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
基本信息
- 批准号:8170894
- 负责人:
- 金额:$ 8.25万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AcidsAlkylationAlzheimer&aposs DiseaseAmidesAmino AcidsAmyloid beta-ProteinAmyloidosisAnalytical ChemistryAntibiotic ResistanceAreaAsparagineBiological ModelsBlindnessBlood capillariesC-terminalCarbonCataractChargeComputer Retrieval of Information on Scientific Projects DatabaseCrystalline LensCrystallinsDetectionDevelopmentDiagnosticDialysis procedureDigestionDiseaseDissociationElectron TransportElectronsFundingGenerationsGlassGlutamic AcidGlutamineGrantHIVHeatingInstitutionIonsIsoaspartic AcidKnowledgeLaboratoriesLinkLongevityManuscriptsMass Spectrum AnalysisMembrane ProteinsMethodsModificationN-terminalNatureNitrogenPaperPathologic ProcessesPathologyPeptide FragmentsPeptidesPhysiologicalPositioning AttributeProcessProtein ConformationProteinsPublicationsPublishingRelative (related person)ReportingResearchResearch PersonnelResistanceResourcesSamplingSeriesSideSiteSolubilitySolutionsSourceSpecificitySubstance PSuccinimidesTimeUnited States National Institutes of HealthVariantWorkagedamino groupanalogbasecapillarycarboxyl groupdeamidationinstrumentionizationlens proteinlens transparencymass spectrometerposterspreventprotein misfoldingprotein structureprotonationresearch studysymposium
项目摘要
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。
在生理条件下,天冬酰胺 (Asn) 残基可以自发脱酰胺,通过琥珀酰亚胺中间体生成天冬氨酸 (Asp) 和异天冬氨酸 (isoAsp) 残基的混合物。此外,isoAsp 残基也可以通过 Asp 异构化形成,尽管其发生速度要慢得多。 Asp 和 isoAsp 残基的分化很重要,因为后者通常会导致蛋白质构象和功能发生更显着的变化,这与许多蛋白质错误折叠疾病和其他病理过程有关。本实验室建立了基于电子捕获解离 (ECD)-MS/MS 的方法,其中来自 isoAsp 残基的诊断性 c+57/z+-57 离子用于两种异构体形式的区分和相对定量。该方法已应用于研究几个模型系统中的 Asn 脱酰胺和 Asp 异构化,总结如下。
A 中的 Asn 脱酰胺和 Asp 异构化
Asn 脱酰胺或 Asp 异构化导致淀粉样β (A¿) 肽中 isoAsp 残基的积累与阿尔茨海默病的病理学有关。通过 ECD 分析了几种合成的 A¿ 片段肽,包括毒性最强的形式 A¿ 1-42。获得了广泛的残基间裂解,并且对于所有含有 isoAsp 的肽都观察到了诊断性 c+57 和/或 z+-57 离子。电子电离解离 (EID) 对 isoAsp 分析的适用性也通过 Asp 异构化 A¿ 片段肽的 EID 谱中存在 isoAsp 诊断离子得到了验证。这些结果发表在最近的一篇分析化学论文中(Sargaeva 等人,2009 年)。
自上而下的 ECD 分析和 ¿2-微球蛋白脱酰胺化的 MS3 研究
尽管 ECD 在肽水平上区分 Asp 和 isoAsp 的方法已经得到很好的证实,但还没有关于扩展该方法以在蛋白质水平上鉴定 isoAsp 残基的报道。自上而下的 ECD 分析比自下而上的方法有几个优点,它消除了与酶消化相关的额外样品处理步骤,这些步骤可能会引入人工脱酰胺(Li et al. 2008)或导致样品损失。
这里选择用于开发自上而下的 isoAsp 分析方法的蛋白质是 ¿2-微球蛋白 (B2M),这是一种 12 kDa 的蛋白质,在透析相关淀粉样变性中具有重要意义。老化的 B2M(还原烷基化后)在其质谱中显示出约 1 Da 的质量位移,表明其中一个 Asn 残基被脱酰胺化。老化 B2M 的 ECD 产生 isoAsp 诊断离子 c16+57,明确将 Asn17 识别为脱酰胺位点。 Asn17 是位于蛋白质表面的快速脱酰胺-NG-序列的一部分,使其成为特别容易的脱酰胺位点。该实验首次证明ECD方法可以直接应用于完整蛋白水平的isoAsp检测。
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残基间裂解的百分比以及 isoAsp 诊断离子形成所需的 C¿-C¿ 裂解的可能性降低。此外,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丰度往往会减少。最后,较大蛋白质中存在的广泛非共价相互作用也可能阻止 ECD 后碎片离子分离,进一步导致诊断离子丰度的降低。应对这些挑战的一种可能的解决方案是对通过传统裂解方法(例如碰撞激活解离 (CAD))生成的较小蛋白质片段进行 ECD 分析。这种 MS3 方法保留了自上而下的优势,因为它也不需要事先进行酶消化,而 ECD 前体离子的较小尺寸增加了观察 isoAsp 诊断离子的几率。作为原理证明,对 CAD 生成的 B2M 的 b22(4+) 离子进行 ECD,观察到诊断性 c16+57 离子,其信噪比比完整蛋白的 ECD 中获得的信噪比更好。这些结果将作为海报在第 58 届 ASMS 年度会议上展示(Li et al. 2010)。
我们目前正在将这项工作扩展到 β-氨基酸和谷氨酰胺脱酰胺研究。现将这方面的研究进展报道如下。
使用 ECD 表征 β 肽
β-肽是含有 ¡-氨基酸残基的肽,其氨基键合到 ¡-而不是 ¡-碳上。 β-肽可以两种不同的形式存在:2连接肽的侧链连接到碳,3连接肽的侧链连接到碳。由于它们在自然界中很少出现并且对蛋白水解降解具有抵抗力,因此人们正在探索它们作为逃避抗生素耐药性的一种方法。由于 ECD 已成功用于识别 isoAsp 残基(一种 3 连接肽),因此它也可用于区分 2 和 3 连接肽。
分别在 12 T Bruker SolariX FT-ICR 质谱仪和 Bruker Amazon 离子阱仪器上对几种合成 β 肽进行 ECD 和电子转移解离 (ETD) 分析。这些仪器的电离源中内置的加热玻璃毛细管可以产生用于 ExD 分析的双电荷 Q06 肽 (V¿2A¿2L¿2V¿3A¿3L¿3) 离子,这在以前是很难获得的。与之前在 P 物质类似物的 ECD 中观察到的结果一致,除了 isoAsp 之外,在 ε-氨基酸位点通常不存在 N-C¿ 和 C¿-C¿ 裂解,这凸显了 isoAsp 中相邻羧基对于自由基稳定的重要性。取代c/z离子,a/y离子的形成在β-氨基酸残基的N端侧大大增强,特别是在HIV包膜蛋白类似物中存在的环化β-氨基酸残基处。这可以通过另一种 ECD 机制来解释,该机制是由质子化酰胺氮上的电子捕获引发的。随后的均裂可以在其 N 端侧发生以产生 a/y 离子,或在其 C 端侧发生以产生 c/z 离子,后者由于所得 z+ 离子的不稳定性而在 ¿-肽中受到抑制。该机制也与较高电荷态肽的 ECD 中较高 a/y 离子丰度的观察结果一致,其中酰胺氮质子化更可能发生。基于增强的 a/y 裂解和减弱的 c/z 裂解,ECD 似乎可用于识别 β-氨基酸的存在。然而,目前的研究尚未实现两种类型 ¿-裂解的区分。这些结果将作为海报在第 58 届 ASMS 年度会议上展示(Sargaeva 等人,2010 年)。
谷氨酸和 ¿-谷氨酸残基的区分
尽管 Asn 脱酰胺是蛋白质中最常见的 PTM,但谷氨酰胺 (Gln) 在生理条件下也可能脱酰胺,生成谷氨酸 (Glu) 和 ¿-谷氨酸 (¿-Glu) 酸的混合物。 Gln 脱酰胺的速度要慢得多,与 Asn 对应物相比,大约慢两个数量级。谷氨酰胺脱酰胺通常在周转时间较长的蛋白质中观察到,例如在眼晶状体蛋白中。晶状体蛋白是高度可溶的结构蛋白,包含 90% 的晶状体蛋白,其在其生命周期中很少发生更新,从而允许积累多种修饰。其中,脱酰胺是最普遍的一种,它会降低晶状体蛋白的溶解度,改变晶状体的透明度,并且还与白内障的形成有关,白内障是导致失明的主要原因。在晶状体蛋白中观察到广泛的谷氨酰胺脱酰胺作用。
在这项研究中,我们基于 isoAsp 研究中获得的知识,探索了扩展 ECD 方法来区分 Glu 和 ¿-Glu 的可能性。继去年对一组含有 Glu 或 ¿-Glu 残基的合成晶状体蛋白肽片段进行 ECD 研究之后,通过 ECD 分析了一组 P 物质变体,以研究 N 末端诊断离子的可能存在。尽管对应于 z+-59 和 z+-72 离子的两组 C 端片段特异性地存在于 ¿-Glu 残基处,但由于含 Glu 的肽中存在 z+-59 离子,因此只有后者可用于识别 ¿-Glu 残基的存在和定位,尽管不具有位点特异性。迄今为止观察到的所有 N 端诊断离子 c+57、c+59 和 c+72 离子均特异于 Pro-isoGlu 序列。与 Asp 对应物不同,在含 Glu 的肽中没有发现诊断性侧链丢失离子。 Glu 残基的存在可以通过观察一系列 zn+-59 离子来推断,尽管它既不是位点特异性的,也没有受到 ¿-Glu 残基的干扰。描述这些结果的手稿已被《分析化学》接受发表(Li et al. 2010)。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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CHI-WEI LIN其他文献
CHI-WEI LIN的其他文献
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{{ truncateString('CHI-WEI LIN', 18)}}的其他基金
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:
8365564 - 财政年份:2011
- 资助金额:
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TRAINING IN FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY
傅里叶变换离子回旋共振质谱培训
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8365521 - 财政年份:2011
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ECD AND EDD OF NATIVE AND PERMETHYLATED GLYCANS
天然和全甲基化聚糖的 ECD 和 EDD
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- 资助金额:
$ 8.25万 - 项目类别:
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:
8365528 - 财政年份:2011
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$ 8.25万 - 项目类别:
CHARGE REMOTE FRAGMENTATION IN ECD AND ETD
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8365565 - 财政年份:2011
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蛋白质/肽棕榈酰化的质谱研究
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8365575 - 财政年份:2011
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- 批准号:
8365576 - 财政年份:2011
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$ 8.25万 - 项目类别:
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- 批准号:
8170939 - 财政年份:2010
- 资助金额:
$ 8.25万 - 项目类别:
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 - 财政年份:2010
- 资助金额:
$ 8.25万 - 项目类别:
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- 资助金额:
$ 8.25万 - 项目类别:
CAREER: Engineered SAM-Dependent Enzymes for Stereoselective Alkylation Reactions
职业:用于立体选择性烷基化反应的工程 SAM 依赖性酶
- 批准号:
2145749 - 财政年份:2022
- 资助金额:
$ 8.25万 - 项目类别:
Standard Grant
Characterizing the role of RNF25 in repair of DNA alkylation in blood cancers
表征 RNF25 在血癌 DNA 烷基化修复中的作用
- 批准号:
10580070 - 财政年份:2022
- 资助金额:
$ 8.25万 - 项目类别:
Alkyl(aryl)iodonium Reagents for Late-Stage Alkylation
用于后期烷基化的烷基(芳基)碘鎓试剂
- 批准号:
EP/W00934X/1 - 财政年份:2022
- 资助金额:
$ 8.25万 - 项目类别:
Research Grant
Ketone Alkylation Using Simple Olefins: A Sustainable Chemistry Approach
使用简单烯烃进行酮烷基化:一种可持续的化学方法
- 批准号:
2154632 - 财政年份:2022
- 资助金额:
$ 8.25万 - 项目类别:
Standard Grant
Development of New Methods for Asymmetric Synthesis and Nitrogen Fixation by using dihydropyridine derivatives as Alkylation Reagents
以二氢吡啶衍生物为烷基化试剂开发不对称合成和固氮新方法
- 批准号:
22J14253 - 财政年份:2022
- 资助金额:
$ 8.25万 - 项目类别:
Grant-in-Aid for JSPS Fellows














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