Tags for oxidized proteins (TOP) useful for assessing the effect of reactive oxygen species on disease proteomics

活性氧簇(ROS)在体内对核酸、脂肪酸、碳水化合物和蛋白质造成氧化损伤。赖氨酸、精氨酸、脯氨酸和苏氨酸残基的侧链容易受到ROS的损伤而发生羰化。已经证明，蛋白质羰基与衰老和许多疾病有很好的相关性，包括糖尿病、退行性脑疾病(阿尔茨海默病和帕金森病)和慢性肺部疾病。然而，这些碳化蛋白与这些疾病之间的关系尚不清楚。在这里，我们建立了一种使用称为TOP(氧化蛋白标签)的亲和标签来研究疾病与蛋白质羰基之间的关系的方法。TOP由三个功能元件组成：亲和标签、同位素编码的连接物和与蛋白质羰基反应的酰肼。该标签使我们能够从粗细胞提取液中富集碳化蛋白质，并随后识别碳化位点。将TOP应用于人工氧化肽的LC-MS研究，成功地从不同氧化程度的多肽中分离纯化出了羰基化多肽。研究发现，蛋氨酸氧化后赖氨酸发生了羰化反应。此外，该标签还用于研究一种人工氧化的蛋白质。

Reactive oxygen species (ROS) causes oxidative damages on nucleic acids, fatty acids, carbohydrates, and proteins in vivo. Side chains of lysine, arginine, proline and threonine residues are susceptible to ROS damages to be carbonylated. It has been demonstrated that protein carbonyls correlates well with aging and many diseases including diabetes, degenerative brain diseases (Alzheimer's disease and Parkinson's disease) and chronic lung disease. However, the relationships between the carbonylated proteins and those diseases have not yet been clarified.Here, we established a method to mass-spectrometrically study the relationships between disease and protein carbonyls using an affinity tag called TOP (tags for oxidized proteins). TOP consists of three functional elements : an affinity tag, an isotope-coded linker, and hydrazide to react with protein carbonyls. The tag enables us to enrich carbonylated proteins from crude cell extracts and to subsequently identify carbonylated sites. Furthermore, TOP lets us quantitatively and directly analyze the protein carbonyls using mass spectroscopy.We applied the TOP to studying states of artificially oxidized peptides by LC-MS and succeeded in purifying the carbonylated peptides from variously oxidized ones. It was found that carbonylation of lysine occurred after oxidation of methionine. Furthermore, this tag was applied to studying an artificially oxidized protein.