DIFFERENTIATION OF ASPARTIC VERSUS ISO-ASPARTIC ACID RESIDUES IN PEPTIDES

肽中天冬氨酸残基与异天冬氨酸残基的区分

• 批准号: 7602007
• 负责人: PETER B. O'CONNOR
• 金额: $ 6.25万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602007
• 关键词: Arginine; Aspartic Acid; Calmodulin; Cells; Characteristics; Charge; Computer Retrieval of Information on Scientific Projects Database; Cytochromes; Data; Detection; Diagnostic; Dissociation; Electrons; Fourier Transform; Funding; Grant; Home environment; Incubated; Institution; Ions; Lysine; Melanocytic nevus; Methods; Mole the mammal; N-terminal; Peptides; Positioning Attribute; Post-Translational Protein Processing; Protein Analysis; Proteins; Range; Relative (related person); Research; Research Personnel; Resources; Series; Side; Site; Source; Structure; Testing; Thinking; United States National Institutes of Health; Vertebral column; Week; base; carbene; dalton; deamidation; mass spectrometer; nano; protein misfolding; research study; synthetic peptide

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. Deamidation of asparaginyl residues in proteins is a post-translational modification resulting in a mixture of aspartyl and isoaspartyl residues and thought to be responsible for the inactivation and misfolding of proteins. Of the two isomeric products, isoaspartyl is thought to be the most damaging to protein activity because the primary structure is shifted by the insertion of a methylene group into the protein backbone thus making differentiation of the two forms important. ECD (electron capture dissociation) has been shown to differentiate the two forms in synthetic peptides based on characteristic fragment ions of each form. Data presented here shows that these ECD fragment ions are reproducible in tryptic peptides from a deamidated protein proving the method?s applicability to protein analysis. ECD analysis was performed on a home built qQq-FTMS (Fourier transform mass spectrometer with mass filtering front-end quadrupoles and CAD cell) equipped with a nano-spray source and 7T actively shielded magnet. For each experiment, the multiply charged precursor ions were isolated in Q1, externally accumulated in Q2 and then transmitted to the ICR cell for ECD and subsequent detection. A tryptic fragment of cytochrome C (H-TGPNLHGLFGR-OH, m/z = 584.8153, 2+) was fully deamidated overnight at 80¿C and pH 12 indicated by a mass shift of approximately 1 dalton. Calmodulin was incubated at 37¿C and pH 8 for two weeks then digested by tryspin and a tryptic peptide (H-VFDKDGNGYISAAELR-OH, m/z = 585.6290, 3+) was shown to be completely deamidated. The ECD spectrum of the cytochrome C deamidated tryptic peptide showed all z? ions within the range of detection although only four c ions were detected (c7-c10) and were in general of lower abundance due to the N-terminal arginine residue. A peak corresponding to the z8-57 (1 ppm) fragment indicated the presence of the isoaspartyl residue. No complimentary fragment ion (c?+58) was found in the spectrum. The deficiency of this ion is most likely due to the position of the arginine residue in concurrence with the fact that the diagnostic isoaspartyl ions are typically of lower abundance than the c/z? series ion. A peak corresponding to the neutral loss of 60 daltons, the loss of the aspartic acid side chain from the reduced precursor ion, was not found indicating that the aspartyl product was of much lower abundance than the isoaspartyl form. The ECD spectrum of the calmodulin tryptic peptide showed 12 c and 12 z? ions, all of which are in similar abundances most likely due to the N-terminal arginine residue and the lysine residue close to the C-terminus. Both the c7?+58 and z8-57 ions were found (1 ppm) indicating the presence of the isoaspartyl residue substituted for the arginine residue. The peak corresponding to the loss of the aspartic acid side chain from the reduced precursor ion for this peptide was of considerable abundance but cannot provide unambiguous evidence of the aspartyl form because of the two aspartyl residues in the peptide. The results above show that the isoaspartyl product from deamidation of asparaginyl residues in peptides and proteins can be detected based on the presence of the c?+58 and z8-57 diagnostic ions. Furthermore, the relative abundance of these diagnostic ions has been shown to be linear with their mole ratio so that quantitation of the asp/isoasp ratio in a site-specific manner is possible. Further testing is necessary to determine if this extends to whole proteins and is ongoing.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白质中天冬酰胺残基的脱酰胺化是一种翻译后修饰，导致天冬氨酸和异天冬氨酸残基的混合物，被认为是导致蛋白质失活和错误折叠的原因。在这两种异构体中，异天冬氨酸被认为对蛋白质活性的损害最大，因为亚甲基插入到蛋白质骨架中会使一级结构发生变化，因此区分这两种形式很重要。电子捕获解离(ECD)已被证明可以根据每种形式的特征碎片离子来区分合成肽中的两种形式。这里提供的数据表明，这些ECD片段离子在脱酰胺化蛋白质的胰酶多肽中是可以重现的，证明了S方法适用于蛋白质分析。 在自制的QQQ-FTMS(带质量过滤前端四极杆和CAD池的傅里叶变换质谱仪)上进行了ECD分析，该仪器配备了纳米喷雾源和7T主动屏蔽磁体。对于每个实验，在第一季度分离出多电荷的前体离子，在第二季度从外部积累，然后传输到ICR池进行ECD和随后的检测。细胞色素C的一个胰酶片段(H-TGPNLHGLFGR-OH，m/z=584.8153，2+)在80℃和pH 12下被完全脱胺，质量位移约为1道尔顿。将钙调蛋白在37℃、pH 8的条件下孵育两周后，用TRYSPIN消化，得到一种完全脱胺的胰蛋白酶多肽(H-VFDKDGNGYISAAELR-OH，m/z=585.6290，3+)。 细胞色素C去酰胺化胰蛋白酶多肽的ECD谱显示所有的z？虽然只检测到四个C离子(C7-C10)，但由于N-端精氨酸残基，通常丰度较低。与z8-57(1ppm)片段对应的峰表明存在异天冬氨酸残基。光谱中未发现补充性碎片离子(c？+58)。该离子的缺乏很可能是由于精氨酸残基的位置与诊断的异天冬氨酸离子的丰度通常低于c/z？系列离子。没有发现与中性损失60道尔顿相对应的峰，即从还原的前体离子中损失天冬氨酸侧链，这表明天冬氨酸基产物的丰度比异天冬氨酸形式低得多。 钙调素胰蛋白酶多肽的ECD谱分别为12c和12z？离子，所有离子的丰度相似，很可能是由于N-末端的精氨酸残基和靠近C-末端的赖氨酸残基。C7+58和z8-57离子都被发现(1ppm)，表明存在异天冬氨酸残基取代精氨酸残基。与该肽的还原前体离子失去天冬氨酸侧链相对应的峰相当丰富，但由于该肽中有两个天冬氨酸残基，因此不能提供明确的天冬氨酸形式的证据。 上述结果表明，基于C+58和Z8-57诊断离子的存在，可以检测到多肽和蛋白质中天冬酰胺残基脱酰胺的异天冬氨酸产物。此外，这些诊断离子的相对丰度已被证明与它们的摩尔比是线性的，因此以特定地点的方式定量asp/isasp比率是可能的。有必要进行进一步的测试，以确定这是否延伸到整个蛋白质，并正在进行中。