcellular level (by fractionation according to protein localization within a cell), on the biochemical level (according to protein pI, M r , solubility, hydrophobicity etc. ), or by a combination of these methods [95,100,[104][105][106][107]. In theory, information gained from screening the subproteomes can then be merged, eventually approximating complete coverage of the brain proteome.…”

“…Chromatography-based approaches, such as SDS-PAGE coupled to LC-MS/MS or multidimensional LC-MS/MS, are rapidly gaining popularity as fast and reliable methods for high-throughput protein identification [97][98][99][100]. Although impervious to many of the 2-DE shortcomings, the number of proteins identified in LC-based studies is usually between several hundred and a few thousand [66,98,101].…”

“…Novel, highly efficient fractionation protocols for the purification of these neuronal subproteomes are required. Despite recent progress, such as the enrichment of total plasma membrane from rat cerebellar tissue by affinity partitioning [100], further refinements are required to isolate and analyze the various plasma membrane microdomains in sufficient purity and yield. A recent proteomic analysis of synaptosomes isolated by differential and density-gradient centrifugation from mouse brain, resulted in more than 1100 protein identifications [132].…”

Different protein profiles in inferior colliculus and cerebellum: A comparative proteomic study

“…Reduction of the sample complexity by pre-fractionation represents a possible solution to this problem. Several pre-fractionation protocols have been developed that are applicable to brain tissue, including those suitable for plasma membrane proteins, which may be most interesting to neuroscientists (Li et al, 2004;Guillemin et al, 2005;Schindler et al, 2006Schindler et al, , 2008. Separating the proteome into less complex sub-fractions is generally limited by the amount of starting tissue available, as each separating step involves loss of proteins.…”