Combining microscale solution-phase isoelectric focusing with Multiplexed Proteomics dye staining to analyze protein post-translational modifications.
Electrophoresis
; 25(15): 2539-44, 2004 Aug.
Article
em En
| MEDLINE
| ID: mdl-15300774
ABSTRACT
Previously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous multisubunit complexes, the strategy is not generally applicable to other complex proteomes. We determined that prefractionating samples by solution-phase isoelectric focusing is an effective alternative to sucrose-gradient fractionation that can be applied equally well to the analysis of mitochondrial and plasma proteins. Fluorescence-based multiplexing dye technologies greatly extend the capacity of SDS-polyacrylamide gel electrophoresis with respect to the investigation of proteome-wide changes in protein expression and post-translational modification, such as phosphorylation and glycosylation [2]. Overall, the prefractionation/Multiplexed Proteomics staining technology permits rapid, higher throughput screening of specimens for the identification of potentially interesting fractions that can subsequently be evaluated more thoroughly by two-dimensional gel electrophoresis.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfoproteínas
/
Processamento de Proteína Pós-Traducional
/
Proteômica
/
Corantes Fluorescentes
/
Mitocôndrias Cardíacas
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Electrophoresis
Ano de publicação:
2004
Tipo de documento:
Article