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Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis
The most popular approach for proteomics analysis is based on the combination of two‐dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated inst...
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| Published in: | Proteomics (Weinheim) 2001-11, Vol.1 (11), p.1378-1389 |
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| Main Authors: | , , , , , , |
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| container_end_page | 1389 |
| container_issue | 11 |
| container_start_page | 1378 |
| container_title | Proteomics (Weinheim) |
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| creator | Norais, Nathalie Nogarotto, Renzo Iacobini, Emilia Tiziana Garaguso, Ignazio Grifantini, Renata Galli, Giuliano Grandi, Guido |
| description | The most popular approach for proteomics analysis is based on the combination of two‐dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two‐dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. The approach is particularly useful when a specific subset of proteins needs to be identified in a complex protein mixture. |
| doi_str_mv | 10.1002/1615-9861(200111)1:11<1378::AID-PROT1378>3.0.CO;2-S |
| format | article |
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Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two‐dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. 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Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two‐dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. The approach is particularly useful when a specific subset of proteins needs to be identified in a complex protein mixture.</description><subject>Animals</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial proteomes</subject><subject>Cloning, Molecular</subject><subject>Computational Biology - methods</subject><subject>Databases as Topic</subject><subject>Electrophoresis, Gel, Two-Dimensional - methods</subject><subject>Genetic Vectors</subject><subject>Hydrogen-Ion Concentration</subject><subject>Image Processing, Computer-Assisted</subject><subject>In vitro transcription</subject><subject>Mass Spectrometry</subject><subject>Meningococcus B</subject><subject>Models, Biological</subject><subject>Neisseria meningitidis - metabolism</subject><subject>Plasmids - metabolism</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Protein Biosynthesis</subject><subject>Proteins - chemistry</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><subject>Transcription, Genetic</subject><subject>translation</subject><subject>Two-dimensional electrophoresis</subject><issn>1615-9853</issn><issn>1615-9861</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><recordid>eNqVUdtu1DAQjRCIlsIvID8hkMjWl1y3CKkEKBVVt3SLingZOfEEDEm82F7K_gDfjUOW5YkH5JE8lzNnRnOi6IjRGaOUH7KMpXFZZOwxp5Qx9oTNGXvGRF7M58enL-OLy8XVGD0XMzqrFkc8Xt6K9nddt3d-Kvaie859CSx5UeZ3oz3GSs7TstiPflamr_WAisi1N730wbuoLknTmUEPn54SPZDv2ltDvJWDa6xeeW2Gw99RJ0efyEERf2NipXscXMjIjmCHTehafTYWnXakNZbUsvFodaiurPFoegytstuE-v3oTis7hw-2_0H0_vWrq-pNfLY4Oa2Oz-ImobyIa5YkGRdJGfYXsi1lI5VUihWIKq1bmpdZq3it0lYILmUrMUnKupWcY8JrVOIgejTxhg2-rdF56LVrsOvkgGbtIGe85DQvAnA5ARtrnLPYwsrqXtoNMAqjPDAeF8ZDwyQPhBcsCAIQ5IE_8oAACtUCOCwD68Pt-HXdo_rLudUjAD5MgBvd4eZ_Zv5j5C4XqOOJWjuPP3bU0n6FLBd5CtfnJ_DiXV6cXy_fwkfxCzh7vms</recordid><startdate>200111</startdate><enddate>200111</enddate><creator>Norais, Nathalie</creator><creator>Nogarotto, Renzo</creator><creator>Iacobini, Emilia Tiziana</creator><creator>Garaguso, Ignazio</creator><creator>Grifantini, Renata</creator><creator>Galli, Giuliano</creator><creator>Grandi, Guido</creator><general>WILEY-VCH Verlag GmbH</general><general>WILEY‐VCH Verlag GmbH</general><scope>BSCLL</scope><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>7X8</scope></search><sort><creationdate>200111</creationdate><title>Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis</title><author>Norais, Nathalie ; Nogarotto, Renzo ; Iacobini, Emilia Tiziana ; Garaguso, Ignazio ; Grifantini, Renata ; Galli, Giuliano ; Grandi, Guido</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4028-b144623491923af9acadadd18eed5bf0796fd2bd5f332aafae449bfa22e42bed3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Animals</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial proteomes</topic><topic>Cloning, Molecular</topic><topic>Computational Biology - methods</topic><topic>Databases as Topic</topic><topic>Electrophoresis, Gel, Two-Dimensional - methods</topic><topic>Genetic Vectors</topic><topic>Hydrogen-Ion Concentration</topic><topic>Image Processing, Computer-Assisted</topic><topic>In vitro transcription</topic><topic>Mass Spectrometry</topic><topic>Meningococcus B</topic><topic>Models, Biological</topic><topic>Neisseria meningitidis - metabolism</topic><topic>Plasmids - metabolism</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Protein Biosynthesis</topic><topic>Proteins - chemistry</topic><topic>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</topic><topic>Transcription, Genetic</topic><topic>translation</topic><topic>Two-dimensional electrophoresis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Norais, Nathalie</creatorcontrib><creatorcontrib>Nogarotto, Renzo</creatorcontrib><creatorcontrib>Iacobini, Emilia Tiziana</creatorcontrib><creatorcontrib>Garaguso, Ignazio</creatorcontrib><creatorcontrib>Grifantini, Renata</creatorcontrib><creatorcontrib>Galli, Giuliano</creatorcontrib><creatorcontrib>Grandi, Guido</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Proteomics (Weinheim)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Norais, Nathalie</au><au>Nogarotto, Renzo</au><au>Iacobini, Emilia Tiziana</au><au>Garaguso, Ignazio</au><au>Grifantini, Renata</au><au>Galli, Giuliano</au><au>Grandi, Guido</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis</atitle><jtitle>Proteomics (Weinheim)</jtitle><addtitle>Proteomics</addtitle><date>2001-11</date><risdate>2001</risdate><volume>1</volume><issue>11</issue><spage>1378</spage><epage>1389</epage><pages>1378-1389</pages><issn>1615-9853</issn><eissn>1615-9861</eissn><notes>istex:DA52EC0421122508C7D1C126129D653E55466FF6</notes><notes>ArticleID:PROT1378</notes><notes>ark:/67375/WNG-BQ78NWSK-Z</notes><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>The most popular approach for proteomics analysis is based on the combination of two‐dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two‐dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. 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| ispartof | Proteomics (Weinheim), 2001-11, Vol.1 (11), p.1378-1389 |
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| subjects | Animals Bacterial Proteins - chemistry Bacterial proteomes Cloning, Molecular Computational Biology - methods Databases as Topic Electrophoresis, Gel, Two-Dimensional - methods Genetic Vectors Hydrogen-Ion Concentration Image Processing, Computer-Assisted In vitro transcription Mass Spectrometry Meningococcus B Models, Biological Neisseria meningitidis - metabolism Plasmids - metabolism Polymerase Chain Reaction - methods Protein Biosynthesis Proteins - chemistry Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Transcription, Genetic translation Two-dimensional electrophoresis |
| title | Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis |
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