N‑Acylated Dipeptide Tags Enable Precise Measurement of Ion Temperature in Peptide Fragmentation

Peptide fragmentations into b- and y-type ions are useful for the identification of proteins. The b ion, having the structure of a N-protonated oxazolone, dissociates to the a-type ion with loss of CO. This CO-loss process affords the possibility of characterizing the temperature of the b ion. Herein, we used N-acylated dipeptide tags, isobaric tags originally developed for protein quantification, as internal standards for the measurement of the ion temperature in peptide fragmentation. Amine-reactive dipeptide tags were attached to the N-termini of sample peptides. Collision-induced dissociation (CID) of the tagged peptides yielded a b-type quantitation signal (b_S) from the tag, which subsequently dissociated into the a_S ion with CO-loss. As the length of alkyl side chain on the dipeptide tag was extended from C₁ to C₈, the yield of a_S ion gradually increased for the 4-alkyl-substituted oxazolone ion but decreased for the 2-alkyl-substituted one. To gain insights into the unimolecular dissociation kinetics, we obtained the potential energy surface from ab initio calculations. Theoretical study suggested that the 4-alkyl substitution on N-protonated oxazolone decreased the enthalpy of activation by stabilizing the productlike transition state, whereas the 2-alkyl substitution increased it by stabilizing the reactant. Resulting potential energy surfaces were used to calculate the microcanonical and canonical rate constants as well as the a_S-ion yield. Arrhenius plots of canonical rate constants provided activation energies and pre-exponential factors for the CO-loss processes in the 600–800 K range. Comparison of experimental a_S-ion yields with theoretical values led to precise determination of the temperature of b_S ion. Thus, the b_S-ion temperature of tagged peptide can be measured simply by combining kinetic parameters provided here and a_S-ion yields obtained experimentally. Although the b-type fragment patterns varied with the chain length and position of alkyl substituent on the N-protonated oxazolone, the y-type fragment patterns were almost identical under these conditions. Furthermore, b_S-ion temperatures were nearly the same with only a few degrees K difference. Our results demonstrate a novel use of N-acylated dipeptide tags as internal temperature standards, which enables the reproducible acquisition of peptide fragment spectra