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 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.
Norais
N
N
Department of Molecular Biology, Chiron SpA, Siena, Italy.
Nogarotto
R
R
Lacobini
E T
ET
Garaguso
I
I
Grifantini
R
R
Gauli
G
G
Grandi
G
G
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D013058:Mass Spectrometry; D008954:Models, Biological; D009345:Neisseria meningitidis / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D011506:Proteins