title: Analysis of histidine phosphorylation using tandem MS and ion-electron reactions.
abstract: Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
authors.forename: Anne J
authors.initials: AJ
authors.lastname: Kleinnijenhuis
authors.forename: Frank
authors.initials: F
authors.lastname: Kjeldsen
authors.forename: Birgitte
authors.initials: B
authors.lastname: Kallipolitis
authors.forename: Kim F
authors.initials: KF
authors.lastname: Haselmann
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004583:Electrons; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D007477:Ions; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*