Analysis of peptide mixtures through convenient isotopic labeling and electrospray ionization-mass spectrometry

Abstract

We have developed a labeling method for sequencing of mixed peptides through modification of peptides and multistage electrospray ionization-mass spectrometry (ESI-MS). Mixed peptides were selectively phosphonylated on amino group through reaction of 2-bromoethyl phenylphosphinate (BEPP-H) or methyl/methyl-d 3 (1:1) phenylphosphinate (MPP-X) with peptides in the presence of tetrachloromethane and triethylamine under mild conditions to provide the labeled phosphonamidate peptides, and their primary ESI mass spectra showed doublet and triplet peaks. The triplet and doublet peaks were corresponding to the protonated molecules of the modified peptides having and having no one lysine residue, respectively, and the singlet peaks were from the non-peptides. Further, tandem mass spectra (MS/MS) of the readily identified protonated molecules unambiguously gave sequence of each member in mixed peptides.