Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Korea.
J Phys Chem B. 2012 Dec 6;116(48):13982-90. doi: 10.1021/jp308697v. Epub 2012 Nov 27.
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.
肽的 b-和 y-型离子片段对于蛋白质的鉴定很有用。具有 N-质子化恶唑酮结构的 b 离子通过失去 CO 解离为 a 型离子。这种 CO 损失过程提供了表征 b 离子温度的可能性。在这里，我们使用了 N-酰化二肽标签，这是最初为蛋白质定量开发的等压标签，作为肽片段化中离子温度测量的内标。胺反应性二肽标签被连接到样品肽的 N-末端。标记肽的碰撞诱导解离 (CID) 从标签产生 b 型定量信号 (b(S))，随后该信号与 CO 损失一起解离为 a(S)离子。随着二肽标签上的烷基侧链长度从 C(1)延伸到 C(8)，4-烷基取代的恶唑酮离子的 a(S)离子的产率逐渐增加，但 2-烷基取代的恶唑酮离子的产率却降低了。为了深入了解单分子离解动力学，我们从从头算计算中获得了势能面。理论研究表明，N-质子化恶唑酮上的 4-烷基取代通过稳定产物样过渡态降低了焓变活化能，而 2-烷基取代则通过稳定反应物增加了它。由此产生的势能面被用于计算微正则和正则速率常数以及 a(S)-离子产率。正则速率常数的 Arrhenius 图在 600-800 K 范围内提供了 CO 损失过程的活化能和指前因子。将实验得到的 a(S)-离子产率与理论值进行比较，可以精确确定 b(S)离子的温度。因此，通过结合此处提供的动力学参数和实验获得的 a(S)-离子产率，可以简单地测量标记肽的 b(S)-离子温度。虽然 b-型片段模式随 N-质子化恶唑酮上的烷基取代基的链长和位置而变化，但在这些条件下，y-型片段模式几乎相同。此外，b(S)-离子温度几乎相同，只有几度的差异。我们的结果证明了 N-酰化二肽标签作为内部温度标准的新用途，这使得可重现地获取肽片段谱成为可能。
