Detection, identification, and quantification of oxidative protein modifications

Abstract

Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidentally (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications-at the amino acid, peptide, or protein level-and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.

Keywords: carbonyl; disulfide; hydroperoxide; oxidative stress; oxygen radicals; post-translational modification; protein chemical modification; protein cross-linking; reactive oxygen species.

Figure 1.

Overview of interconversion processes of common biological oxidants. The extent of these reactions depends on the circumstances and reaction conditions and is therefore only intended as guide to the complexity of examining oxidant reactions. Adapted from Ref. .

Figure 2.

Summary of O₂-dependent reactions of carbon, peroxyl, and alkoxyl radical reactions on proteins and the occurrence of short-chain reactions. In this scheme, R, R′, and R″ are used to designate carbon-centered species with different chemical structures.

Figure 3.

Chemical structures of some of the most abundant and/or commonly examined side-chain oxidation products.

Figure 4.

Overview of radical reactions resulting in cleavage of the protein backbone. This can arise from both direct reactions at backbone sites (principally at the α-carbon) and also indirectly via initial oxidation at side-chain sites with subsequent radical transfer to the backbone, either intra- or intermolecularly. For further details see main text and Refs. , , , .

Figure 5.

Initial oxidation at electron-rich sites (e.g. Tyr and Trp residues but also Met, His, and Cys) can result in rapid electron transfer both within, and between, protein molecules. This can result in subsequent reactions and products being formed at sites that were not the initial site of oxidation and at locations remote from the initial site.

Figure 6.

Workflow to assess protein amino acid composition and their associated modifications. Proteins isolated and purified before digestion or hydrolysis to their constituent amino acids. Free amino acids and/or related oxidation products are then separated by LC. For some applications, pre-column or post-column derivatization of the amino acids and related products is required before separation to enable detection and quantification using one or more detection methods, which typically include MS, fluorescence, UV or visible absorption, or electrochemical (EC) detection. Abbreviations used are as follows: MSA, methane sulfonic acid; NFK, N-formylkynurenine.

Figure 7.

Approaches and experimental methods to detect reactive intermediates on proteins.

Figure 8.

Overview of methods for the detection and analysis of carbonyls (both protein-bound and low-molecular mass) arising from protein oxidation.

Figure 9.

Workflow to assess protein modifications by peptide mass–mapping approaches. Proteins were isolated and purified before digestion to peptides with trypsin or other protease enzymes. Peptides analyzed by MS following cleanup and LC separation. Peptide mass fingerprinting analysis and/or peptide fragmentation analysis by MS-MS with appropriate database searches using fixed or variable modifications followed by validation to limit artifacts.

Figure 10.

Overview of approaches to detect cross-links on proteins.

Figure 11.

Outline of isotope-labeling method to detect and characterize protein cross-links using MS.