Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases

Abstract

Generation of reactive oxygen species and related oxidants is an inevitable consequence of life. Proteins are major targets for oxidation reactions, because of their rapid reaction rates with oxidants and their high abundance in cells, extracellular tissues, and body fluids. Additionally, oxidative stress is able to degrade lipids and carbohydrates to highly reactive intermediates, which eventually attack proteins at various functional sites. Consequently, a wide variety of distinct posttranslational protein modifications is formed by protein oxidation, glycoxidation, and lipoxidation. Reversible modifications are relevant in physiological processes and constitute signaling mechanisms ("redox signaling"), while non-reversible modifications may contribute to pathological situations and several diseases. A rising number of publications provide evidence for their involvement in the onset and progression of diseases as well as aging processes. Certain protein oxidation products are chemically stable and formed in large quantity, which makes them promising candidates to become biomarkers of oxidative damage. Moreover, progress in the development of detection and quantification methods facilitates analysis time and effort and contributes to their future applicability in clinical routine. The present review outlines the most important classes and selected examples of oxidative protein modifications, elucidates the chemistry beyond their formation and discusses available methods for detection and analysis. Furthermore, the relevance and potential of protein modifications as biomarkers in the context of disease and aging is summarized.

Keywords: Aging; Biomarker; Oxidative stress; Protein modification; Protein oxidation.

Graphical abstract

Fig. 1

Oxidation of sulfur-containing amino acids. Cysteine is oxidized in a multi-step reaction to the respective sulfenic, sulfinic, and sulfonic acid modifications or oxidatively conjugated with glutathione (GSH) or another cysteine residue (A). Methionine is reversibly oxidized to methionine sulfoxide and irreversibly oxidized to methionine sulfone (B).

Fig. 2

Oxidation of aromatic amino acids. Oxidation of tyrosine is favored by the intermediary tyrosyl radical and leads to the stable modifications dihydroxyphenylalanine and dityrosine (A). Unusual tyrosine isomers like ortho-tyrosine are generated by oxidation of phenylalanine residues (B). Oxidation of tryptophan is the initial step of the N-formyl kynurenine reaction cascade (C). The stable modification 2-oxohistidine is formed by histidine oxidation (D).

Fig. 3

Formation of glycoxidation products. A special subset of advanced glycation endproducts is exclusively formed under oxidative conditions including the prominent modifications carboxymethyl lysine and pentosidine. The novel modifications glyoxylyl and formyl lysine are potential biomarkers of oxidative stress based on their formation pathways.

Fig. 4

Formation of lipoxidation products. Complex reaction cascades of malondialdehyde (A) and 4-hydroxynonenal (B) are briefly summarized and important protein modifications presented.

Fig. 5

Pathways of protein carbonylation. Lysine and proline residues are vitally important precursors of protein carbonylation by oxidative formation of aminoadipic semialdehyde (A) and glutamic semialdehyde (B), respectively. Oxidative cleavage of peptide backbone results in protein carbonylation via diamide and α-amidation pathways (C). Formation of 2-pyrrolidone by oxidation of prolyl containing peptide (D).

Fig. 6

Generation of reactive nitrogen species (RNS). Nitric oxide synthase (NOS) catalyzes the generation of nitric oxide, which is the precursor of S-nitrosylation and nitration.

Fig. 7

Analysis of oxidative protein modifications. Proteins are oxidized by ROS. Subsequent molecular changes can be analyzed for individual proteins, peptides or modified amino acids. Depending on the choice of analytes, different analysis techniques are available. Among the presented methods, mass spectrometry is the most accurate technique and its application shall be further increased for biological samples to advance the knowledge regarding protein oxidation.

Fig. 8

Quantitation of protein carbonylation. Carbonyl groups are stabilized by derivatization with 2,4-dinitrophenylhydrazine (DNPH). The resulting hydrazones are quantified using a spectrophotometer, specific antibodies, or are analyzed using LC-MS.

Fig. 9

Schematic overview of frequently used biomarkers of protein oxidation. Their strengths, limitations, and relevance as biomarkers in human diseases and aging are discussed in sections 4.1–4.6, based on the current state of research. For further details see also Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 3-NT – 3-Nitrotyrosine, 4-HNE – 4-Hydroxy-2-nonenal, AD – Alzheimer's disease, AGE – Advanced glycation endproduct, ALD – Alcoholic liver disease, AOPP – Advanced oxidation protein product, AS – Amino acids, CAD – Coronary artery disease, CML – Carboxymethyl lysine, COPD – Chronic obstructive pulmonary disease, CSF – Cerebrospinal fluid, Cys-SO3H – Cysteine sulfonic acid, ELISA – Enzyme linked immunosorbent assay, HIV – Human immunodeficiency virus, HPLC – High pressure liquid chromatography, IHC – Immunohistochemistry, NAFLD – Non-alcoholic fatty liver disease, MCI – Mild cognitive impairment, MDA – Malondialdehyde, MetS – Metabolic syndrome, MetSO – Methionine sulfoxide, MS – Mass spectrometry, PBMC – Peripheral blood mononuclear cells, PD – Parkinson's disease, RA – Rheumatoid arthritis, Skin AF – Skin autofluorescence, T2D – Type 2 diabetes, TBARS – Thiobarbituric acid reactive substances, WB – Western Blot. *among others.