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Electrophoresis. 2004 Aug;25(15):2526-32.

Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry.

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1
Proteomics Department, Molecular Probes, Inc., Eugene, OR 97402, USA. birte.schulenberg@probes.com

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, NADH:ubiquinone 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.

PMID:
15300772
DOI:
10.1002/elps.200406007
[Indexed for MEDLINE]
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