Study on the Nitrosation of Peptide and Proteins with Nitrous Acid Using Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry
Nitrite anions are key small molecules for regulating many physiological processes,and are abundant in blood and tissues.In the present work,the reactivity of α-NH2 of N-terminus and e-NH2 of lysine of peptides and proteins toward nitrous acids was studied using matrix assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS).The synthesized standard peptides and standard protein(myoglobin) were used as model system to investigate the effect of different reaction system and different time on the reaction degree of peptides with nitrous acid.The results show that the diazo-reaction occurs between amino group of N-teminal of peptides and nitrous acid,and the intermediate formed in this reaction is a diazonium ion that is unstable to give nitrogen gas and a carbocation.The carbocation proceeds either to nucleophilic substitution or to elimination.The product of M+1 is formed by substitution.The other major product of M-17 observed in the present study is the alkene derivative formed by an elimination reaction.When the reaction system concludes acetic acid and sodium nitrite,the nitrous acid can completely react with amino group of N-termial peptides in 5 min.However,in the same situation,e-NH2 of lysine of peptides could not react with nitrous acids.The results of this study can provide useful information for understanding the interaction of nitrous acid and proteins in depth.