Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
J Am Chem Soc. 2009 Dec 30;131(51):18272-82. doi: 10.1021/ja9030837.
The chemistry of peptide fragmentation by collision-induced dissociation (CID) is currently being reviewed, as a result of observations that the amino acid sequence of peptide fragments can change upon activation. This rearrangement mechanism is thought to be due to a head-to-tail cyclization reaction, where the N-terminal and C-terminal part of the fragment are fused into a macrocycle (= cyclic peptide) structure, thus "losing" the memory of the original sequence. We present a comprehensive study for a series of b fragment ions, from b(2) to b(8), based on the simplest amino acid residue glycine, to investigate the effect of peptide chain length on the appearance of macrocycle fragment structures. The CID product ions are structurally characterized with a range of gas-phase techniques, including isotope labeling, infrared photodissociation spectroscopy, gas-phase hydrogen/deuterium exchange (using CH(3)OD), and computational structure approaches. The combined insights from these results yield compelling evidence that smaller b(n) fragments (n = 2, 3) exclusively adopt oxazolone-type structures, whereas a mixture of oxazolone and macrocycle b fragment structures are formed for midsized b(n) fragments, where n = 4-7. As each of these chemical structures exchanges at different rates, it is possible to approximate the relative abundances using kinetic fits to the H/D exchange data. Under the conditions used here, the "slow"-exchanging macrocycle structure represents approximately 30% of the b ion population for b(6)-b(7), while the "fast"-exchanging oxazolone structure represents the remainder (70%). Intriguingly, for b(8) only the macrocycle structure is identified, which is also consistent with the "slow" kinetic rate in the HDX results. In a control experiment, a protonated cyclic peptide with 6 amino acid residues, cyclo(Gln-Trp-Phe-Gly-Leu-Met), is confirmed not to adopt an oxazolone structure, even upon collisional activation. These results demonstrate that in some cases larger macrocycle structures are surprisingly stable. While more studies are required to establish the general propensity for cyclization in b fragments, the implications from this study are troubling in terms of faulty sequence identification.
肽的碰撞诱导解离（CID）碎片化的化学目前正在被综述，这是由于观察到肽片段的氨基酸序列在激活时可能会发生变化。这种重排机制被认为是由于头到尾的环化反应，其中片段的 N 末端和 C 末端融合成一个大环（=环状肽）结构，从而“失去”了原始序列的记忆。我们提出了一个全面的研究，针对一系列 b 片段离子，从 b(2)到 b(8)，基于最简单的氨基酸残基甘氨酸，研究肽链长度对大环片段结构出现的影响。CID 产物离子通过一系列气相技术进行结构表征，包括同位素标记、红外光解光谱、气相氢/氘交换（使用 CH(3)OD）和计算结构方法。这些结果的综合见解提供了令人信服的证据，表明较小的 b(n)片段（n = 2,3）仅采用噁唑酮型结构，而中等大小的 b(n)片段（n = 4-7）形成噁唑酮和大环 b 片段结构的混合物。由于这些化学结构以不同的速率交换，因此可以通过对 H/D 交换数据进行动力学拟合来近似相对丰度。在使用的条件下，对于 b(6)-b(7)，“慢”交换的大环结构代表 b 离子群体的大约 30%，而“快”交换的噁唑酮结构代表其余部分（70%）。有趣的是，对于 b(8)，仅鉴定到大环结构，这也与 HDX 结果中的“慢”动力学速率一致。在对照实验中，经证实，具有 6 个氨基酸残基的质子化环状肽，cyclo(Gln-Trp-Phe-Gly-Leu-Met)，即使在碰撞激活时也不采用噁唑酮结构。这些结果表明，在某些情况下，较大的大环结构出人意料地稳定。虽然需要进行更多的研究来确定 b 片段中环化的普遍倾向，但从这项研究中得出的结果在序列鉴定错误方面令人感到不安。
