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: 7955921
• 负责人: PETER B. O'CONNOR
• 金额: $ 9.44万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955921
• 关键词: Acids; Affect; Age; Aging; Agreement; Alanine; Alcohols; Alkylation; Alzheimer' s Disease; Amino Acids; Amyloid beta-Protein; Animals; Antibiotic Resistance; Antibiotics; Area; Asparagine; Aspartate; Behavior; Binding; Biological; Biological Models; Biology; Biotechnology; Blindness; Brain; Buffers; Calcium-Binding Proteins; Calmodulin; Carbon; Carboxylic Acids; Cataract; Characteristics; Charge; Cleaved cell; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystalline Lens; Crystallins; DNA Sequence Rearrangement; Diagnostic; Digestion; Disease; Dissociation; Drug Formulations; EF Hand Motifs; Electrons; Electrospray Ionization; Environment; Filament; Funding; Glutamic Acid; Glutamine; Grant; Hour; Human; Hydrogen; Hydrolysis; Incubated; Institution; Ions; Isoaspartic Acid; Knowledge; Laboratories; Lead; Life; Link; Location; Longevity; Mass Spectrum Analysis; Medicine; Metals; Methods; Modification; Monitor; N-terminal; Nature; Pathologic Processes; Patients; Pattern; Peptide Fragments; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Play; Positioning Attribute; Process; Proline; Protein Conformation; Proteins; Relative (related person); Reporting; Reproduction; Research; Research Personnel; Resolution; Resources; Role; Sampling; Senile Plaques; Series; Side; Site; Solubility; Solutions; Somatropin; Source; Structure; Substance P; Succinimides; System; Therapeutic; Time; Trypsin; United States National Institutes of Health; Work; adduct; aged; amino group; ammonium bicarbonate; analog; base; beta pleated sheet; carboxyl group; cell growth; deamidation; disulfide bond; divalent metal; ionization; lens protein; lens transparency; prevent; protein aminoacid sequence; protein misfolding; protein structure; reaction rate; research study

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 beta Amyloid beta (A beta) is a very hydrophobic 40-42 amino acid peptide that is the main constituent of amyloid plaques in the brain of Alzheimer's patients. There have been several reports on the isomerization of Asp residues in A beta in amyloid plaques at Asp(1), Asp(7), Asp(23) positions. IsoAsp formations have been associated with increases in beta-sheet structures that are found more abundantly in the filaments and plaques. The applicability of the ECD based method to the study of isoAsp formation in A beta was investigated (Sargaeva et al., 2009). ECD of the synthetic A beta peptide fragment 17-28 with isoAsp at position 23 showed the characteristic z6+-57 peak that defines the isoAsp location. Since artificial deamidation may also produce isoAsp during enzymatic digestion processes, particularly in a basic environment when using trypsin, it is sometimes desirable to analyze the whole protein with the top-down approach. A beta 1-40 with Asn substitution at position 7 was allowed to deamidate which generated a mixture of Asp and isoAsp residue. ECD of the deamidated A beta 1-40 produced 75% of the predicted fragments, including the z+-57/c+57 diagnostic peaks for the isoAsp form, clearly demonstrating the potential of extending this method to top-down analysis of intact A beta peptides. The next step is to apply this method to study isoAsp formation in biological samples from Alzheimer's patients. Electron ionization dissociation (EID) of the isomerized A beta 17-28 fragment was also performed, which showed abundant fragmentation and intense fragment ion peaks. However, in these higher energy studies, the diagnostic peaks were of low intensity, limiting the ability to detect the isomerized aspartate predicted in the Alzheimer's plaques, even though EID cleaved >97% of the inter-residue bonds. Nevertheless, this experiment demonstrated that EID may be used in isoaspartomics research alternative to ECD. This could be advantageous for analysis of singly charged ions where ECD is not applicable. Asn deamidation and Asp isomerization in Calmodulin The 148 amino acid Calmodulin (CaM) is a calcium-binding protein with multiple Asn-Gly and Asp-Gly sequences in its four EF-motif hands, which are labile deamidation and isomerization sites in absence of Ca2+. The ESI mass spectrum of the aged CaM (pH 8.0, 14 days, no Ca2+) showed a 2 Da mass shift, indicating the presence of deamidation at two Asn residues (Yao et al., 2009). Top-down analysis of the aged CaM with activated ion (AI)-ECD resulted in cleavages of 66 out of a total of 147 inter-residue bonds. The observation of the 1 Da mass shift in fragment ion z524+, the 2 Da mass shift in y969+, and no mass shifts in any N-terminal fragments smaller than c59 unambiguously identified the deamidation sites at Asn 60 and Asn 97. However, no diagnostic ions for isoAsp were observed, possibly due to the low ion abundance as the result of fragment ions being spread out over many dissociation channels. As an alternative, a bottom-up approach was employed to monitor the isoAsp formation in CaM, where the aged CaM was first digested by trypsin, followed by ECD analysis of the relevant peptides. One Da mass shift was observed for peptide (38-74) and peptide (91-106), identifying the Asn 60 and Asn 97 as the deamidation sites, in agreement with the top-down results. Isomerization of Asp has not been previously detected using the ECD method, presumably because of its much slower reaction rate compared with that of the Asn deamidation. In the current study, diagnostic c+57/z+-57 ions were observed in several tryptic peptide ECD spectra, indicating the formation of isoAsp not only at residues Asn 60 and Asn 97 as the result of deamidation, but also at Asp 22, Asp 24 and Asp 95 due to Asp isomerization. The presence of Ca2+ during incubation appeared to inhibit the deamidation process. Aging of CaM at pH 8 for 2 weeks with Ca2+ produced far less deamidations. This is evident in the ESI spectra of the resulted tryptic peptide (91-106), with its isotopic pattern indicating that less than 20% of Asn 97 were converted to acidic products. Amino acid residues 91-106 are in domain III of CaM, with its -DGNG- sequence serving as the Ca2+ binding motif. Asn deamidation in HGH Not only does deamidation play an important role in many diseases, it also affects the potency and shelf-life of therapeutic drugs. The 191 amino acid protein human growth hormone (HGH) is the first biotechnology drug that is used for stimulation of growth and cell reproduction in humans and other animals. HGH is known to deamidate in the pharmaceutical formulations that are sold. We have recently initiated a collaboration with a local start-up pharmaceutical company to study modifications of HGH, including deamidation (Cui et al., 2009). The disulfide bonds in HGH were reduced and alkylated before the ESI-MS and MS/MS analysis, which resulted in a shift towards higher charge states. Collision-activated dissociation (CAD) at 25 eV of the mass selected 18+ charge state of the native HGH yielded 14 y-type and 7 b-type as well as numerous internal fragment ions; while the 21+ charge state generated 39 y-type and 8 b-type fragments. Increasing the collision energy from 25 eV to 35 and 45 eV did not produce more sequence specific ions for the 21+ charge state, instead, more internal fragment ions were observed. Top-down analysis of the aged HGH using CAD and ECD is ongoing. Bottom-up study of the aged HGH has also been carried out. After the reductive alkylation, HGH was allowed to incubate in 0.1 M ammonium bicarbonate buffer solution (pH ~8) at 37oC for 5 days. The aged HGH was then digested by trypsin for one hour, and the resulted peptide mixture was purified by self-packed POROS column and analyzed by ESI-MS and ECD. One peptide (147-FDTNSHNDDALLK-159) was observed with a mass shift of +1 Da, indicative of deamidation occurring at one or both of the two Asn residues. In the ECD spectrum of this peptide, z7 ion displayed a significant shift in its isotopic pattern, indicating that Asn 153 is partially deamidated. Because the intra-complex hydrogen transfer may also lead to a +1 Da mass shift to the z ion isotopic pattern (z+ to z2), it was difficult to quantify the percentage of deamidation at Asn 153 site, or to determine if Asn 150 also deamidated. High resolution tandem MS experiment should help to differentiate these two effects, as the deamidation leads to a +0.984 Da mass shift, while the z2 ion formation leads to a +1.008 Da shift. No diagnostic ions for the isoAsp form were observed, which was a little surprising, but not completely unexpected. Since the deamidation was carried out prior to enzymatic digestion, the tertiary structure of the HGH might have prevented the formation of isoAsp residue when the succinimide intermediate underwent hydrolysis. However, there also existed another possibility that the diagnostic ion formation was suppressed in this acidic residue rich peptide due to abundant intramolecular interactions. It is thus imperative to carry out the aging study after the HGH is digested. Since isoAsp formation would be expected to be favored during the deamidation process of a random coil peptide, ECD of the resulted deamidated peptide should be able to discern the two possibilities. This experiment will be performed next. 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 Isoaspartic acid is a beta-linked amino acid, whose amino group is bonded to the ¿ carbon rather than the ¿ carbon. With the sole exception of ¿ alanine, other beta amino acids rarely appear in nature. Because of this, ¿ peptide based antibiotics are being explored as ways of evading antibiotic resistance. ¿ 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. Since ECD has been implemented successfully to identify isoAsp residues, based on the diagnostic ions generated via C¿-C¿ bond cleavage, it may also be used to differentiate the ¿2 and ¿3 linked peptides. An effort to extend the ECD method for the characterization of other beta amino acid residues produced mixed results (Sargaeva et al., 2008, 2009). Electrospray ionization (ESI) of the Q06 peptide (V¿2A¿2L¿2V¿3A¿3L¿3) generated predominantly singly charged precursor ions, which are not suitable for ECD analysis. Three methods have been applied to increase the charge state of the Q06 peptide. Addition of p-nitrobenzyl alcohol (p-NBA) to the ESI solution often led to an up-shift of the charge state distribution; although, in the case of the Q06 peptide, the resulted doubly charged precursor ions were still of too low an abundance to be analyzed by ECD. Covalent attachment of cholamine to the carboxylic acid is much more efficient in producing doubly charged ions, but ECD of these ions produced mostly losses of the cholamine tag and b/y cleavages, with just one N-C¿ and no C¿-C¿ cleavages. Finally, divalent metal ion (Ca2+) adduction also produced abundant doubly charged Q06 ions. However, no C¿-C¿ cleavages were produced in ECD of these metal adducted ions either. Since the addition of a fixed charge group or a metal ion may alter the ECD fragmentation behavior, a substance P analogue with the Gln 5 and Leu 10 replaced by beta-homoGln and beta-homoLeu, respectively, was synthesized and analyzed by ECD. Surprisingly, the doubly protonated beta-substance P generated neither N-C¿ nor C¿-C¿ cleavage at sites containing beta amino acid residues. The disparity between the ECD fragmentation pattern of the isoAsp peptides and that of other beta-linked peptides demonstrated the importance of side-chain groups in ECD. It suggested that the adjacent carboxyl group might be instrumental in stabilizing the radical formed in ECD of isoAsp containing peptides. Such stabilization effect is, in general, absent in other beta-linked peptides. 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 (Li et al., 2009), we developed an ECD-based method to differentiate the Glu and ¿-Glu residues, based on the knowledge from the isoAsp research. A set of synthetic crystallin peptide fragments containing either Glu or ¿-Glu residues were analyzed by ECD. Similar to the c+57/z+-57 diagnostic ions observed in isoAsp containing peptides, characteristic c+72/z+-72 ions were also observed in ¿-Glu peptides. Some z+-72 ions were also present in the ECD spectra of Glu containing peptides, although they never appeared at cleavage sites immediately preceding the Glu residues. These ions likely resulted from the charge remote fragmentation initiated by the radical site on the z+ ion, which is a subject currently being investigated. Since the z+-72 ions in ECD of ¿-Glu peptides are formed only when the initial N-C¿ bond cleavage occurred on the N-terminal side of the ¿-Glu residue, the z+-72 ions can be used for differentiation of Glu and ¿-Glu residues, provided that the sequence of peptide is known. N-terminal diagnostic ions, c+57, c+59 and c+72 ions were also observed in ECD spectra of ¿-Glu containing peptides, but not in those of Glu containing peptides. It appears that the ring opening of proline may be involved in the formation of these N-terminal diagnostic ions, although the number of systems investigated here is too small to make a conclusive statement. The exact mechanism to generate these ions is not yet clear, but is expected to involve radical rearrangements similar to that which generates the isoaspartic acid diagnostic ion series.

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。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 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，异天冬氨酸残基也可以通过天冬氨酸异构化形成，尽管这发生在Asp 和 isoAsp 残基的分化速度要慢得多，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。该实验室建立了一种基于电子捕获解离 (ECD)-MS/MS 的方法，其中来自 isoAsp 残基的诊断性 c+57/z+-57 离子用于区分和相对。该方法已应用于研究几种模型系统中的 Asn 脱酰胺和 Asp 异构化，如下所述。 A beta 中的 Asn 脱酰胺和 Asp 异构化 β 淀粉样蛋白 (Aβ) 是一种高度疏水性的 40-42 个氨基酸肽，是阿尔茨海默病患者大脑中淀粉样斑块的主要成分。已有一些关于淀粉样斑块中 Aβ 中的 Asp 残基异构化的报道。 (1)、Asp(7)、Asp(23) 位置与 β-折叠结构的增加有关，β-折叠结构在丝和丝中更为丰富。研究了基于 ECD 的方法在 Aβ 中 isoAsp 形成研究中的适用性（Sargaeva 等人，2009），对 23 位具有 isoAsp 的合成 Aβ 肽片段 17-28 进行的 ECD 显示了特征 z6+-。 57 峰定义了 isoAsp 位置，因为人工脱酰胺也可能在酶消化过程中产生 isoAsp，特别是在使用碱性环境时。在胰蛋白酶中，有时需要采用自上而下的方法分析整个蛋白质，其中第 7 位具有 Asn 取代，从而产生脱酰胺 A beta 1 的 Asp 和 isoAsp 残基的混合物。 -40 产生 75% 的预测片段，包括 isoAsp 形式的 z+-57/c+57 诊断峰，清楚地证明了将该方法扩展到自上而下分析的潜力下一步是应用这种方法来研究阿尔茨海默病患者生物样品中 isoAsp 的形成。 还对异构化的 A beta 17-28 片段进行了电子电离解离 (EID)，显示出丰富的碎片和强烈的片段离子峰。然而，在这些较高能量的研究中，诊断峰强度较低，限制了检测能力。尽管 EID 裂解了 > 97% 的残基间键，但该实验证明 EID 可用于阿尔茨海默病斑块中预测的异构化天冬氨酸。同天冬氨酸组学研究可替代 ECD，这对于分析不适用 ECD 的单电荷离子可能是有利的。 钙调蛋白中的 Asn 脱酰胺和 Asp 异构化 148 个氨基酸的钙调蛋白 (CaM) 是一种钙结合蛋白，其四个 EF 基序具有多个 Asn-Gly 和 Asp-Gly 序列，这些序列在没有 Ca2+ 的情况下是不稳定的脱酰胺和异构化位点。老化的 CaM（pH 8.0，14 天，无 Ca2+）显示出 2 Da 质量位移，表明两个 Asn 残基处存在脱酰胺作用（Yao 等人，2009）使用活化离子 (AI)-ECD 对老化的 CaM 进行自上而下的分析，结果观察到 147 个残基间键中的 66 个发生断裂。碎片离子 z524+、y969+ 中的 2 Da 质量偏移，以及小于 c59 的任何 N 端片段中没有质量偏移，明确识别了脱酰胺位点位于 Asn 60 和 Asn 97。然而，没有观察到 isoAsp 的诊断离子，这可能是由于碎片离子分布在许多解离通道上导致离子丰度较低。 作为替代方案，采用自下而上的方法来监测 CaM 中 isoAsp 的形成，其中老化的 CaM 首先被胰蛋白酶消化，然后对相关肽进行 ECD 分析，观察到肽 (38-74) 的一个 Da 质量位移。 ) 和肽 (91-106)，将 Asn 60 和 Asn 97 识别为脱酰胺位点，与 Asn 自上而下的异构化结果一致。以前使用 ECD 方法检测到，可能是因为与 Asn 脱酰胺反应相比，其反应速率要慢得多。在当前的研究中，在几个胰蛋白酶肽 ECD 光谱中观察到诊断性 c+57/z+-57 离子，表明形成了 。 isoAsp 不仅存在于因脱酰胺而导致的 Asn 60 和 Asn 97 残基上，而且还存在于因 Asp 而导致的 Asp 22、Asp 24 和 Asp 95 上异构化。 孵育过程中 Ca2+ 的存在似乎抑制了 CaM 在 pH 8 下老化 2 周，产生的脱酰胺作用要少得多，这在所得胰蛋白酶肽 (91-106) 及其同位素的 ESI 光谱中很明显。该模式表明少于 20% 的 Asn 97 被转化为酸性产物，氨基酸残基 91-106 位于结构域 III 中。 CaM，其 -DGNG- 序列作为 Ca2+ 结合基序。 HGH 中的 Asn 脱酰胺 脱酰胺不仅在许多疾病中发挥重要作用，而且还会影响治疗药物的效力和保质期。191 个氨基酸的蛋白质人类生长激素 (HGH) 是第一种用于刺激生长和细胞的生物技术药物。众所周知，HGH 在出售的药物制剂中会脱酰胺。我们最近与当地一家初创制药公司合作，研究 HGH 的修饰，包括脱酰胺（Cui 等人， 2009）。在 ESI-MS 和 MS/MS 分析之前，HGH 中的二硫键被还原并烷基化，导致所选 18+ 电荷质量在 25 eV 处向更高电荷态转变。天然 HGH 的 21+ 电荷态产生 14 个 y 型和 7 个 b 型以及大量内部碎片离子，而 21+ 电荷态产生 39 个 y 型和 7 个 b 型离子。将碰撞能量从 25 eV 增加到 35 和 45 eV 并没有产生更多 21+ 电荷态的序列特异性离子，相反，使用老化的 HGH 进行了更多的内部碎片离子分析。 CAD 和 ECD 正在进行中。 还对老化的 HGH 进行了自下而上的研究。还原烷基化后，将 HGH 在 0.1 M 碳酸氢铵缓冲溶液（pH ~ 8）中于 37°C 孵育 5 天，然后用胰蛋白酶消化老化的 HGH。 1小时，所得肽混合物通过自装POROS柱纯化，并通过ESI-MS和ECD分析一种肽。 (147-FDTNSHNDDALLK-159) 观察到质量偏移 +1 Da，表明两个 Asn 残基之一或两个处发生脱酰胺化。在该肽的 ECD 谱中，z7 离子 a 显示其同位素模式发生显着变化。 ，表明 Asn 153 部分脱酰胺，因为复合物内氢转移也可能导致 +1 Da 质量转移到 z 离子。同位素模式（z+ 到 z2），很难量化 Asn 153 位点的脱酰胺百分比，或确定 Asn 150 是否也脱酰胺，高分辨率串联 MS 实验应该有助于区分这两种效应，因为脱酰胺会导致+0.984 Da 质量位移，而 z2 离子的形成导致 +1.008 Da 位移，未观察到 isoAsp 形式的诊断离子。这有点令人惊讶，但并非完全出乎意料。由于脱酰胺是在酶消化之前进行的，因此当琥珀酰亚胺中间体进行水解时，HGH 的三级结构可能会阻止异天冬氨酸残基的形成。由于丰富的分子内相互作用，诊断离子的形成在这种富含酸性残基的肽中受到抑制，因此在 HGH 被消化后进行老化研究是必要的。预计在无规卷曲肽的脱酰胺过程中会受到青睐，所得脱酰胺肽的 ECD 应该能够辨别这两种可能性。接下来将进行该实验。 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，该领域的研究进展如下。 使用 ECD 表征 β 肽 异天冬氨酸是一种 β 连接氨基酸，其氨基与 ¿碳而不是 ¿碳除外。丙氨酸，其他β氨基酸很少出现在自然界中。人们正在探索基于肽的抗生素作为避免抗生素耐药性的方法。肽可能以两种不同的形式存在：a ¿ 2 连接肽的侧链连接到 ¿碳和 ¿ 3 连接肽的侧链连接到 ¿由于 ECD 已成功实施，基于通过 C¿ 生成的诊断离子来识别 isoAsp 残留物。 -C¿键断裂，它也可用于区分 ¿ 2 和 ¿ 3 个相连的肽。 扩展 ECD 方法来表征其他 β 氨基酸残基的努力产生了不同的结果（Sargaeva 等人，2008，2009）Q06 肽（V¿2A¿2L¿2V¿3A¿）。 3L¿3）主要产生单电荷前体离子，不适合 ECD 分析，已应用三种方法来增加电荷态。在 ESI 溶液中添加对硝基苯甲醇 (p-NBA) 通常会导致电荷态分布上移；尽管在 Q06 肽的情况下，所得的双电荷前体离子仍然是丰度太低，无法通过 ECD 进行分析。 胆胺与羧酸的共价连接在产生双电荷离子方面要有效得多，但这些离子的 ECD 造成了大部分胆胺标签的损失。 b/y 裂解，只有一个 N-C¿并且没有C?? -C¿最后，二价金属离子 (Ca2+) 加合也产生了丰富的双电荷 Q06 离子，但没有 C¿ -C¿这些金属加合离子的 ECD 中也会产生裂解，因为添加固定电荷基团或金属离子可能会改变 ECD 断裂行为，因此用 β-homoGln 和 β- 取代具有 Gln 5 和 Leu 10 的 P 物质类似物。令人惊讶的是，双质子化的β-物质P 分别合成并分析了N-C¿。也不是 C¿ -C¿ isoAsp 肽和其他 β 连接肽的 ECD 断裂模式之间的差异证明了侧链基团在 ECD 中的重要性。稳定含有 isoAsp 的肽的 ECD 中形成的自由基，这种稳定作用通常在其他 β 连接肽中不存在。 谷氨酸和 ¿ 的区别-谷氨酸残基 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) 的 Gln 脱酰胺化速度要慢得多，与 Asn 对应物相比，Gln 脱酰胺化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，占晶状体蛋白的 90%，在其生命周期中很少发生更新，从而允许积累多种修饰，其中脱酰胺是最重要的修饰之一。普遍存在，它会降低晶状体蛋白溶解度，改变晶状体透明度，并且还参与白内障的形成，白内障是导致失明的主要原因。 在这项研究中（Li et al., 2009），我们开发了一种基于 ECD 的方法来区分 Glu 和 ¿ -Glu 残基，基于 isoAsp 研究的知识，一组包含 Glu 或 ¿ 的合成晶状体蛋白肽片段。 -Glu 残基通过 ECD 进行分析，与在含有 isoAsp 的肽中观察到的 c+57/z+-57 诊断离子类似，在 ¿ 中也观察到了特征性 c+72/z+-72 离子。 -Glu 肽。一些 z+-72 离子也存在于含 Glu 的肽的 ECD 光谱中，尽管它们从未出现在紧邻 Glu 残基之前的裂解位点上，这些离子可能是由基团上的自由基位点引发的电荷远程断裂产生的。 z+ 离子，这是目前正在研究的一个主题，因为 ¿ 的 ECD 中的 z+-72 离子。 -Glu肽仅在初始N-C¿时形成键断裂发生在 ¿ 的 N 端侧-Glu 残基，z+-72 离子可用于区分 Glu 和 ¿ -Glu 残基，前提是肽的 N 端诊断离子序列已知，在 ¿ 的 ECD 光谱中也观察到了 c+57、c+59 和 c+72 离子。 -Glu 含肽，但不在含 Glu 肽中 看来脯氨酸的开环可能参与这些 N 末端诊断离子的形成，尽管此处研究的系统数量太少，无法得出结论。生成这些离子的确切机制尚不清楚，但预计涉及类似于生成异天冬氨酸诊断离子系列的自由基重排。