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。 beta中的ASN死亡化和ASP异构化 淀粉样蛋白β（beta）是一种非常疏水的40-42氨基酸肽，是阿尔茨海默氏症患者大脑中淀粉样蛋白斑块的主要结构。关于ASP（1），ASP（7），ASP（23）位置的淀粉样蛋白斑块中ASP残基的异构化的几份报告。 ISOASP地层与β-片结构的增加有关，这些结构在细丝和斑块中更丰富。研究了基于ECD的方法在Beta中研究ISOASP形成的研究（Sargaeva等，2009）。合成的ECD Aβ胡椒片段17-28在位置23显示了定义ISOASP位置的特征Z6+-57峰。由于人工死亡还可以在酶促消化过程中产生ISOASP，尤其是在使用胰蛋白酶时的基本环境中，因此有时希望使用自上而下的方法分析整个蛋白质。允许在第7位的ASN取代的Beta 1-40销毁，从而产生ASP和ISOASP居住的混合物。脱膜的ECDβ1-40产生了75％的预测片段，包括ISOASP形式的Z+-57/C+57诊断峰，清楚地证明了将这种方法扩展到完整的Beta辣椒的自上而下的潜力。下一步是应用这种方法研究阿尔茨海默氏症患者的生物样品中的ISOASP形成。 还执行了异构化的电子离子化解离（EID）17-28片段，该片段显示出广泛的片段化和强烈的片段化离子峰。但是，在这些较高的能量研究中，诊断峰的强度较低，限制了在阿尔茨海默氏菌的斑块中检测到异构天冬氨酸的能力，即使EID裂解了> 97％的层间粘结键。然而，该实验表明，EID可以用于ECD的同性恋者研究替代方案。这对于不适用ECD的单一充电离子的分析可能是有利的。 钙调蛋白中的ASN死亡化和ASP异构化 148个氨基酸钙调蛋白（CAM）是一种钙结合蛋白，在其四只EF-MOTIF手中具有多个ASN-GLY和ASP-GLY序列，它们在没有Ca2+的情况下标记为死亡和异构化位点。老年凸轮的ESI质量谱（pH 8.0，14天，无CA2+）显示出2个DA质量转移，表明在两个ASN残基处存在死亡（Yao等，2009）。对具有活化离子（AI）-ECD的老年CAM的自上而下分析，导致总共147个占用间键中的分裂为66。观察碎片离子Z524+，Y969+的2次质量转移的1 DA质量转移，并且在任何N端碎片中没有质量变化，而在ASN 60和ASN 97上明确鉴定出的n末端碎片中的任何N末端碎片均未识别出死亡。但是，由于诊断的诊断范围并未被诊断出来。 作为替代方案，采用自下而上的方法来监测CAM中的ISOASP组，在该组合中首先通过胰蛋白酶消化老化的CAM，然后对相关肽进行ECD分析。肽（38-74）和肽（91-106）观察到一个DA质量转移，将ASN 60和ASN 97识别为死亡部位，与自上而下的结果一致。 ASP的异构化以前尚未使用ECD方法检测到，这大概是因为与ASN死亡的反应速率要慢得多。在当前的研究中，在几种胰蛋白酶肽ECD光谱中观察到诊断C+57/Z+-57离子，这表明由于死亡导致的残留物ASN 60和ASN 97在ASP 22，ASP 22，ASP 24和ASP 95中，ISOASP不仅形成了ISOASP，因此也形成了ISOASP。 孵育过程中Ca2+的存在似乎抑制了死亡过程。 Ca2+的CAM在pH 8处老化2周产生的死亡较少。这是所产生的胰蛋白酶肽（91-106）的ESI光谱中的证据，其同位素模式表明，ASN 97中只有不到20％的ASN 97转化为酸性产物。氨基酸保留91-106在CAM的域III中，其-dgng-序列用作Ca2+结合基序。 HGH中的ASN脱氨 死亡不仅在许多疾病中起着重要作用，还会影响治疗药物的效力和保质期。 191氨基酸蛋白人类生长马（HGH）是第一种用于人类和其他动物的生长和细胞繁殖的生物技术药物。众所周知，HGH会在出售的药品公式中销毁。我们最近与当地的一家初创企业制药公司合作研究了HGH的修改，包括死亡（Cui等，2009）。在ESI-MS和MS/MS分析之前，HGH中的二硫键降低和烷基化，从而导致向高电荷状态转移。在质量的25 eV处碰撞激活的解离（CAD）所选的18+天然HGH的电荷状态产生14 Y型和7 B型以及许多内部片段离子；而21+电荷状态产生了39 Y型和8个B型片段。将碰撞能量从25 eV增加到35和45 eV并未产生21+电荷态的序列特异性离子，而是观察到更多的内部碎片离子。使用CAD和ECD对老年HGH的自上而下分析正在进行中。 还进行了老化HGH的自下而上研究。降低烷基化后，允许HGH在37oC下在0.1 m碳酸氢铵缓冲液溶液（pH〜8）中孵育5天。然后用胰蛋白酶消化老化的HGH一小时，并通过自包装的波罗柱纯化所得的肽混合物，并通过ESI-MS和ECD分析。观察到一种肽（147-FDTNSHNDDALLK-159），质量转移为+1 Da，表明在两个ASN残留物中的一个或两个处发生死亡。在该肽的ECD光谱中，Z7离子在其同位素模式上显示出显着的转移，表明ASN 153被部分破坏。由于复合氢转移也可能导致+1 DA质量转移向Z离子同位素模式（Z +至Z2），因此很难量化ASN 153位点的死亡百分比，或确定ASN 150是否也被摧毁。高分辨率串联MS实验应有助于区分这两种效应，因为死亡导致+0.984 DA质量转移，而Z2离子形成导致+1.008 DA偏移。没有观察到ISOASP形式的诊断离子，这有点令人惊讶，但并非完全出乎意料。由于死亡剂是在酶促消化之前进行的，因此HGH的三级结构可能阻止了琥珀酰亚胺中间体进行水解时的ISOASP形成。但是，由于丰富的分子内相互作用，这种酸性保留率富含肽的肽也抑制了诊断离子形成的另一种可能性。因此，在消化HGH后，必须进行老化研究。由于在随机线圈肽的死亡过程中，预计ISOASP形成将受到青睐，因此所得的脱膜肽的ECD应该能够辨别这两种可能性。该实验将接下来进行。 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 等素酸是一种与β相关的氨基酸，其氨基群粘合到碳而不是碳。除丙氨酸的唯一外，其他β氨基酸在自然界中很少出现。因此，正在探索基于肽的抗生素作为逃避抗生素耐药性的方式。肽可能以两种不同的形式存在：A»2个链接的肽具有连接到碳的侧链，并且3链链接的肽具有连接到碳的侧链。由于已经成功实施了ECD以识别ISOASP残基，这是基于通过C键裂解产生的诊断离子，因此它也可用于区分2和3链接肽。 扩展ECD方法以表征其他β氨基酸的一种努力会产生混合的结果（Sargaeva等，2008，2009）。 Q06肽的电喷雾电离（ESI）（V¿2A¿2L¿2L¿2V¿3A¿3L¿3）产生了主要单一充电的前体离子，这不适合ECD分析。已经应用了三种方法来增加Q06肽的电荷状态。在ESI溶液中添加P-硝基苯酒精（P-NBA）通常会导致电荷状态分布的上班；尽管在Q06肽的情况下，所得的双重前体离子仍然太低，无法通过ECD分析。在产生双电荷离子方面，胆胺的共价附着在羧酸上更有效，但是这些离子中的ECD主要产生的胆胺标签和b/y裂解的主要损失，只有一个n-c？，没有一个n-c？，没有c¿-c。-c¿裂解。最后，二价金属离子（Ca2+）添加也产生了绝对双重电荷的Q06离子。但是，在这些金属添加离子的ECD中也没有产生c¿-c。也没有产生裂解。由于添加固定电荷组或金属离子可能会改变ECD碎片化行为，因此通过ECD综合并分析了由Beta-Homogln和Beta-Homoleu代替的GLN 5和LEU 10的物质类似物。令人惊讶的是，双倍的质子化β-固定p在含有β氨基酸保留的位点既没有产生n-c？也不产生c¿-c？裂解。 ISOASP Petides的ECD碎片模式与其他与Beta相关的Petides的ECD碎片模式之间的差异证明了侧链组在ECD中的重要性。它表明，相邻的羧基可能有助于稳定含有宠物的ISOASP ECD中形成的自由基。通常，这种稳定效应在其他β连锁宠物中不存在。 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（Li等，2009）中，我们根据ISOASP研究的知识开发了一种基于ECD的方法来区分GLU和� -Glu残基。通过ECD分析了一组含有GLU或� -glu残基的合成结晶蛋白去皮片段。类似于在含有肽的ISOASP中观察到的C+57/Z+-57诊断离子，在�-glu Pepperides中也观察到了特征C+72/z+-72离子。含有辣椒的GLU的ECD光谱中也存在一些Z+-72离子，尽管它们从未出现在GLU残差之前的裂解位点。这些离子可能是由于激进位点在Z+离子上引发的电荷远程碎片而产生的，Z+离子是当前正在研究的受试者。由于仅当初始n-C键裂解发生在�-Glu居住的N端侧时，才会形成 - Glu肽的ECD中的Z+-72离子，因此可以将Z+-72离子用于分化GLU和®-GLU残基，并提供了Pepper的序列。在含有娇小的glu的ECD光谱中，还观察到N端诊断离子，C+57，C+59和C+72离子，但在含Glu含有小物质的葡萄糖的光谱中也观察到。看来，脯氨酸的环开口可能参与了这些N末端诊断离子的形成，尽管此处研究的系统数量太小而无法做出结论性陈述。生成这些离子的确切机制尚不清楚，但预计将涉及类似于产生同甲酸诊断离子系列的根本重排。