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: 7369273
• 负责人: PETER B. O'CONNOR
• 金额: $ 4.05万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369273
• 关键词: DIFFERENTIATION; ASPARTIC; VERSUS; ISO; ACID

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。蛋白质中天冬酰胺残基的脱酰胺是一种翻译后修饰，导致天冬酰胺基和异天冬酰胺基残基的混合物，并被认为是蛋白质失活和错误折叠的原因。在这两种异构产物中，异戊酰基被认为对蛋白质活性的破坏最大，因为通过将亚甲基插入蛋白质骨架中而使一级结构发生变化，从而使两种形式的区分变得重要。ECD（电子捕获解离）已被证明可以根据每种形式的特征碎片离子区分合成肽中的两种形式。这里提供的数据表明，这些ECD片段离子是可重复的胰蛋白酶肽从脱酰胺蛋白质证明的方法？适用于蛋白质分析。 ECD分析在自制的qQQ-FTMS（具有质量过滤前端四极杆和CAD单元的傅里叶变换质谱仪）上进行，该质谱仪配备有纳米喷雾源和7 T主动屏蔽磁体。对于每个实验，多电荷前体离子在Q1中分离，在Q2中外部积累，然后传输到ICR池进行ECD和后续检测。细胞色素C的胰蛋白酶片段（H-TGPNLHGLFGR-OH，m/z = 584.8153，2+）在80 ℃和pH 12下完全脱酰胺过夜，质量位移约为1道尔顿。将钙调蛋白在37 ℃和pH 8下孵育两周，然后用trypin消化，胰蛋白酶肽（H-VFDKDGNGYISAAELR-OH，m/z = 585.6290，3+）显示完全脱酰胺。 的ECD光谱的细胞色素C脱酰胺胰蛋白酶肽显示所有z？在检测范围内的离子，尽管仅检测到四种C离子（c7-c10），并且由于N-末端精氨酸残基，通常具有较低的丰度。对应于z 8 -57（lppm）片段的峰表明异丙基残基的存在。无互补碎片离子（c？+）58)在光谱中被发现。这种离子的缺乏是最有可能的，由于精氨酸残基的位置，同时与事实，即诊断异戊酰离子通常是较低的丰度比c/z？系列离子没有发现对应于60道尔顿中性损失的峰，即从还原的前体离子损失天冬氨酸侧链，表明乙酰基产物的丰度比异乙酰基形式低得多。 钙调素胰蛋白酶肽的ECD光谱显示12 c和12 z？离子，所有这些离子的丰度相似，最有可能是由于N-末端精氨酸残基和靠近C-末端的赖氨酸残基。C7+？发现z 8 - 58和z 8 -57离子（1 ppm），表明存在取代精氨酸残基的异丙基残基。该肽的还原前体离子导致天冬氨酸侧链丢失的峰具有相当大的丰度，但由于肽中有两个乙酰基残基，因此无法提供乙酰基形式的明确证据。 上述结果表明，肽和蛋白质中天冬酰胺残基脱酰胺的异戊酰产物可以基于c？+的存在而被检测到。58和Z 8 -57诊断离子。