title: Chemical derivatization of peptides containing phosphorylated serine/threonine for efficient ionization and quantification in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: We describe a useful method for the efficient ionization and relative quantification of peptides containing serine/threonine phosphorylation sites. This method is based on beta-elimination of the phosphate group from serine/threonine phosphorylation sites under alkaline conditions, followed by Michael addition reaction with N-(2-mercaptoethyl)-6-methylnicotinamide (MEMN). As a result of the derivatization reaction, the negatively charged phosphate group is substituted with the nicotinoyl moiety to improve the ionization efficiency of the derivatized peptide. The combination of d(3)-labeled MEMN (d(3)-MEMN) and MEMN (d(0)-MEMN) generates a 3 Da mass difference between d(3)-MEMN-labeled and d(0)-MEMN-labeled peptides, which is a useful signature for the identification of peptides containing serine/threonine phosphorylation sites in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum. Moreover, the mass difference is useful for the quantitative analysis of serine/threonine phosphorylation in proteins. In this paper, we describe the synthesis of d(0)/d(3)-labeled MEMN and an application of our approach to model peptides and proteins.
authors.affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tsumoto
authors.forename: Moonjin
authors.initials: M
authors.lastname: Ra
authors.forename: Keijiro
authors.initials: K
authors.lastname: Samejima
authors.forename: Ryo
authors.initials: R
authors.lastname: Taguchi
authors.forename: Kohfuku
authors.initials: K
authors.lastname: Kohda
mesh_terms: D000465:Algorithms; D003198:Computer Simulation; D008956:Models, Chemical; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D010766:Phosphorylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012694:Serine / Q000737:chemistry*; D013048:Specimen Handling / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013912:Threonine / Q000737:chemistry*
chemical_list: D010455:Peptides; D013912:Threonine; D012694:Serine