Oxidative redox processes can cause human disease. During periods of oxidative stress, reactive oxygen and nitrogen species can structurally alter and impair the function of cellular proteins, lipids and DNA. While these highly reactive and short-lived species are hard to measure directly, researchers monitor them through the footprints they leave behind on certain proteins. Identifying these residues in turn may help to pinpoint disease biomarkers.

In a detailed review article of redox proteomics, Mermelekas et al.1 define the challenges of monitoring reactive oxygen and nitrogen species along with their targets. They provide a detailed description of the methods used to capture this information and offer examples of how these methods have been applied to investigate disease-specific biomarkers. By summarizing the pre-analysis workflow, the authors describe the different factors that need to be considered before and during analysis.

Sample preparation challenges

The transient nature of redox-related post-translational modifications is a key challenge. Mermelekas et al. describe the complex experimental procedures required to ensure that protein thiols do not oxidize during sample processing and preparation. They recommend degassing all reagents to remove oxygen and using chelating agents to remove metal ions such as copper, iron and nickel. Samples need to be prepared under acid reactions or in the presence of alkylating agents to “trap” the thiol-disulphide status of the proteins of interest.

Protein labeling and enrichment

Both labeling and non-labeling approaches can be used. Mermelekas describes the specific advantages, limitations and applications of the different protein labeling techniques used with different analytical methods. Fluorescent labeling, biotin labeling, stable isotope labeling, metabolic labeling and isobaric labeling are all described in detail. While these labeling methods and kits seem direct and easy, their cost can be high.

Protein enrichment is necessary to select for the redox-modified proteins. Classical approaches, such as biotin enhancement and activated thiol chromatography, can be used, and newer experimental affinity methods are being developed. One downside to enrichment is that it increases the number of experimental steps required, which can impact the variability and reduce reproducibility.

Analysis methods

The two methods used to analyze redox modifications are gel-based proteomics and mass spectrometry. The author provides examples to illustrate how scientists have applied each to identify disease biomarkers. One example describes the effects of berberine on MCF7 breast cancer cells using fluorescent labeling and 2D DIGE (two-dimensional difference in gel electrophoresis).2 The research team found 22 proteins with redox alterations. Further workup revealed that the HSP27 (already implicated in breast cancer) was one of the proteins modified. They confirmed their results using western blotting and immunoprecipitation.

Future directions and challenges

The authors anticipate that redox proteomics will increasingly be used to identify therapeutic targets. Label-free quantitative MS-based techniques will likely dominate in the future, as they are fast, inexpensive and relatively easy to perform. The ongoing challenge will be to verify that the identified redox-modifications have biological significance and functional implications.

This review provides a very detailed summary of the different labeling options and is a must-read for researchers in search of new ways to identify elusive redox-modified peptides. In addition to 74 references, the review features a list of key issues, an expert commentary, and a forward-looking five-year view.

  1. Mermelekas G, Makridakis M, Koeck T, Vlahou A. Redox proteomics: from residue modifications to putative biomarker identification by gel- and LC-MS-based approaches. Expert Rev Proteomics. 2013 Dec;10(6):537-49. doi: 10.1586/14789450.2013.855611.
  1. Chou HC, Lu YC, Cheng CS, et al. Proteomic and redox-proteomic analysis of berberine-induced cytotoxicity in breast cancer cells. J Proteomics. 2012 Jun 18;75(11):3158-76. doi: 10.1016/j.jprot.2012.03.010. Epub 2012 Mar 21.