PROBING THE B-ION STRUCTURES BY ECD AND THEORETICAL MODELING

通过 ECD 和理论建模探测 B 离子结构

• 批准号: 8170938
• 负责人: CHI-WEI LIN
• 金额: $ 1.48万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170938
• 关键词: Amides; Amino Acid Sequence; Basic Amino Acids; Behavior; C-terminal; Charge; Computer Retrieval of Information on Scientific Projects Database; Dissociation; Eledoisin; Funding; Grant; Hand; Institution; Ions; Laboratories; Link; Lysine; N-terminal; Neurokinin A; Oxazolone; Peptide Sequence Determination; Peptides; Positioning Attribute; Proteins; Pyrrolidonecarboxylic Acid; Relative (related person); Research; Research Personnel; Resources; SHFM1 gene; Series; Source; Staging; Structure; Substance P; Theoretical model; United States National Institutes of Health; Work; amino group; base; carbonyl group; numb protein; peptide structure; posters; prevent; protein aminoacid sequence; research study; symposium; theories

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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).

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 肽 b 离子的结构一直是争论的主题。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型和红外光谱研究的支持。然而，最近越来越多的证据表明，b-离子也可能呈现大环形式，其N端氨基与其C端形成酰胺键。宏环可能会重新打开，有时在与原始环不同的位置，导致广泛的序列扰乱，并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来说是个问题，特别是当涉及 CID 的多个阶段时。我们的实验室最近证明了 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，研究当在 MS3 实验的第二阶段采用 ECD 时序列置乱是否也会出现问题具有实际意义。在本研究中，我们研究了来自 N 末端附近具有多个碱性残基的几种肽的双电荷 b 离子的 ECD 行为。 来自 α-神经激肽 (HKTDSFVGLM-NH2) 的 b8 和 b9 离子的 ECD 生成了几乎完整的 c 离子系列，这似乎与恶唑酮结构一致。然而，这些 c-离子仅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连接C端羰基和N端氨基的大环，然后在不同位置开环而产生的。此外，来自 α-神经激肽的较小 b-离子的 ECD 主要产生序列杂乱的碎片离子，没有 c-离子，表明尺寸对 b-离子结构的影响。 在此研究的来自香芹素 (pEPSKDAFIGLM-NH2) 的任何 b-离子的 ECD 中均未观察到序列扰乱。由于焦谷氨酸残基的存在，香菇素的N端氨基被封闭，从而阻止了环状结构的形成。 尽管存在未封闭的 N 端氨基，但几乎没有证据表明 P 物质 (RPKPQQFFGLM-NH2) 的一系列 b 离子 (b5-b10) 的 ECD 中存在序列扰乱。然而，除了正常的 c-离子系列之外，这些 b-离子的 ECD 还产生了一系列与具有额外赖氨酸侧链丢失 (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端羰基与赖氨酸侧链氨基形成酰胺键。 b 离子的 ECD 光谱中这些 c-Lys 离子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。 为了更好地了解这些 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，以检查恶唑酮和环状结构的相对稳定性。由于其尺寸相对较大，因此应用 RHF/3-21G 水平的理论，无需 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，这解释了为什么观察到 c 和 c-Lys 离子具有相似的丰度。另一方面，b8 离子的 Lys 环状结构预计比恶唑酮形式稳定 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，我们发现 N 端环状结构的能量明显高于相应的 Lys 环状形式（b8 为 52 kcal/mol，b6 为 28 kcal/mol），这可能就是几乎观察不到序列扰乱的原因这里。 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序列扰乱的证据。这可能是由于不利的熵因子使 N 端和 C 端足够接近以形成大环结构。 这些结果表明，肽 b 离子可能以几种不同形式的混合物存在，其倾向受其大小、N 末端以及可能的碱性氨基酸残基侧链的影响。尽管间接序列离子也可能在 CID/ECD 实验中形成，但这对于较大的 b 离子预计不会是一个重大问题。这项研究将作为海报在第 58 届 ASMS 年度会议上展示（Lin 等人，2010 年）。