DIFFERENTIATION OF ISOMERIC AMINO ACID RESIDUES IN PEPTIDES USING ECD

使用 ECD 区分肽中的异构氨基酸残基

• 批准号: 8170894
• 负责人: CHI-WEI LIN
• 金额: $ 8.25万
• 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/8170894
• 关键词: Acids; Alkylation; Alzheimer' s Disease; Amides; Amino Acids; Amyloid beta-Protein; Amyloidosis; Analytical Chemistry; Antibiotic Resistance; Area; Asparagine; Biological Models; Blindness; Blood capillaries; C-terminal; Carbon; Cataract; Charge; Computer Retrieval of Information on Scientific Projects Database; Crystalline Lens; Crystallins; Detection; Development; Diagnostic; Dialysis procedure; Digestion; Disease; Dissociation; Electron Transport; Electrons; Funding; Generations; Glass; Glutamic Acid; Glutamine; Grant; HIV; Heating; Institution; Ions; Isoaspartic Acid; Knowledge; Laboratories; Link; Longevity; Manuscripts; Mass Spectrum Analysis; Membrane Proteins; Methods; Modification; N-terminal; Nature; Nitrogen; Paper; Pathologic Processes; Pathology; Peptide Fragments; Peptides; Physiological; Positioning Attribute; Process; Protein Conformation; Proteins; Publications; Publishing; Relative (related person); Reporting; Research; Research Personnel; Resistance; Resources; Sampling; Series; Side; Site; Solubility; Solutions; Source; Specificity; Substance P; Succinimides; Time; United States National Institutes of Health; Variant; Work; aged; amino group; analog; base; capillary; carboxyl group; deamidation; instrument; ionization; lens protein; lens transparency; mass spectrometer; posters; prevent; protein misfolding; protein structure; protonation; research study; symposium

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. Under physiological conditions, asparagine (Asn) residues can deamidate spontaneously, generating a mixture of aspartic (Asp) and isoaspartic acid (isoAsp) residues via a succinimide intermediate. In addition, isoAsp residue may also be formed through Asp isomerization, although this occurs at a much slower rate. Differentiation of Asp and isoAsp residues is important as the latter often causes more significant changes in protein conformation and functions, which has been linked to many protein misfolding diseases and other pathological processes. An electron capture dissociation (ECD)-MS/MS based method was established in this laboratory, where diagnostic c+57/z+-57 ions from isoAsp residues were used for the differentiation and relative quantification of the two isomeric forms. This method has been applied to study the Asn deamidation and Asp isomerization in several model systems as summarized below. Asn deamidation and Asp isomerization in A¿ Accumulaion of isoAsp residues in amyloid beta (A¿) peptides either as a result of Asn deamidation or Asp isomerization has been linked to the pathology of Alzheimer's disease. Several synthetic A¿ fragment peptides, including the most toxic form, A¿ 1-42, were analyzed by ECD. Extensive inter-residue cleavages were obtained, and the diagnostic c+57 and/or z+-57 ions were observed for all isoAsp containing peptides. The applicability of electron ionization dissociation (EID) to isoAsp analysis was also validated by the presence of the isoAsp diagnostic ion in the EID spectrum of an Asp-isomerized A¿ fragment peptide. These results have been published in a recent Analytical Chemistry paper (Sargaeva et al. 2009). Top-down ECD analysis and MS3 study of ¿2-microglobulin protein deamidation Although differentiation of Asp and isoAsp by ECD at the peptide level has been well established, there have been no reports on extending this method to identify isoAsp residue at the protein level. A top-down ECD analysis has several advantages over the bottom-up approach, by eliminating the additional sample processing steps associated with the enzymatic digestion that may introduce artificial deamidations (Li et al. 2008) or cause sample losses. The protein chosen here for the development of a top-down approach for isoAsp analysis is ¿2-microglobulin (B2M), a 12 kDa protein that has significant implication in dialysis-related amyloidosis. The aged B2M (after reductive alkylation) showed ~1 Da mass shift in its mass spectrum, suggesting that one of the Asn residues was deamidated. ECD of the aged B2M produced the isoAsp diagnostic ion, c16+57, unambiguously identifying the Asn17 as the deamidation site. Asn17 is part of a fast deamidating -NG- sequence located on the surface of the protein, making it a particularly facile deamidating site. This experiment demonstrated, for the first time, that the ECD method can be applied directly to the detection of isoAsp at the intact protein level. The top-down approach has its own limitations. An increase in protein size often leads to a decrease in the percentage of inter-residue cleavages, as well as the likelihood of the C¿-C¿ cleavage necessary for the isoAsp diagnostic ion formation. In addition, as more fragmentation channels become available for larger proteins, ion abundance in any given channel tends to decrease. Finally, the extensive noncovalent interactions present in larger proteins may also prevent fragment ion separation after ECD, further contributing to a decrease in the diagnostic ion abundance. One possible solution to these challenges is to perform ECD analysis on a smaller piece of the protein, generated by a traditional fragmentation method, such as collisionally activated dissociation (CAD). This MS3 approach retains the top-down advantage since it, too, does not require prior enzymatic digestion, while the smaller size of the ECD precursor ion increases the odds for the observation of the isoAsp diagnostic ions. As a proof of principle, ECD was performed on the b22(4+) ion of B2M generated by CAD, and the diagnostic c16+57 ion was observed, with a better S/N ratio than that obtained in ECD of the intact protein. These results will be presented as a poster at the 58th annual ASMS conference (Li et al. 2010). We are currently extending this work to beta-amino acids and to glutamine deamidation studies. Research progresses made in this area are reported below. Characterization of beta-peptides using ECD Beta-peptides are peptides containing ¿-amino acid residues, with their amino groups bonded to the ¿- rather than the ¿-carbon. Beta-peptides may exist in two different forms: a ¿2 linked peptide has its side chain connected to the ¿ carbon, and a ¿3 linked peptide has its side chain connected to the ¿ carbon. Because of their rare occurrence in nature and resistance to proteolytic degradations, they are being explored as a way to evade antibiotic resistance. Since ECD has been implemented successfully to identify isoAsp residues, which is a ¿3 linked peptide, it may also be applicable to differentiate the ¿2 and ¿3 linked peptides. ECD and electron transfer dissociation (ETD) analyses of several synthetic beta-peptides were performed on a 12 T Bruker solariX FT-ICR mass spectrometer and a Bruker AmaZon ion trap instrument, respectively. The heated glass capillary incorporated in the ionization source of these instruments has made possible generation of doubly charged Q06 peptide (V¿2A¿2L¿2V¿3A¿3L¿3) ions for ExD analysis, which was previously difficult to obtain. Consistent with previous observations in ECD of a substance P analogue, N-C¿ and C¿-C¿ cleavages at the ¿-amino acid sites were generally absent with the sole exception of isoAsp, highlighting the importance of the adjacent carboxyl group in isoAsp for radical stabilization. In place of the c/z ions, a/y ion formation was greatly enhanced at the N-terminal side of the ¿-amino acid residues, particularly at cyclized ¿-amino acid residues as present in an HIV envelop protein analogue. This could be explained by an alternative ECD mechanism, which is initiated by an electron capture at a protonated amide nitrogen. Subsequent homolytic cleavage can occur either at its N-terminal side to produce a/y ions, or at its C-terminal side to produce c/z ions, the latter of which is inhibited in ¿-peptides because of the instability of the resulting z+ ions. This mechanism is also consistent with the observation of higher a/y ion abundances in ECD of peptides of higher charge states, where amide nitrogen protonation is more likely to occur. It appears that ECD can be used to identify the presence of ¿-amino acids, based on the enhanced a/y cleavage and the diminished c/z cleavage. However, differentiation of the two types of ¿-cleavages was not achieved in the current study. These results will be presented as a poster at the 58th annual ASMS conference (Sargaeva et al. 2010). Differentiation of Glutamic and ¿-glutamic acid residues Although Asn deamidation is the most commonly observed PTM in proteins, glutamine (Gln) may also deamidate under physiological conditions to generate a mixture of glutamic acid (Glu) and ¿-glutamic acid (¿-Glu) acid. Gln deamidates at a much slower rate, about two orders of magnitude slower compared with its Asn counterpart. Gln deamidation is usually observed in proteins with long turn-over time, such as in eye lens crystallins. Crystallins are highly soluble structure proteins and comprise 90% of lens proteins, which undergo little turnover during their life spans, allowing accumulation of many kinds of modifications. Among these, deamidation is one of the most prevalent, which decreases crystallin solubility, alters lens transparency, and is also involved in cataract formation, a leading cause of blindness. Extensive Gln deamidation has been observed in crystallin proteins. In this study, we explored the possibility of extending the ECD method to differentiate Glu and ¿-Glu, based on the knowledge obtained in isoAsp studies. Continuing from last year's ECD study on a set of synthetic crystallin peptide fragments containing either Glu or ¿-Glu residues, a set of substance P variants were analyzed by ECD to investigate the possible presence of N-terminal diagnostic ions. Although two sets o C-terminal fragments, corresponding to z+-59 and z+-72 ion, exist site specifically at the ¿-Glu residues, only the latter can be used to identify the presence and locate the position of the ¿-Glu residues, because of the presence of z+-59 ions in Glu-containing peptides, albeit without the site-specificity. All N-terminal diagnostic ions, c+57, c+59 and c+72 ions, observed so far, were specific to the Pro-isoGlu sequence. Unlike its Asp counterpart, no diagnostic side-chain loss ions were found in Glu-containing peptides. The presence of Glu residue(s) may be inferred from the observation of a series of zn+-59 ions, although it was neither site specific, nor without interference from the ¿-Glu residues. A manuscript describing these results has been accepted for publication in Analytical Chemistry (Li et al. 2010).

该副本是使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，这是调查员的机构。 在物理条件下，天冬酰胺（ASN）幸存下来可以自发破坏，产生天冬氨酸（ASP）和异质酸（ISOASP）的混合物通过琥珀酰亚胺中间体生存。此外，尽管速率要慢得多，但也可以通过ASP异构化形成ISOASP保留率。 ASP和ISOASP保留的分化很重要，因为后者通常会导致蛋白质构象和功能的更大变化，这与许多蛋白质错误折叠疾病和其他病理过程有关。在该实验室中建立了电子捕获解离（ECD）-MS/MS方法，在该实验室中，使用ISOASP响应的诊断C+57/Z+-57离子用于两种异构体形式的分化和相对数量。如下所述，该方法已应用于研究几个模型系统中的ASN死亡衍生和ASP异构化。 a。的ASN脱氨酸和ASP异构化 ASN死亡或ASP异构化导致淀粉样蛋白β（A peptides）中ISOASP残基的积聚与阿尔茨海默氏病的病理有关。通过ECD分析了几种合成的片段肽，包括最具毒性的形式A a。1-42。对于所有含有肽的所有ISOASP，都观察到了广泛的残留间切割，并观察到诊断C+57和/或Z+-57离子。电子离子化解离（EID）在ISOASP分析中的适用性也通过在ASP异构化A片段肽的EID光谱中存在ISOASP诊断离子的存在验证。这些结果已发表在最近的分析化学论文中（Sargaeva等，2009）。 自上而下的ECD分析和MS3研究»2-微球蛋白蛋白质死亡 尽管已经确定了ecd在胡椒水平上对ASP和ISOASP的分化，但尚无关于扩展这种方法以识别蛋白质水平识别ISOASP残基的报道。自上而下的ECD分析通过消除与可能引入人造死亡蛋白的酶消化相关的其他样品处理步骤（Li等，2008）或导致样本损失的其他样本处理步骤，具有多个优点。 该蛋白质选择用于开发自上而下的ISOASP分析方法是2-微球蛋白（B2M），这是一种12 kDa蛋白，对透析相关的淀粉样变性具有显着意义。老化的B2M（烷基化降低后）显示质量光谱〜1 Da质量转移，这表明其中一个ASN残基被脱膜。老年B2M的ECD产生了ISOASP诊断离子C16+57，明确地将ASN17识别为脱氨酸位点。 ASN17是位于蛋白质表面上的快速脱氨基-NG-序列的一部分，使其成为特别容易的脱膜位点。该实验首次证明了ECD方法可以直接应用于在完整的蛋白质水平上检测ISOASP。 自上而下的方法有其自身的局限性。蛋白质大小的增加通常会导致分层裂解的百分比降低，以及ISOASP诊断离子形成所需的c¿-c。裂解的可能性。另外，随着更多的碎裂通道可用于较大的蛋白质，任何给定通道中的离子抽象往往会减少。最后，较大蛋白质中存在的广泛的非共价相互作用也可能防止ECD后碎片离子分离，进一步导致诊断离子抽象的减少。解决这些挑战的一种可能解决方案是，通过传统的碎片化方法（例如碰撞激活的解离（CAD））对较小的蛋白质进行ECD分析。这种MS3方法保留了自上而下的优势，因为它也不需要先前的酶消化，而较小的ECD前体离子的大小增加了观察ISOASP诊断离子的几率。作为原理的证明，在CAD产生的B2M的B22离子（4+）离子上进行了ECD，并且观察到诊断性C16+57离子，其S/N比比完整蛋白的ECD中获得的S/N比更好。这些结果将在第58届年度ASM会议上作为海报（Li等，2010）。 我们目前正在将这项工作扩展到β-氨基酸和谷氨酰胺死亡研究。下面报告了该领域的研究进度。 使用ECD对β肽的表征 β肽是含有``氨基酸的宠物''，其氨基基团粘合到€ - 而不是 - 碳。 β肽可能以两种不同的形式存在：A»2链接的肽具有连接到碳的侧链，并且3链链肽的侧链与碳连接在一起。由于它们在性质中很少发生和对蛋白水解降解的抗性，因此正在探索它们是逃避抗生素耐药性的一种方式。由于ECD已成功实施以识别ISOASP残基（这是3个链接的肽），因此它也可以适用于区分2和3链接的肽。 在12吨Bruker Solarix FT-ICR质谱仪和Bruker Amazon Ion Trap仪器上，对几种合成β肽进行了ECD和电子转移分离（ETD）分析。这些仪器的电离源中的加热玻璃毛细管掺入使双电荷Q06肽（V�2A¿2L¿2V¿3A¿3L¿3）离子的生成可能产生，以进行EXD分析，以前很难获得。与先前在ecd中观察到的p类似物，n -c¿和c¿-c。在�-氨基酸位点的切割通常不存在ISOASP除外，突显了ISOASP除外，强调了ISOASP中相邻羧基的重要性，以进行自由基稳定。代替C/Z离子，在»氨基酸残留物的N端侧的A/Y离子形成大大增强，尤其是在环化的»氨基酸中，如HIV包膜蛋白类似物所存在的那样存在。这可以通过一种替代的ECD机制来解释，该机制是由质子酰胺氮的电子捕获引发的。随后的均质裂解可以在其N末端发生以产生A/Y离子，也可以在其C末端产生C/Z离子，因为所得Z+离子的不稳定，后者在肽中抑制了后者。这种机制也与观察到较高电荷状态的ECD的较高的a/y离子丰度一致，在较高的电荷状态下，酰胺氮质子更可能发生。看来，根据增强的A/Y裂解和C/Z裂解的减少，可以使用ECD来鉴定 - 氨基酸的存在。但是，在当前研究中未实现两种类型的 - 分解的区分。这些结果将在第58届年度ASM会议上作为海报（Sargaeva等，2010）。 谷氨酸和 - 谷氨酸残基的分化 尽管ASN死亡是蛋白质中最常见的PTM，但谷氨酰胺（GLN）也可能在物理条件下破坏，以产生谷氨酸（GLU）和 - 谷氨酸（� -Glu）酸的混合物。 GLN以较慢的速度破坏，与ASN的ASN相比，大约两个数量级。 GLN通常在具有长时间转换时间的蛋白质中观察到，例如在眼镜结晶蛋白中。结晶蛋白是高度溶解的结构蛋白，占90％的晶状体蛋白质，它们在其生命跨度跨越几乎没有营业额，从而加速了许多类型的修饰。其中，脱氨酸是最普遍的一种，它降低了结晶蛋白的溶解度，改变了晶状体的透明度，并且还参与白内障形成，这是失明的主要原因。在结晶蛋白蛋白中已经观察到了广泛的GLN死亡。 在这项研究中，我们探讨了根据ISOASP研究中获得的知识扩展ECD方法以区分GLU和� -Glu的可能性。从去年的ECD研究继续对包含GLU或� -Glu残基的一组合成结晶蛋白胡椒片段，通过ECD分析了一组物质P变体，以研究N末端诊断离子的可能存在。尽管两组O c末端片段对应于Z+-59和Z+-72离子，现有位点特别是在�-glu残基上，但只能使用后者来识别存在并定位ood-glu残基的位置，因为Z+-59离子在glu contives for petides中的存在，而无需使用该站点。到目前为止观察到的所有N末端诊断离子，C+57，C+59和C+72离子均针对亲异数序列。与其ASP对应物不同，在含GLU的Petides中未发现诊断性侧链损失离子。可以通过观察一系列Zn+-59离子来推断GLU居住地的存在，尽管它既不是位点特异性的，也不是不受€-GLU残差的干扰。描述这些结果的手稿已被接受用于分析化学的出版（Li等，2010）。