Proteomic Analysis of Brain Plasma Membranes Isolated by Affinity Two-phase Partitioning
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 compartm...
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| Veröffentlicht in: | Molecular & cellular proteomics 2006-02, Vol.5 (2), p.390-400 |
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| container_title | Molecular & cellular proteomics |
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| creator | Schindler, Jens Lewandrowski, Urs Sickmann, Albert Friauf, Eckhard Nothwang, Hans Gerd |
| description | 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 γ-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. |
| doi_str_mv | 10.1074/mcp.T500017-MCP200 |
| format | Article |
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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 γ-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.</description><identifier>ISSN: 1535-9476</identifier><identifier>EISSN: 1535-9484</identifier><identifier>DOI: 10.1074/mcp.T500017-MCP200</identifier><identifier>PMID: 16249173</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Alkaline Phosphatase - metabolism ; Amino Acid Sequence ; Animals ; Biomarkers ; Brain - cytology ; Cell Membrane - chemistry ; Chromatography, Liquid ; Female ; Male ; Mass Spectrometry ; Membrane Proteins - analysis ; Membrane Proteins - chemistry ; Membrane Proteins - isolation & purification ; Mitochondria - metabolism ; Molecular Sequence Data ; NADH, NADPH Oxidoreductases - metabolism ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A - analysis ; Succinate Dehydrogenase - metabolism</subject><ispartof>Molecular & cellular proteomics, 2006-02, Vol.5 (2), p.390-400</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c466t-c11a7dcc7d9b24931d78834c96409f572b6e2d3e8e62708ed0f51f28ce6a162f3</citedby><cites>FETCH-LOGICAL-c466t-c11a7dcc7d9b24931d78834c96409f572b6e2d3e8e62708ed0f51f28ce6a162f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16249173$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Schindler, Jens</creatorcontrib><creatorcontrib>Lewandrowski, Urs</creatorcontrib><creatorcontrib>Sickmann, Albert</creatorcontrib><creatorcontrib>Friauf, Eckhard</creatorcontrib><creatorcontrib>Nothwang, Hans Gerd</creatorcontrib><title>Proteomic Analysis of Brain Plasma Membranes Isolated by Affinity Two-phase Partitioning</title><title>Molecular & cellular proteomics</title><addtitle>Mol Cell Proteomics</addtitle><description>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 γ-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.</description><subject>Alkaline Phosphatase - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Biomarkers</subject><subject>Brain - cytology</subject><subject>Cell Membrane - chemistry</subject><subject>Chromatography, Liquid</subject><subject>Female</subject><subject>Male</subject><subject>Mass Spectrometry</subject><subject>Membrane Proteins - analysis</subject><subject>Membrane Proteins - chemistry</subject><subject>Membrane Proteins - isolation & purification</subject><subject>Mitochondria - metabolism</subject><subject>Molecular Sequence Data</subject><subject>NADH, NADPH Oxidoreductases - metabolism</subject><subject>Proteomics</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Receptors, GABA-A - analysis</subject><subject>Succinate Dehydrogenase - metabolism</subject><issn>1535-9476</issn><issn>1535-9484</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkD1PwzAQhi0E4vsPMIAnthTbcexkLBUflYroUCQ2y3HO1CiJi52q6r_HqBWMTHfD8766exC6omREieR3nVmNFgUhhMrsZTJnhBygU1rkRVbxkh_-7lKcoLMYPwlhCS2O0QkVjFdU5qfofR78AL5zBo973W6ji9hbfB-06_G81bHT-AW6OugeIp5G3-oBGlxv8dha17thixcbn62WOgKe6zC4wfne9R8X6MjqNsLlfp6jt8eHxeQ5m70-TSfjWWa4EENmKNWyMUY2VZ1uymkjyzLnphKcVLaQrBbAmhxKEEySEhpiC2pZaUDo9IXNz9HtrncV_Nca4qA6Fw20bTrYr6MSUvAq9fwLUkmqgkuaQLYDTfAxBrBqFVynw1ZRon7EqyRe7cWrnfgUut63r-sOmr_I3nQCbnbA0n0sNy6Aqp03S-hUoZjKK5J_A5QEijU</recordid><startdate>20060201</startdate><enddate>20060201</enddate><creator>Schindler, Jens</creator><creator>Lewandrowski, Urs</creator><creator>Sickmann, Albert</creator><creator>Friauf, Eckhard</creator><creator>Nothwang, Hans Gerd</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20060201</creationdate><title>Proteomic Analysis of Brain Plasma Membranes Isolated by Affinity Two-phase Partitioning</title><author>Schindler, Jens ; 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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 γ-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.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>16249173</pmid><doi>10.1074/mcp.T500017-MCP200</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Alkaline Phosphatase - metabolism Amino Acid Sequence Animals Biomarkers Brain - cytology Cell Membrane - chemistry Chromatography, Liquid Female Male Mass Spectrometry Membrane Proteins - analysis Membrane Proteins - chemistry Membrane Proteins - isolation & purification Mitochondria - metabolism Molecular Sequence Data NADH, NADPH Oxidoreductases - metabolism Proteomics Rats Rats, Sprague-Dawley Receptors, GABA-A - analysis Succinate Dehydrogenase - metabolism |
| title | Proteomic Analysis of Brain Plasma Membranes Isolated by Affinity Two-phase Partitioning |
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