Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification

Abstract

Covalent crosslinks within or between proteins play a key role in determining the structure and function of proteins. Some of these are formed intentionally by either enzymatic or molecular reactions and are critical to normal physiological function. Others are generated as a consequence of exposure to oxidants (radicals, excited states or two-electron species) and other endogenous or external stimuli, or as a result of the actions of a number of enzymes (e.g., oxidases and peroxidases). Increasing evidence indicates that the accumulation of unwanted crosslinks, as is seen in ageing and multiple pathologies, has adverse effects on biological function. In this article, we review the spectrum of crosslinks, both reducible and non-reducible, currently known to be formed on proteins; the mechanisms of their formation; and experimental approaches to the detection, identification and characterization of these species.

Keywords: aggregation; crosslink; di-tryptophan; di-tyrosine; dimerization; disulfides; mass spectrometry; protein oxidation; proteomics; radicals; thiols.

Figure 1

Overview of crosslinks formed on proteins, their nature and mechanisms of formation.

Figure 2

Mechanisms of traditional ‘thiol–disulfide exchange’ (top) and ‘oxidant-mediated thiol–disulfide exchange’ (bottom) reactions.

Figure 3

Generation of crosslinks via oxidized thiol residues. Similar reactions of the ‘activated’ thiols (RS–OH, RS–Cl, RS–Br, RS–SCN, RS–NO) can occur with nitrogen nucleophiles (e.g., RNH₂) to give new S–N bonded species (see text for further details).

Figure 4

Michael addition reactions of nucleophiles to αβ-unsaturated carbonyl compounds.

Figure 5

Formation and reactions of Tyr phenoxyl radicals (Tyr^•). Tyr^• self-react to produce di-Tyr (o,o’-di-Tyr, red; iso-di-Tyr, black) or react with O₂ to generate oxygenated products. Kinetic data from [130].

Figure 6

Formation and reactions of Trp indolyl radicals (Trp^•). Self-reactions of Trp^• produce carbon–carbon (C3–C3) and carbon–nitrogen (C3–N1) di-Trp crosslinks. It should be noted that multiple stereoisomers are potentially formed for both di-Trp dimers. Kinetic constants for self-reactions of Trp^• and their reaction with O₂ are from [159] and [36], respectively.

Figure 7

Michael addition reactions of amino acid side-chains to oxidized His and Tyr residues.

Figure 8

Overview of methods to detect and characterize crosslinked proteins and the sites/types of modifications. Abbreviations used: CD: circular dichroism, SANS: small angle neutron scattering, SAXS: small angle X-ray scattering, H–D: hydrogen–deuterium exchange mass spectrometry.