DIFFERENTIATION OF ASPARTIC VERSUS ISO-ASPARTIC ACID RESIDUES IN PEPTIDES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。蛋白质中天冬酰胺残基的脱酰胺是一种翻译后修饰，导致天冬氨酸和异天冬氨酸残基的混合物，被认为是蛋白质失活和错误折叠的原因。在这两种异构体产物中，异天冬氨酸被认为是对蛋白质活性最具破坏性的，因为通过在蛋白质主链中插入亚甲基而改变了初级结构，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片段离子来区分合成肽的两种形式。这里提供的数据表明，这些ECD片段离子在脱酰胺蛋白的色氨酸肽中是可复制的，证明了该方法？S对蛋白质分析的适用性。ECD分析在配备纳米喷雾源和7T主动屏蔽磁铁的自制qQq-FTMS（带有质量滤波前端四极和CAD单元的傅立叶变换质谱仪）上进行。在每个实验中，在Q1中分离出带多重电荷的前体离子，在Q2中外部积累，然后传递到ICR细胞进行ECD和随后的检测。细胞色素C的色氨酸片段（H-TGPNLHGLFGR-OH, m/z = 584.8153,2 +）在80°C和pH为12的条件下过夜完全脱酰胺，质量位移约为1道尔顿。钙调素在37℃和pH 8条件下孵育2周后，用tryspin消化，得到一个色氨酸（H-VFDKDGNGYISAAELR-OH, m/z = 585.6290, 3+）完全脱酰胺。细胞色素C脱酰胺色氨酸的ECD谱显示全部为z？虽然仅检测到4个c离子（c7-c10），但由于n端精氨酸残基，通常丰度较低。z8-57 （1 ppm）片段对应的峰表明异天冬氨酸残留的存在。无互补片段离子(c？+58)在光谱中被发现。这种离子的缺乏很可能是由于精氨酸残基的位置，同时诊断性异天冬氨酸离子的丰度通常低于c/z？系列离子。没有发现60道尔顿中性损失的峰，即还原前体离子的天冬氨酸侧链损失，表明天冬氨酸产品的丰度远低于异天冬氨酸形式。钙调蛋白色氨酸的ECD谱显示为12c和12z ？所有这些离子的丰度相似，很可能是由于n端精氨酸残基和靠近c端的赖氨酸残基。两架c7？发现+58和z8-57离子（1 ppm），表明存在异天冬氨酸残留取代精氨酸残留。从该肽的还原前体离子中对应的天冬氨酸侧链损失的峰相当丰富，但由于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。以上结果表明，基于c?的存在，可以检测肽和蛋白质中天冬酰胺残基脱酰胺的异天冬酰胺产物。+58和z8-57诊断离子。

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.