Speaker: Jeffrey Kowalak, Laboratory of Neurotoxicology, National Institute of Mental Health, Bethesda, MD

Topic: Derivatization of Phosphopeptides For Affinity Chromatography

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Tuesday, February 13, 2001, at 2:00 PM

Abstract: Phosphorylation of polypeptides has been shown to be a fundamental mechanism for modulation of cellular events. The phosphorylation state of a given polypeptide is dynamic and hence the phosphorylated form is often sub-stoichiometric. Therefore, characterization of phosphorylation sites in the presence of a large background of non-phosphorylated polypeptide can be analytically challenging.

We have developed a strategy designed to simplify characterization of (potentially) sub-stoichiometric phosphorylation sites by removing non-phosphopeptides from a mixture of peptides. The strategy is based on the chemical conversion of phosphoserine and phosphothreonine to cysteine and beta-methylcysteine. Peptides containing these nascent residues are coupled via a disulfide linkage to a biotinylated affinity tag and subsequently purified by chromatography on immobilized avidin. Affinity captured peptides can be released by the addition of a reducing agent to the eluent, thus facilitating subsequent mass spectrometric analysis. Data from synthetic phosphopeptides and model phosphoproteins will be presented.