title: N-acylated dipeptide tags enable precise measurement of ion temperature in peptide fragmentation.
abstract: 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 C(1) to C(8), 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.
authors.affiliation: Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Korea.
authors.forename: Jongcheol
authors.initials: J
authors.lastname: Seo
authors.forename: Min-Soo
authors.initials: MS
authors.lastname: Suh
authors.forename: Hye-Joo
authors.initials: HJ
authors.lastname: Yoon
authors.forename: Seung Koo
authors.initials: SK
authors.lastname: Shin
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D004151:Dipeptides / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013816:Thermodynamics
chemical_list: D004151:Dipeptides; D007477:Ions; D010446:Peptide Fragments