Redox proteomics: from residue modifications to putative biomarker identification by gel- and LC-MS-based approaches

Abstract

Quantitative determination of reactive oxygen species and reactive nitrogen species in body fluids, tissues or cells has always been problematic due to their high chemical reactivity and the resulting short half-life. This high reactivity may involve reversible and/or irreversible protein modifications, in particular the covalent oxidative modification of specific amino acid residues. Thus, the occurrence of reactive oxygen species and reactive nitrogen species can be monitored indirectly from the identification of specific protein-chemical footprints. In combination with classical gel-based proteomics or liquid chromatography labeling or label-free techniques, mass spectrometry has emerged as a powerful tool to identify these protein modifications in biological samples. In this review, we present the main methodological approaches for gel-based proteomics and quantitative mass spectrometry applied to oxidative protein modifications, mainly Cys. Representative examples from their application in identifying respective biomarkers in diseases related to oxidative stress are also presented.

Keywords::

• 2DE
• Cys
• mass spectrometry
• modifications
• redox proteomics
• thiol group

Financial & competing interests disclosure

G Mermelekas, M Makridakis and A Vlahou were supported in part by the European Community’s Framework Programme (FP7/2007-2013) under grant agreement number 278611 (Neurinox). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• • Reactive oxygen series and reactive nitrogen series modified proteins may consist disease biomarkers and therapeutic targets.

• • Trapping the thiol proteome and avoiding ex vivo oxidation is a key step in redox proteomics.

• • Proteins in body fluids and biological samples can be analyzed for their redox modifications using gel- and mass spectrometry based proteomics approaches.

• • The general workflow involves trapping of redox status via acidification or alkylation, selective enrichment of peptides having the modified residues, followed by detection thereof via the use of labeling and/or label-free mass spectrometry based approaches.

• • Cys is the most common target for reactive oxygen series and reactive nitrogen series modifications.

• • Enrichment for Cys containing peptides can be regularly performed via various chromatographic approaches.

• • Isotopic reagents have been developed for specific labeling of the redox modified Cys and other residues.

• • Following up on selected findings remains a bottleneck as it involves demonstration of oxidative modification impact on protein function.

Notes