Oxidation generates multiple diverse post-translational modifications resulting in changes in protein structure and function, associated with a wide range of diseases. Of these modifications, carbonylations have often been used as hallmarks of oxidative damage. However, accumulating evidence supports the hypothesis that other oxidation products may be quantitatively more important under physiological conditions. To address this issue we have developed a holistic mass spectrometry-based approach for simultaneous identification, localization and quantification of a broad range of oxidative modifications based on so-called ‘dependent peptides’. The strategy involves unrestricted database searches with rigorous filtering focusing on oxidative modifications. The approach was applied to bovine serum albumin and human serum proteins subjected to metal ion-catalyzed oxidation resulting in identification of more than sixty different types of oxidative modifications. The most common modification in the oxidized samples is hydroxylation, but carbonylation, decarboxylation and dihydroxylation, are also abundant. Carbonylation however showed the largest increase in abundance relative to non-oxidized control samples. Site specific localization of modified residues reveals several ‘oxidation hotspots’ showing high levels of modification occupancy, including specific histidine, tryptophan, methionine, glutamate and aspartate residues, even though the majority of the modifications occur at low occupancy levels on a diversity of side-chains.

中文翻译：

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用于鉴定，定位和定量氧化蛋白修饰的无限制质谱数据分析

氧化产生多种多样的翻译后修饰，导致蛋白质结构和功能的改变，与多种疾病有关。在这些修饰中，羰基化经常被用作氧化损伤的标志。但是，越来越多的证据支持这样的假设，即在生理条件下其他氧化产物在数量上可能更为重要。为了解决这个问题，我们开发了一种基于整体质谱的方法，可以同时识别，定位和定量基于所谓“依赖肽”的多种氧化修饰。该策略涉及不受限制的数据库搜索，并以氧化修饰为重点进行严格过滤。该方法应用于经过金属离子催化氧化的牛血清白蛋白和人血清蛋白，从而鉴定出六十多种不同类型的氧化修饰。氧化样品中最常见的修饰是羟基化，但是羰基化，脱羧和二羟基化也很丰富。然而，羰基化显示相对于未氧化的对照样品，丰度增加最大。修饰残基的位点特异性定位揭示了几个“氧化热点”，显示出较高的修饰率，包括特定的组氨酸，色氨酸，蛋氨酸，谷氨酸和天冬氨酸残基，即使大多数修饰发生在低占用率下，也存在多种侧面变化。链。