Chemical derivatization of peptides containing phosphorylated serine/threonine for efficient ionization and quantification in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

• Published in: Rapid communications in mass spectrometry Vol. 22; no. 7; pp. 965 - 972
• Main Authors: Tsumoto, Hiroki, Ra, Moonjin, Samejima, Keijiro, Taguchi, Ryo, Kohda, Kohfuku
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 15.04.2008
• Subjects: Algorithms; Computer Simulation; Models, Chemical; Peptides - analysis; Peptides - chemistry; Phosphorylation; Reproducibility of Results; Sensitivity and Specificity; Serine - chemistry; Specimen Handling - methods; Spectrometry, Mass, Electrospray Ionization - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Threonine - chemistry
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.3451

We describe a useful method for the efficient ionization and relative quantification of peptides containing serine/threonine phosphorylation sites. This method is based on β‐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 d3‐labeled MEMN (d3‐MEMN) and MEMN (d0‐MEMN) generates a 3 Da mass difference between d3‐MEMN‐labeled and d0‐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 d0/d3‐labeled MEMN and an application of our approach to model peptides and proteins. Copyright © 2008 John Wiley & Sons, Ltd.