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SYMB: Massenspektrometrie in der Bioanalytik

SYMB 1: Massenspektrometrie in der Bioanalytik

SYMB 1.4: Hauptvortrag

Freitag, 17. März 2006, 12:00–12:30, HV

Analyse der Protein Phosphorylierung mittels Massenspektrometrie — •Wolf Dieter Lehmann — Central Spectroscopy, German Cancer Research Center (DKFZ), Heidelberg

Mass Spectrometry has evolved as the key technology in the practice of finding and characterizing new protein phosphorylation sites. The set-up of a multidimensional analytical method optimized for structural and quantitative protein phosphorylation analysis is described. Liquid chromatography-element mass spectrometry (LC-ICP-MS) or laser ablation ICP-MS with 31-P detection is used as an intial screening method to determine the presence and relative quantity of phosphoproteins, e.g. in cytosolic protein fractions, or in isolated proteins. Phospho-specific chemoaffinity enrichment procedures applied at the phosphoprotein or phosphopeptide level are necessary for molecular characterization of sites with low stoichiometry of phosphorylation. The pinpoting of phosphorylation sites is best done with electrospray tandem mass spectrometry in the positive ion mode, which allows reliable sequencing of phosphopeptides. The specific fragmentation features of phosphopeptide cations and anions are described. Applications of these techniques to the analysis of reference phosphoproteins (ovalbumin, protein kinase A) and to proteins with previously unknown phosphorylation sites is demonstrated.