Abstract:

Edman degradation is the earliest technique used for the peptide N-terminal sequencing. Moreover, this method is still widely applied in multiple disciplines of the life science research community. With the development and application of robust proteomic techniques, the reaction of phenyl isothiocyanate (PITC) in this method developed by Edman has been used to modify the amines of peptides, and the identification of proteolytic cleavages of proteins occurring in cell signaling events, in a high throughput manner. However, there is no report about whether PITC can introduce modifications on the other amino acids, affecting the analysis and identification of proteins and peptide sequences. In order to explore the possibility of PITC modification on other amino acids, we used matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to investigate the reaction products between PITC and a model peptide under two different PITC conditions in this work. Through MS and MS/MS analyses, we found that at a high PITC concentration, PITC could additionally generate a new modification on an amino acid, histidine. The discovery of this modification may help us to more accurately analyze protein and peptide sequences during the application of Edman degradation and proteomics analysis after the optimization of experimental conditions and improvement of analytical methods.

Key words: Edman degradation, histidine, liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), model peptide, phenyl isothiocyanate