Abstract
Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.
Highlights
Introduction to Protein OxidationMany diseases have an oxidative aetiology resulting from activation of the immune system, mitochondrial dysfunction or environmentally-induced oxidative stress
While often these are normal, physiological processes, there is evidence that they can be dysregulated in disease or aging, and there have been some excellent reviews on this topic recently [103,114], including the application of mass spectrometry to support these studies [113,115]
It is clear that MS-based strategies have greatly underpinned the increase in knowledge in this area, and are confidently expected to continue to do so
Summary
Many diseases have an oxidative aetiology resulting from activation of the immune system, mitochondrial dysfunction or environmentally-induced oxidative stress. Various different reactive and oxidizing species exist and vary in their reactivity to protein residues and sites. Protein oxidation is often measured as a marker of oxidative damage and cellular stress, and a wide variety of methods exist, varying from simple global methods to specific approaches to detecting individual modified residues [3]. A commonly measured modification is carbonyl formation, which can occur on lysine, arginine, serine, threonine and proline residues following metal-catalysed oxidation or attack by hypochlorous acid. Total digestion followed by HPLC, LC-MS or LC-MS/MS has been used to investigate a wide range of oxidized amino acids [5], but these approaches do not provide information on the specific protein that has been modified, or the exact site of modification. This article gives an overview of advances and limitations of LC-MS/MS approaches for detecting specific non-enzymatic oxidative modifications to proteins, and summarizes their recent application in studies of disease
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