PROBING THE B-ION STRUCTURES BY ECD AND THEORETICAL MODELING

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 肽b离子的结构一直是长期争论的主题。早期的实验工作的基础上，其解离行为提出恶唑酮结构的最稳定的形式，这一结论是支持的理论建模和红外光谱研究。然而，最近有越来越多的证据表明，b-离子也可能呈现大环形式，其N-末端氨基与其C-末端形成酰胺键。宏观环可能重新打开，有时在与原始环不同的位置，导致广泛的序列扰乱和随后在碰撞诱导解离（CID）条件下形成间接序列离子。这对于肽测序和蛋白质鉴定是有问题的，特别是当涉及CID的多个阶段时。最近在我们的实验室中证明了CID/ECD MS 3方法在蛋白质测序和PTM分析中的实用性（Theberge 2010，Li 2010）。因此，它是具有实际意义的调查是否序列加扰也提出了一个问题，而不是在第二阶段的MS 3实验时，采用ECD。在这项研究中，我们研究了双电荷的b-离子的ECD行为，从几个肽与多个碱性残基附近的N-末端。 来自α-神经激肽（HKTDSFVGLM-NH 2）的b8和b 9离子的ECD产生了几乎完整的一系列c-离子，这似乎与恶唑酮结构一致。然而，这些c离子仅包括产物离子的一小部分。大多数碎片离子只能用序列混乱来解释，序列混乱是由连接C-末端羰基和N-末端氨基的大环形成，然后在不同位置开环引起的。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，这表明尺寸对b-离子结构的影响。 在本文研究的任一来自eledoisin（pEPSKDAFIGLM-NH 2）的b-离子的ECD中未观察到序列扰乱。由于焦谷氨酸残基的存在，eledoisin的N-末端氨基被封闭，从而防止环状结构的形成。 尽管存在未封闭的N-末端氨基，但几乎没有证据表明来自P物质（RPKPQQFFGLM-NH 2）的一系列b-离子（b5-b10）在ECD中的顺序擦除。然而，除了正常的c-离子系列之外，这些b-离子的ECD还产生了一系列碎片离子，其对应于具有额外的赖氨酸侧链损失（c-Lys）的c-离子，这表明另一种类型的环状b-离子结构，其中C-末端羰基与赖氨酸侧链氨基形成酰胺键。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到b7的增加而增加，然后从b8到b10而减少。 为了更好地理解这些b-离子的ECD行为，对来自物质P的两个双电荷B离子（b6和b8）进行从头计算，以检查恶唑酮和环状结构的相对稳定性。由于它们相对较大的尺寸，在没有ZVPE校正的情况下应用RHF/3- 21 G理论水平。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，这解释了为什么观察到的c和c-Lys离子具有相当的丰度。另一方面，预测b8离子的Lys-环状结构比恶唑酮形式稳定15 kcal/mol，这与b8离子ECD中c-Lys离子相对于正常c离子的优势一致。在这两种情况下，发现N-末端环状结构的能量显著高于相应的Lys-环状形式（b8为52 kcal/mol，b6为28 kcal/mol），这可能是这里几乎没有观察到序列扰乱的原因。 最后，还对来自许多蛋白质的较大B（n）离子（n>20）进行了ECD。初步结果显示，几乎没有证据表明序列扰乱。这可能是由于不利的熵因素使N-和C-末端足够接近以形成大环结构。 这些结果表明，一个肽b-离子可能存在的几种不同形式的混合物，其倾向的影响，其大小，N-末端，并可能侧链的碱性氨基酸残基。虽然在CID/ECD实验中也可能形成间接序列离子，但预计这对于较大的B离子不会是显著的问题。这项研究将在第58届ASMS年会上以海报的形式展示（Lin et al. 2010）。 2010-2011年最新情况： 描述这项工作的手稿，题为“Structural Heteroatom of Doubly-Charged Peptide b-Ions”，已经发表在Journal of the American Society for Mass Spectrometry，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.