Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples.
Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples.
复制标题
对生物样品中异天冬氨酰残基进行蛋白质组规模的鉴定和定量。
DOI：
10.1021/pr900428m
复制
发表时间：
2009
影响因子：
4.4
通讯作者：
Zubarev,RomanA
中科院分区：
文献类型：
--
作者：
Yang,Hongqian;Fung,EvaYM;Zubarev,AlexanderR;Zubarev,RomanA
天冬酰胺的脱酰胺和蛋白质中天冬氨酸残基的异构化产生天冬氨酸基和异天冬氨基残基的混合物，后者参与蛋白质的老化和失活。电子捕获解离结合傅立叶变换质谱仪可以通过cn·+58.0054(C_2H_2O_2)和zl-n−_(56.9976)(C_2H_2O_2)的独特片段来区分异天冬氨酸多肽，其中C_2H_2O_2是天冬氨酸残基的位置，C_2H_2是多肽的长度。在本研究中，我们使用特定片段的准确质量来测试isAsp检测的特异性。为此，我们分析了从人类细胞和组织样本中获得的32个完整和部分蛋白质组，并通过ECD 466候选异天冬氨酸肽进行了鉴定。详细的检查显示，这些候选人中有许多人不可靠。为了提高isAsp的检测特异性，必须使用额外的标准，例如相邻的C/z片段、还原物种的特定损失以及色谱峰的形状。对候选人进行最严格的筛选得出了几种情况，其中isAsp的存在是毋庸置疑的。在已鉴定的具有等天冬氨酸的蛋白质中，肌动蛋白、热休克同源71 kDa蛋白和丙酮酸激酶已被鉴定为异天冬氨酸甲基转移酶的底物，异天冬氨酸甲基转移酶是一种将异天冬氨酸转化为天冬氨酸的重要修复酶。用无标记法对相对异构化程度进行了定量。这是首次尝试以高通量的方式分析人类异天冬氨酸组。开发的工作流程可进一步提高生物样品中异天冬氨酸残留量的检测率。
Deamidation of asparaginyl and isomerization of aspartyl residues in proteins produce a mixture of aspartyl and isoaspartyl residues, the latter being involved in protein aging and inactivation. Electron capture dissociation (ECD) combined with Fourier transform mass spectrometry (FT MS) are known to be able to distinguish the isoaspartyl peptides by unique fragments of cn• + 58.0054 (C2H2O2) and zl-n− 56.9976 (C2HO2), wherenis the position of the aspartyl residue andlis the peptide length. In the present study, we tested the specificity of isoAsp detection using the accurate masses of the specific fragments. For this purpose, we analyzed 32 whole and partial proteomes obtained from human cells as well as tissue samples and identified by ECD 466 isoaspartyl peptide candidates. Detailed inspection revealed that many of these candidates were unreliable. To increase the isoAsp detection specificity, additional criteria had to be used, for example, adjacent c/z fragments, specific losses from the reduced species, and the shape of the chromatographic peak. Most stringent filtering of candidates yielded several cases where the presence of isoAsp was beyond doubt. Among the identified proteins with isoAsp, actin, heat shock cognate 71 kDa protein and pyruvate kinase have previously been identified as substrates forl-isoaspartyl methyltransferase, an important repair enzyme converting isoaspartyl to aspartyl. Quantification of relative isomerization degree was performed by the label-free approach. This is the first attempt to analyze the human isoaspartome in a high-throughput manner. The developed workflow allows for further enhancement of the detection rate of isoaspartyl residues in biological samples.