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.

Coupled Unimolecular Dissociation Kinetics of Bromotoluene Radical Cations

“…Importantly, Arrhenius parameters ( E a and A ) listed in Table can be used in the measurement of the ion temperature of the o -, m -, and p -bromotoluene radical cations from their dissociation yields in the temperature range 700–1300 K, as previously shown in the measurement of the ion temperature from the peptide fragmentation yield …”

Multi‐functional MBIT for peptide tandem mass spectrometry

“…These signal‐variable MBITs allow the multiplexed quantification of peptides and proteins, enabling the ( N + 1)‐plex quantitation using N different sets of 2‐plex MBITs (Suh et al, ). Furthermore, the b S ion (N‐protonated oxazolone) enables a precise measurement of ion temperature from the reaction yield of its subsequent unimolecular dissociation to the a S ion (Seo et al, ), because the dissociation rate depends on the internal energy of the b S ion which varies with the temperature of the peptide ion. Most importantly, the isotope‐coded high‐mass H/L y S ions (protonated Ala‐peptides), unlike their complementary low‐mass L/H b S ions, afford the isobaric quantification of peptides in the quadrupole ion trap (QIT) where a 1/3 mass cutoff problem hinders the use of other isobaric tags based on low‐mass signals (Seo, Yoon, & Shin, ).…”

“…Note that the C 1 ‐tag is the Ala‐tag. The isomeric form of the aliphatic C n ‐tag, N ‐acyl‐Ala‐Ala, is called the acyl C n ′‐tag, where a linear alkyl chain is placed at the N ‐acyl group (C n H 2 n +1 CO) and the 1 H/ 2 H‐isotopes are differentially encoded at the methyl groups of the first and second alanines (Seo et al, ). The acyl C n ′‐tag ( n = 1–8) presents the b S doublet at the same position as the aliphatic C n ‐tag; however, their b S ions are structural isomers.…”

“…The MS/MS spectra of MBIT‐linked peptides show both a S and b S ions as low‐mass quantitation signals. Since the b S ion dissociates to the a S ion, the a S ‐ion yield obtained from this unimolecular CO‐loss process can be used to measure the temperature of the b S ion (Seo et al, ).…”

“…The signal‐variable MBITs can be combined together for multiplexed protein quantification (Suh et al, ). MBITs can also be used as internal temperature standards for the ion temperature measurement (Seo et al, ). This review focuses on these multi‐functionalities of MBITs which have been demonstrated with a number of model peptides and proteins as well as yeast heat shock proteins.…”