PROBING THE B-ION STRUCTURES BY ECD AND THEORETICAL MODELING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 多肽b-离子的结构是一个长期争论的主题。基于其解离行为的早期实验工作表明，恶唑酮结构是最稳定的形式，这一结论得到了理论模拟和红外光谱研究的支持。然而，最近有越来越多的证据表明，b-离子也可能呈现大环状，其N-末端的氨基与其C-末端形成酰胺键。在碰撞诱导解离(CID)条件下，宏环可能重新打开，有时在不同的位置，导致广泛的序列扰乱和随后形成间接序列离子。这对于多肽测序和蛋白质鉴定是有问题的，特别是当涉及多个阶段的CID时。最近，我们的实验室展示了CID/ECD MS3方法在蛋白质测序和PTM分析中的实用性(Theberge 2010，Li 2010)。因此，当在MS3实验的第二阶段中使用ECD时，研究序列加扰是否也存在问题具有重要的实际意义。在这项研究中，我们研究了几个N-端附近有多个碱性残基的多肽中双电荷b-离子的ECD行为。 来自α-神经激肽的b8和b9离子(HKTDSFVGLM-NH2)的电子捕获产生了几乎完整的c-离子系列，这似乎与恶唑酮的结构一致。然而，这些C离子只占产物离子的一小部分。大多数碎片离子只能通过序列扰乱来解释，该序列扰乱是由于形成了一个连接C-末端羰基和N-末端氨基的大环，随后在不同位置开环。此外，来自α-神经激肽的较小b离子的ECD主要产生序列混乱的碎片离子，没有c离子，这表明大小对b离子结构的影响。 在ECD中没有观察到来自本研究的任何b离子的序列扰乱。由于焦谷氨酸残基的存在，香豆素的N-末端氨基被封闭，从而阻止了环状结构的形成。 尽管存在未被阻断的N-末端氨基，但在ECD中几乎没有证据表明来自P物质(RPKPQQFFGLM-NH2)的一系列b-离子(b5-b10)存在序列拼接。然而，除了正常的C-离子系列外，这些b-离子的ECD还产生了一系列与具有额外赖氨酸侧链损失(c-Lys)的C-离子相对应的碎片离子，这表明了另一种类型的环状b-离子结构，其中C-末端的羰基与赖氨酸侧链氨基形成了酰胺键。这些c-Lys离子在b离子的ECD谱中的相对丰度随着其尺寸从b5增加到b7而增加，然后从b8减少到b10。 为了更好地理解这些b离子的ECD行为，对P物质中的两个双电荷b离子(b6和b8)进行了从头计算，以考察恶唑酮和环状结构的相对稳定性。由于其相对较大的尺寸，采用了RHF/3-21G水平的理论，没有进行ZVPE校正。结果表明，对于b6离子，恶唑酮结构和赖氨酸连接的环状结构具有相似的稳定性，这解释了为什么c和c-Lys离子具有相似的丰度。另一方面，预测b8离子的Lys环结构比恶唑酮形式更稳定，这与b8离子ECD中c-Lys离子比正常c离子占优势相一致。在这两种情况下，N-末端环结构的能量都显著高于相应的Lys-环形式(b8为52kcal/mol，b6为28kcal/mol)，这可能是这里几乎没有观察到序列扰乱的原因。 最后，我们还对一些蛋白质中较大的b(N)离子(n&gt；20)进行了电子捕获。初步结果显示，几乎没有序列扰乱的证据。这可能是由于不利的熵因素使N-末端和C-末端足够接近，从而形成宏观环结构。 这些结果表明，多肽b-离子可能以几种不同形式的混合物存在，其倾向性受到其大小、N-末端以及可能的碱性氨基酸残基侧链的影响。虽然在CID/ECD实验中也可能形成间接序列离子，但对于较大的b离子来说，这预计不会是一个大问题。这项研究将作为海报在第58届ASMS年会上公布(Lin等人)。2010)。 2010-2011年更新： 描述这项工作的手稿，题为《双电荷肽b-离子的结构异质性》，已发表在《美国社会质谱学杂志》2011，22,245-254上。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The structure of peptide b-ions has been the subject of a long-standing debate. Early experimental work based on its dissociation behavior suggested the oxazolone structure as the most stable form, a conclusion that is supported by both the theoretical modeling and the IR spectroscopic study. However, there have been growing evidences lately, showing that b-ions may also assume a macro-cyclic form, with its N-terminal amino group forming an amide bond with its C-terminus. The macro ring may reopen, sometimes at a different position from the original one, leading to extensive sequence scramblings and subsequent formation of indirect sequence ions under collision-induced dissociation (CID) conditions. This is problematic for peptide sequencing and protein identification, particularly when multiple stages of CID are involved. The utility of an CID/ECD MS3 approach in protein sequencing and PTM analysis has recently been demonstrated in our laboratory (Theberge 2010, Li 2010). It is thus of practical importance to investigate whether sequence scrambling also presents a problem when ECD is employed instead in the second stage of an MS3 experiment. In this study, we investigated the ECD behavior of doubly charged b-ions from several peptides with multiple basic residues near their N-termini. ECD of the b8 and b9 ions from alpha-neurokinin (HKTDSFVGLM-NH2) generated a near complete series of c-ions, which seems to be consistent with the oxazolone structure. However, these c-ions comprise only a small portion of the product ions. Most fragment ions can only be explained by sequence scrambling resulting from the formation of a macro ring connecting the C-terminal carbonyl group to the N-terminal amino group, followed by ring openings at different positions. Further, ECD of smaller b-ions from alpha-neurokinin produced predominantly sequence scrambled fragment ions, with no c-ions, indicative of the size influence on b-ion structures. Sequence scramblings were not observed in ECD of any b-ion from eledoisin (pEPSKDAFIGLM-NH2) studied here. The N-terminal amino group of eledoisin is blocked due to the presence of the pyroglutamic acid residue, thus preventing the formation of the cyclic structure. Despite the presence of an unblocked N-terminal amino group, there was little evidence for sequence scrabmlings in ECD of a series of b-ions (b5-b10) from substance P (RPKPQQFFGLM-NH2). However, in addition to the normal c-ion series, ECD of these b-ions also produced a series of fragment ions that correspond to c-ions with additional lysine sidechain loss (c-Lys), which suggested another type of cyclic b-ion structure, where the C-terminal carbonyl group formed an amide bond with the lysine sidechain amino group. The relative abundance of these c-Lys ions in ECD spectra of b ions increases as its size increases from b5 to b7, then decreases from b8 to b10. To better understand the ECD behavior of these b-ions, ab initio-calculations were preformed on two doubly charged b ions (b6 and b8) from substance P to examine the relative stability of oxazolone and cyclic structures. Due to their relatively large sizes, RHF/3-21G level of theory was applied without ZVPE correction. The results showed that for b6 ion, the oxazolone structure and the lysine-linked cyclic structure have similar stability, which explains why both c and c-Lys ions were observed with comparable abundances. On the other hand, the Lys-cyclic structure for b8 ion was predicted to be 15 kcal/mol more stable than the oxazolone form, consistent with the predominance of c-Lys ions over normal c ions in b8 ion ECD. In both cases, the N-terminus cyclic structure were found to be significantly higher in energy than the corresponding Lys-cyclic form (52 kcal/mol for b8 and 28 kcal/mol for b6), which is probably why sequence scrambling was hardly observed here. Finally, ECD on larger b(n) ions (n>20) from a number of proteins were also performed. The preliminary results showed little evidence of sequence scrambling. This might be due to a disfavored entropic factor to bring the N- and C-termini close enough to form the macro ring structure. These results suggested that a peptide b-ion may exist as a mixture of several different forms, with their propensities influenced by its size, N-terminus, and possibly the sidechains of basic amino acid residues. Although indirect sequence ions may also be formed in CID/ECD experiments, this is not expected to be a significant problem for larger b ions. This research will be presented as a poster at the 58th annual ASMS conference (Lin et al. 2010). Update for 2010-2011: A manuscript describing this work, entitled "Structural Heterogeneity of Doubly-Charged Peptide b-Ions", has been published on Journal of the American Society for Mass Spectrometry, 2011, 22, 245-254.