title: A new derivatization strategy for the analysis of phosphopeptides by precursor ion scanning in positive ion mode.
abstract: Although numerous strategies have been devised to analyze protein phosphorylation, an abundant intracellular protein modification, there is still a need for different methods for the analysis of this modification. A method to both detect and localize the phosphorylation within a protein/peptide is especially required. In this paper, a new strategy is described, which makes use of beta-elimination/Michael addition reactions to introduce a functional group at the original site of phosphorylation, which gives rise to a dimethylamine-containing sulfenic acid derivative with a unique m/z value. This enables the detection of the phosphorylated species within peptide mixtures by sensitive and specific precursor ion scanning in positive ion mode. Working under acidic conditions in positive ion mode has the added advantage that subsequent normal peptide sequencing for the exact localization can be performed. No other peptide derived fragment ion is observed at the m/z value of the sulfenic acid derivative formed, thus specific precursor ion experiments can also be carried out on instruments with low fragment ion resolution and lends itself to LC-MS/MS approaches when skimmer fragmentation routines or triple quadrupole mass spectrometers are used.
authors.affiliation: Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense.
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D002147:Calmodulin / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D010047:Ovalbumin / Q000737:chemistry; D010196:Pancreatic Elastase / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D012996:Solutions
chemical_list: D002147:Calmodulin; D007202:Indicators and Reagents; D010748:Phosphopeptides; D012996:Solutions; D010047:Ovalbumin; D010196:Pancreatic Elastase