Pinpointing oxidative modifications in proteins-recent advances in analytical methods

Oxidation is one of the key degradation pathways in proteins and is of relevance to analyze in a wide variety of research disciplines. The types of different modifications occurring during oxidation by reactive oxygen species (ROS) are almost as many as the number of available analytical methods. Protein oxidation in biological samples has traditionally been analyzed by various protein carbonyl assays, sometimes combined with mass spectrometry (MS) for identification of the carbonylated proteins. MS is now increasingly used also for the determination of the exact position of the oxidation in the amino acid sequence, an approach that is aided by recent developments within the field of proteomics. The following review summarizes the effects of ROS on proteins, describes methods for labeling and separation of oxidized proteins in complex mixtures, and provides insight into various MS-based methods to localize the modifications within the protein primary structure. Pitfalls with the different techniques are given, as well as examples from various applications within biological studies, protein therapeutics, plant science, the food industry and industrial biotechnology.