Analysis of oxidative modification of proteins.
Analysis of oxidative modification of proteins.
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DOI：
10.1002/0471140864.ps1404s56
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发表时间：
2009-04-01
影响因子：
--
通讯作者：
Yan, Liang-Jun
中科院分区：
文献类型：
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作者：
Yan, Liang-Jun
蛋白质是氧化修饰的目标。本单元描述了分析常见蛋白质氧化指标的详细程序，包括蛋白质羰基形成、蛋白质硫醇损失、硝基酪氨酸和二酪氨酸形成以及异天冬氨酸形成。详细介绍了用 2,4-二硝基苯肼、氚化硼氢化钠和生物素酰肼标记的蛋白质羰基的分析程序，然后根据每种标记化学物质的显着特征进行检测测量。概述了通过使用放射性标记的[(14)C]-碘乙酰胺定量游离蛋白质硫醇的损失来测定蛋白质半胱氨酸氧化的方法。描述了通过气相色谱/质谱法测量蛋白质二酪氨酸的方案，以及通过竞争性 ELISA 方法检测蛋白质硝基酪氨酸的详细信息。最后，描述了使用蛋白质-L-异天冬氨酰甲基转移酶定量蛋白质结合的异天冬氨酸的方法，该酶将肽和蛋白质中异常的L-异天冬氨酰残基转化为正常的天冬氨酰残基。
Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.