Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis.
Proteomics
; 8(23-24): 4862-72, 2008 Dec.
Article
em En
| MEDLINE
| ID: mdl-19003865
ABSTRACT
Complex protein mixtures have traditionally been separated by 2-DE. Görg introduced IPGs as the first dimension of protein separation. In recent years, MS-based proteomics has increasingly become the method of choice for identifying and quantifying large number of proteins. In that technology, to decrease analyte complexity, proteins are often separated by 1-D SDS-gel electrophoresis before online MS analysis. Here, we investigate a recently introduced device for peptide separation with IPGs (Agilent OFFGEL). Loading capacity for optimal peptide focusing is below 100 microg and--similar to 2-D gels--IEF is more efficient in the acidic than the basic pH region. The 24-well fractionation format resulted in about 40% additional peptide identifications but less than 20% additional protein identifications than the 12-well format. Compared to in-gel digestion, peptide IEF consistently identified a third more proteins with equal number of fractions. Low protein starting amounts (10 microg) still resulted in deep proteome coverage. Advantages of the in-gel format include better reliability and robustness. Considering its superior performance, diminished sample and work-up requirements, peptide IEF will become a method of choice for sample preparation in proteomics.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Fitas Reagentes
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Proteínas
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Proteoma
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Proteômica
Limite:
Humans
Idioma:
En
Revista:
Proteomics
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Alemanha