Redox homeostasis is a delicate cellular balance between reactive oxygen species (ROS), reactive nitrogen species (RNS), and the cell’s own antioxidant capacity. Disequilibrium, or oxidative stress, often results in selective modifications of target proteins. Because oxidative modifications generally occur on cys residues when a molecular entity undergoes structural or functional change, monitoring alterations in oxidative processes may offer insight into the diagnosis and progression of various human diseases, including Alzheimer’s disease, diabetes and cancer. For this reason, the field of redox proteomics represents an emerging, biologically significant area of study.
Recently, Mermelekas et al. (2013) reviewed current methodologies in gel-based proteomics and quantitative mass spectrometry as used to evaluate oxidative protein modifications.1 They describe specific applications of these approaches in the identification of biomarkers for diseases marked by oxidative stress. The authors also offer expert insight into the limitations of available methods and the likely foci of future studies.
Perhaps the most challenging aspect of redox proteomics is capturing the thiol proteome while minimizing artificial oxidation during experimental processes. This requires sample acidification and/or thiol alkylation, the two primary approaches to “trap” the thiol-disulfide status. The authors indicate that combining these two methods often produces optimal results, depending on the cell samples. For example, in experiments involving microorganisms, acidification destroys the cell wall while alkylating agents cannot; however, mammalian cell membranes are readily penetrated by thiol-blocking reagents.
Other experimental methods reviewed by Mermelekas et al. include reduction and labeling for differential selection of modified proteins, including direct, indirect and double labeling of thiol groups by a fluorophore, as well as the biotin switch technique. The authors present examples of fluorescent labeling, biotin labeling and direct capture techniques used in conjunction with gel-based analysis. They also review mass-spectrometry–specific stable isotope labeling, including isotope code affinity tag (ICAT), isobaric tags for relative and absolute quantitation (ITRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), as well as label-free quantitative mass spectrometry. Based on their assertion that label-free approaches provide efficiency and ease of use, the authors anticipate increased reliance upon these strategies. They indicate that a direct comparison of labeling and non-labeling methods within redox experiments would benefit the research community.
The authors assert that a fundamental issue with these methods is that the complex nature of enrichment, reduction and labeling may diminish the experiment’s reproducibility; this issue is of particular concern in comparative experiments and biomarker studies. Other limitations include a lack of adequate reagents for the identification of modified protein isoforms and limited scholarship on the relationships between particular modifications and protein function.
Despite this, the researchers describe current methodology as both selective and highly sensitive. They call for additional research into the redox-analysis–specific benefits and drawbacks of various methods of fragmentation, as well as available quantitation software packages.
In the immediate future, Mermelekas et al. predict a steady increase in the amount of research dedicated to redox proteomics, particularly given the therapeutic potential inherent in biomarker discovery. They note that maintaining strict guidelines for mass spectrometric data reporting will allow new research to coalesce with existing scholarship and enable research teams to draw meaningful conclusions from their data.
1. Mermelekas, G., et al. (2013) “Redox proteomics: From residue modifications to putative biomarker identification by gel- and LC-MS-based approaches,” Expert Reviews Proteomics, 10(6) (pp. 537–49).
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.