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Submitted on October 3, 2003
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Odense 5230
Corresponding Author: mrl{at}bmb.sdu.dk
A common strategy in proteomics to improve the number and quality of peptides detected by mass spectrometry (MS) is to desalt and concentrate proteolytic digests using reversed phase (RP) chromatography prior to analysis. However, this does not allow for detection of small or hydrophilic peptides, or peptides altered in hydrophilicity such as phosphopeptides. We used micro-columns to compare the ability of RP resin or graphite powder to retain phosphopeptides. A number of standard phosphopeptides and a biologically relevant phosphoprotein, dynamin I, were analysed. MS revealed that some phosphopeptides did not bind the RP resin but were retained efficiently on the graphite. Those that did bind the RP resin often produced much stronger signals from the graphite powder. In particular, the method revealed a doubly phosphorylated peptide in a tryptic digest of dynamin I purified from rat brain nerve terminals. The detection of this peptide was greatly enhanced by graphite micro-purification. Sequencing by tandem mass spectrometry confirmed the presence of phosphate at both Ser-774 and Ser-778, while a singly phosphorylated peptide was predominantly phosphorylated only on Ser-774. The method further revealed a singly and doubly phosphorylated peptide in dynamin III, analogous to the dynamin I sequence. A pair of dynamin III phosphorylation sites were found at Ser-759 and Ser-763 by tandem mass spectrometry. The results directly define the in vivo phosphorylation sites in dynamins I and III for the first time. The findings indicate a large improvement in the detection of small amounts of phosphopeptides by MS and the approach has major implications for both small- and large-scale projects in phosphoproteomics.
Revised on January 30, 2004
Accepted on February 2, 2004
Improved detection of hydrophilic phosphopeptides using graphite powder micro-columns and mass spectrometry: Evidence for in vivo doubly phosphorylated dynamin I and dynamin III
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