LAUSR

Abstract The protonated basic side chains in tryptic peptide ions can form intramolecular hydrogen bonds with backbone amide carbonyls, which inhibit the backbone cleavage and hinder the peptide sequencing using mass spectrometry. To study the strength of intramolecular hydrogen bonds and its effect on the backbone cleavage, we carried out the energy-dependent collision-induced dissociation (CID) of tryptic peptides and their modified ones. The N-acylation of lysine-terminated peptides was used to convert the primary amines at both lysine and the N-terminus into amides in order to block the primary protonation sites. The guanidination was employed to transform lysine into homoarginine in order to imitate arginine-terminated peptides. Then, N-acylation was applied to the guanidinated peptides to prepare N-acylated, homoarginine-terminated peptides. Singly charged peptide ions were generated by electrospray ionization in a Q-TOF instrument and their CID spectra were obtained as a function of collision energy under nearly single-collision conditions with Xe as the target gas. The energy-dependent survival yield of the precursor ion provided information on the change in dissociation energy of peptides after chemical modifications. We also identified possible sites and numbers of hydrogen bonds that stabilized a charge-solvated structure by comparing the variation in product yields with the location of backbone cleavage. By taking these data together, we estimated the relative strength of intramolecular hydrogen bonds formed in lysine- and homoarginine-terminated peptides before and after N-acylation. Our results demonstrate that N-acylation lowers the dissociation energy of tryptic peptides and facilitates the backbone cleavage by preventing the formation of intramolecular hydrogen bonds, enabling accurate identification of peptides.