Redox proteomics: understanding oxidative stress in the progression of age-related neurodegenerative disorders

Redox proteomics is an approach that can be applied to the brain to delineate mechanisms of pathogenesis of age-related neurodegenerative disorders. In addition, redox proteomics can also play roles in biomarkers discovery and efficacy of pharmaceutical interventions in these disorders.

Analogous to the term genomics, proteomics is defined as the analysis of the entire protein complement expressed by a genome. However, proteomics is more diverse than genomics, since the proteome differs from cell to cell, and proteins undergo a number of post-translational modifications, such as glycosylation and phosphorylation, as well as alternate splicing [1,2]. Proteomics provides a useful tool for qualitative, quantitative and functional characterization of the entire protein profile of a given cell, tissue and/or organism, enabling the analysis of not just native proteins, but also the profiling and identification of isoforms, splice variants, mutants and post-translationally modified species, as well as insight into protein–protein interactions.

Although age-related neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis and Huntington’s disease, among others, are characterized by different etiologies and pathogenesis, each of these disorders demonstrates oxidative stress in the CNS [3]. Hence, identification of oxidatively modified brain proteins in these disorders may shed light on unique molecular mechanisms involved for each disorder. This is the province of redox proteomics [4].

One approach to proteomics couples 2D polyacrylamide gel electrophoresis (2D-PAGE) separation of proteins with mass spectrometric techniques. This involves the separation of complex protein mixtures based on two important physicochemical properties, such as isoelectric focusing that separates proteins based on their isoelectric points, followed by separation of proteins based on their relative mobility on SDS-PAGE in the second dimension. A single spot on the 2D gel nearly always represents a single protein, and this property enables the separation of thousands of different protein spots on a single gel. Furthermore, 2D-PAGE is used to catalog proteins and create databases.

In redox proteomics, the same principles for separation and identification of proteins are employed, but a 2D western blot is also used to immunochemically detect oxidatively modified proteins [4]. Typical oxidative modifications of brain proteins involve protein carbonyls, protein-bound HNE (a product of lipid peroxidation), 3-NT and glutathionylation [5]. Antibodies to protein-bound HNE, 3-NT and protein-bound glutathione exist. To immunochemically detect protein carbonyls, protein carbonyl groups are derivatized to hydrazones by chemicals such as 2,4-dinitrophenylhydrazine before the separation of proteins or following the 2D separation of proteins. Computer-assisted detection of these derivatized proteins on 2D western blots is coupled to mass spectrometric analysis to identify oxidatively modified proteins in the sample of interest [6–11]. In this method, a parallel analysis is employed: first the 2D western blots and 2D gel images are matched by computer-assisted image analysis, and the anti-DNP immunoreactivity of individual proteins is normalized to protein content that is obtained by measuring the intensity of colloidal Coomassie blue staining or SYPRO^® Ruby-stained spots [6,7,9,10]. Normalization enables comparison of levels of oxidatively modified brain proteins in disease versus control subjects. Once the protein is identified as oxidatively modified, the protein is digested in-gel with a protease (typically trypsin) and peptide mass fingerprints are obtained using MALDI mass spectroscopy. The mass fingerprints are characteristic of a particular protein, which facilitates the identification of proteins using a suitable database, such as SwissProt and other databases. These databases utilize computer algorithms and are available gratis through the internet. Furthermore, the proteomics-determined identity of the proteins should be consistent with the isoelectric points and molecular weight observed on the 2D gel, and validation of the identity should be performed, usually by immunochemical means [6,12].

Redox proteomics methods have enabled the identification of a number of oxidatively modified brain proteins in various neurodegenerative disorders and their models, with the result that new insights into these disorders have emerged [5,6,13–15]. In AD and mild cognitive impairment, a number of oxidatively modified brain proteins were identified using redox proteomics, and these proteins are involved in different important cellular processes that correlated with the AD pathology [6,9,10,15]. For example, many of the proteins that were identified as oxidatively modified in brain from subjects with AD or mild cognitive impairment belong to the glycolytic or TCA pathways, or are mitochondrial proteins. Since oxidatively modified proteins are generally dysfunctional [3,5,8–10], these findings are consistent with PET studies that demonstrate decreased energy metabolism in AD and mild cognitive impairment compared with that of the control [16,17].

Prior to redox proteomics, immunoprecipitation methods were used for the identification of oxidatively modified proteins [12]. However, this method has serious limitations, including the requisite prior knowledge of the identity of the protein of interest, the availability of a specific antibody for that protein, the time-consuming and laborious processes involved, and the fault that only one protein can be analyzed at a time. Furthermore, post-translational modification of proteins may alter their structure, thereby altering or inhibiting antigen–antibody complex formation. Redox proteomics evolved to overcome all these hindrances, thereby enabling the simultaneous identification of a large number of oxidatively modified proteins in cells, tissues and other biological samples, which were previously undetected. Redox proteomics offers a broad spectrum of information that provides insights into the mechanisms of diseases, identification of disease-associated markers, and may also help to identify selected targets for specific therapy [15].

However, 2D gel-based proteomics methods still pose a number of challenges, despite being a sensitive and reliable method with high reproducibility. These limitations include: less than ideal solubilization processes for membrane proteins, difficulty in identification of highly basic proteins, and an inability to detect low-abundance proteins. Studies are ongoing to resolve all the aforementioned problems. Immobilized pH gradient strips, with their immobilized ampholyte-mediated fixed pH gradients, improve the reproducibility of protein maps and eliminate the typical cathodic drift associated with the previously used tube gels. In addition, the availability of narrow-range immobilized pH gradient strips facilitates high-resolution protein separation over a wide pH range, but within one pH unit.

As noted, solubilization of membrane proteins has been one of the biggest problems associated with 2D-PAGE [18]. Accordingly, solubilization techniques for redox proteomics are still mainly limited to cytosolic proteins, resulting in difficulties in obtaining gel maps of membrane proteins although 2D gel maps including membrane proteins have been produced [19–23]. A new method using tributylphosphine in combination with dithiothreitol improved the resolution of protein spots in 2D gels [24]. This method also used cup-loading to guide membrane proteins into immobilized pH gradient strips and added Tris 20 mM to the sample to improve solubilization of membrane proteins. Other methods to increase membrane protein solubilization involve the use of detergents such as 2% ASB-14 and 4% CHAPS [25]. Membrane protein solubilization is also achieved using SDS. Although SDS helps better solubilization of membrane proteins, this detergent is not the optimum choice, because it will impart negative charge to the protein, thereby interfering with isoelectric focusing [26].

“Redox proteomics provides a broad spectrum of information that provides insights into the mechanisms of diseases, identification of disease-associated markers, and may also help to identify selected targets for specific therapy.”

Proteomics can be used to screen biomarkers from a variety of complex biological samples, including blood, urine, serum and plasma [27]. Likewise, redox proteomics may have utility in biomarker development. However, one of the biggest challenges is to remove relatively abundant proteins, such as immunoglobulins and albumin, from samples such as plasma that will otherwise mask the remaining proteins that are present in relatively low concentrations when separated by 2D-PAGE. Commercial columns are available that deplete albumin and immunoglobulins from plasma, and it is also possible to narrow research by just focusing on a limited type of proteins by using affinity columns (i.e., columns to isolate glycoproteins and phosphorylated proteins from plasma). The removal of high-abundance proteins and restricting research to one particular class of proteins may make it possible to detect proteins from a low-abundance protein group that may be involved in the pathogenesis of a neurodegenerative disorder.

Although 2D-PAGE is the most commonly employed method for protein separation for use in protemics, other non-SDS-PAGE methods also are employed, including 2D HPLC that is coupled directly to mass spectrometric analysis [28]. The samples can be differentially labeled using different isotopes or fluorophores; a modification of methods that use isotopically coded affinity tags [29]. These alternative techniques are introduced to overcome the limitations of the 2D-PAGE method [30]. Furthermore, to improve the sensitivity of detection of oxidatively modified proteins and to work with small quantities of samples, researchers are working towards developing specific and sensitive stains for protein oxidation, without a derivatization step.

The field of redox proteomics is rapidly expanding and evolving, and it is now being developed for clinical diagnosis and in establishing biomarkers for disease and drug discovery [31,32]. Redox proteomics applied to age-related neurodegenerative disorders will continue to provide new insights into pathogenesis and potential pharmacological interventions.

Financial & competing interests disclosure

This work was supported, in part, by National Institutes of Health grants to D Allan Butterfield [AG-05119; AG-10836]. 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.

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