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Submitted on February 18, 2002
Center for Experimental Bioinformatics, University of Southern Denmark, Odense M DK-5230
Corresponding Author: pandey{at}cebi.sdu.dk
Although proteins phosphorylated on tyrosine residues can be enriched by immunoprecipitation with anti-phosphotyrosine antibodies, it has been difficult to identify proteins that are phosphorylated on serine/threonine residues due to lack of immunoprecipitating antibodies. In this report, we describe several antibodies that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting. Importantly, these antibodies can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation as well. Using these antibodies, we have immunoprecipitated proteins from untreated cells or those treated with a calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine phosphorylated proteins including drebrin 1, alpha-actinin 4 and filamin-1. We also identified a protein, poly (A)-binding protein 2 (PABP2), which was previously not known to be phosphorylated in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated to be a protein kinase A substrate in vitro. We identified several in vivo phosphorylation sites by tandem mass spectrometry using Frigg protein immunoprecipitated from cells. Our method should be applicable as a generic strategy for enrichment and identification of serine/threonine phosphorylated substrates in signal transduction pathways.
Revised on July 5, 2002
Accepted on July 22, 2002
A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: Identification of a novel protein, Frigg, as a protein kinase A substrate
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