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).

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 肽B型离子的结构一直是一场长期辩论的主题。基于其解离行为的早期实验工作表明，沙发唑酮结构是最稳定的形式，这一结论得到了理论建模和IR光谱研究的支持。但是，最近有越来越多的证据表明，B-ISON也可能采用宏观循环形式，其N末端氨基组与其C端形成酰胺键。宏环可能会重新打开，有时与原始环不同，导致广泛的序列拼凑而成，随后在碰撞引起的解离（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，尤其是在涉及多个CID阶段的情况下。最近在我们的实验室中证明了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，研究序列在MS3实验的第二阶段使用时，序列是否也提出问题是实际的重要性。在这项研究中，我们调查了来自几个肽的双重电荷B型离子在其N末端附近的多个基本残基的ECD行为。 来自α-Neurokinin（HKTDSFVGLM-NH2）的B8和B9离子的ECD产生了几乎完整的C-ION系列，这似乎与恶唑龙结构一致。但是，这些c-ions仅包含一个产品离子的一小部分。大多数碎片离子只能通过由将C末端羰基与N末端氨基组连接到N端氨基组的宏环形成导致的序列争吵，然后在不同位置进行环形开口。此外，来自α-神经激素的较小b-ions的ECD主要产生序列的序列拼写片段，没有C型离子，表明大小对B-ION对B-ION的影响。 在此处研究的Eledoisin（Pepskdafiglm-NH2）的任何B-ION的ECD中未观察到序列炒。由于存在焦谷氨酸残基的存在，将N-末端氨基氨基氨基群阻塞，从而阻止了环状结构的形成。 尽管存在未阻止的N末端氨基群，但几乎没有证据表明来自物质P（RPKPQQQFFGLM-NH2）的一系列B-IOS（B5-B10）的ECD序列scrabmlings。然而，除了正常的C-ION系列外，这些B-IOS的ECD还产生了一系列片段离子，这些离子与具有其他赖氨酸侧链损失（C-LYS）相对应，该离子表明另一种类型的环状B-ION结构，其中C-末端羰基组形成了与裂解蛋白局部局部局部局部局部抗体抗体组成的酰胺键。这些c-透析离子在B离子的ECD光谱中的相对丰度随其大小从B5增加到B7而增加，然后从B8降低到B10。 为了更好地理解这些B-IOS的ECD行为，从物质P中预先构造了Ab tribious估计值（B6和B8），以检查奥沙唑酮和环状结构的相对稳定性。由于它们相对较大的大小，因此在没有ZVPE校正的情况下应用了RHF/3-21G理论水平。结果表明，对于B6离子，恶唑龙结构和赖氨酸连接的循环结构具有相似的稳定性，这解释了为什么观察到C和C-Lys离子的含量可比。另一方面，预计B8离子的LYS循环结构被预测为15 kcal/mol比恶唑酮形式更稳定，这与B8离子ECD中c-液体离子的优势一致。在这两种情况下，都发现N末端循环结构的能量明显高于相应的LYS环状形式（B8的52 kcal/mol，B6的28 kcal/mol），这可能是为什么在此几乎不观察到序列的序列。 最后，还进行了来自许多蛋白质的较大B（n）离子（n> 20）上的ECD。初步结果几乎没有序列争夺的证据。这可能是由于不利的熵因素使N-和C-termini足够接近以形成宏环结构。 这些结果表明，肽B-ION可能是几种不同形式的混合物，其倾向受其大小，N末端以及可能的碱性氨基酸残基的侧链影响。尽管在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，这不是一个重要的问题。这项研究将在第58届年度ASMS会议上作为海报（Lin等，2010）。