Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry.
Electrophoresis
; 25(15): 2526-32, 2004 Aug.
Article
em En
| MEDLINE
| ID: mdl-15300772
ABSTRACT
Protein phosphorylation plays a vital role in the regulation of most aspects of cellular activity, being key to propagating messages within signal transduction pathways and to modulating protein function. Pro-Q Diamond phosphoprotein gel stain is suitable for the fluorescence detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The technology is especially appropriate for profiling steady-state and dynamic phosphorylation on a proteome-wide scale, as demonstrated through detection of the native phosphorylation of cardiac mitochondrial phosphoproteins and changes in this profile arising from the activity of a protein kinase. For example, Pro-Q Diamond phosphoprotein gel stain was employed to demonstrate that among the 46 subunits of the mitochondrial respiratory chain complex, NADHubiquinone oxidoreductase (complex I), a 42 kDa subunit is phosphorylated in the steady-state. However, exposure of mitochondria to cAMP-dependent protein kinase increases phosphorylation of this 42 kDa subunit and results in de novo phosphorylation of an 18 kDa subunit as well. Since Pro-Q Diamond dye binds to phosphorylated residues noncovalently, the staining technology is fully compatible with modern microchemical analysis procedures, such as peptide mass profiling by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and post-source decay analysis of peptide phosphorylation.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Subunidades Proteicas
/
Complexo I de Transporte de Elétrons
/
Corantes Fluorescentes
/
Mitocôndrias Cardíacas
Limite:
Animals
/
Female
/
Humans
Idioma:
En
Revista:
Electrophoresis
Ano de publicação:
2004
Tipo de documento:
Article