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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Bibliographic Details
Published in:Proteomics (Weinheim) 2001-11, Vol.1 (11), p.1378-1389
Main Authors: Norais, Nathalie, Nogarotto, Renzo, Iacobini, Emilia Tiziana, Garaguso, Ignazio, Grifantini, Renata, Galli, Giuliano, Grandi, Guido
Format: Article
Language:English
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
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Summary: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.
ISSN:1615-9853
1615-9861
DOI:10.1002/1615-9861(200111)1:11<1378::AID-PROT1378>3.0.CO;2-S