Originally published In Press as doi:10.1074/mcp.M700278-MCP200 on August 4, 2007.
Molecular & Cellular Proteomics 6:2032-2042, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
Research
Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment*,S
Xumin Zhang ,
Juanying Ye ,
Ole N. Jensen and
Peter Roepstorff¶
From the Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark
A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. The method is based on calcium phosphate precipitation of the phosphopeptides prior to further enrichment with established affinity enrichment methods. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Introduction of a two-step IMAC enrichment procedure after calcium phosphate precipitation resulted in observation of an increased number of phosphopeptides.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Tel.: 45-6550-2404; Fax: 45-6593-2661; E-mail: roe{at}bmb.sdu.dk

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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
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