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Reactive oxygen species (ROS) are molecules containing oxygen that are highly reactive due to the presence of unpaired valence electrons. ROS are formed through the course of many metabolic processes including energy production by the mitochondrial respiratory chain (1). These ROS are often the cause of oxidative stress in which high levels of ROS result in damage to cell structure and loss of function (2), but may also be involved as redox signals (3). Endogenous ROS lead to reversibly modified mitochondrial proteins containing thiol groups. A technique based on difference gel electrophoresis (DIGE) was developed to allow the detection of the small number of proteins modified by these ROS. Samples are labeled with fluorescent tags specific to thiol groups in order detect modifications to the redox sensitive thiol groups. This method, redox-DIGE, allowed relative changes in fluorescence between samples to be used in identifying thiol-containing proteins that are reversibly modified by ROS of the mitochondrial respiratory chain (3).
B. Background and Significance
Reactive oxygen and nitrogen species (ROS and RNS) possess the ability to behave randomly and destructively, but have recently been shown to be tightly regulated with specific targets (4). As such, it is believed that ROS and RNS serve as signaling molecules in redox regulation (3). Hydrogen peroxide (H2O2) is an example of a ROS species which can react with specific cellular targets including thiol-containing proteins (5). Glutathione (GSH) and thioredoxin are proteins which act as antioxidants to help protect cells from oxidative stress caused by ROS (3). Many of the modifications caused by ROS are reversible through interaction with these specific antioxidant species. The major source of ROS production at the cellular level occurs through the mitochondrial respiratory chain whereby adenosine triphosphate (ATP) is produced for energy. The identification of mitochondrial thiol-containing proteins modified in response to ROS is difficult due to their low levels, and therefore, the role of these proteins in redox regulation is unclear (3).
To gain an understanding into the role of these proteins in redox regulation, redox-DIGE was developed. This technique allows for the detection of thiol-containing proteins which are modified by ROS. Protein thiols are targeted with different fluorescent dyes containing thiol-reactive maleimide groups (3). These dyes contain fluorophores exhibiting different emission wavelengths allowing for detection of relative fluorescence changes between samples. Protein spots showing relative fluorescence differences are excised from the gel and subjected to peptide mass fingerprinting by mass spectrometry in order to identify the proteins of interest. Upon application of redox-DIGE to mitochondrial protein samples exposed to ROS, a small number of thiol-containing proteins were identified as selectively oxidized. Through identification, the proteins were found to be involved in fatty acid oxidation and in regulation of pyruvate dehydrogenase leading to the belief that protein thiol modification may be important in redox signaling and regulation (3).
C. Specific Aims
To determine the effects of exogenous hydrogen peroxide (H2O2) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) exposure on the redox state of mitochondrial thiol-containing proteins. Solvent-exposed thiol-containing proteins were studied since these proteins represent those most likely to be modified during redox challenge. Proteins were exposed to varying concentrations of ROS and RNS in order to determine which concentrations would lead to the highest difference in total mitochondrial thiol content.
To develop a sensitive technique which would allow for the identification of thiol-containing proteins modified upon exposure to ROS/RNS. Since changes to thiol-containing protein redox state may occur at low levels, a technique needed to be developed to allow the visualization or measurement of these changes. Measurement of total mitochondrial thiol content was insensitive to changes in redox state of thiol-containing proteins, and as such, a technique that would allow for identification of low abundant thiol-containing proteins was developed.
To characterize the functional significance of modification to thiol-containing mitochondrial proteins. Modification to thiol-containing proteins does not necessarily indicate the involvement of those proteins in redox regulation. Further studies needed to be performed in order to assess whether or not the modification affected a protein's ability to function.
Proteomics is a technique that allows for the characterization of protein expression occurring within a specific protein mixture (6). The study of this expression leads to an increased understanding of the proteins involved in specific cellular processes as protein expression is highly dependent upon the environmental conditions to which the proteins are exposed. Numerous proteins must be reversibly modified in order to function properly. These modifications are often based on oxidation of peptide functional groups in which the modifications may be reversed through antioxidant species such as glutathione (2). ROS/RNS play a crucial role in production of these modifications. When the level of ROS is elevated, there is no longer proper functioning, and the body is said to be under oxidative stress (1). This condition of oxidative stress has been linked to processes involved in normal aging as well as diseases such as cancer and neurodegeneration (4). Despite the negative aspect stemming from overproduction of ROS, ROS and RNS are needed for proper bodily function as the species can act as cellular signals to other molecules in a variety of pathways.
A major problem in proteomics involves the separation of the large number of proteins that can be identified at any one time at the cellular level. The number of proteins expressed at any given time can exceed one thousand with even greater numbers for eukaryotic organisms (7). The technique most often used in the study of protein expression is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). This technique relies on multidimensional separation based on protein isoelectric point (charge) in one dimension followed by molecular weight in the second dimension. 2D-PAGE has the ability to resolve more than 5,000 proteins simultaneously depending on the size of the gel and pH gradient used (7). The technique also provides for quantitation between samples run on different gels by measuring relative abundances of proteins based on spot intensity (8). A strength of 2D-PAGE involves the ability to study post translational modifications which can be visualized through the existence of distinct spot trains on the gel (7). The technique can also be coupled to blotting techniques involving antibodies used to specifically detect sites of protein carbonylation for example (1). Major drawbacks exist for 2D-PAGE despite its ability to separate large numbers of proteins. Data analysis is often time consuming and difficult owing to the variability that exists between gels (9, 10). In order to study protein expression or post translational modifications between samples, different gels must be run and directly compared. The downfall comes from the fact that no two gels are identical due to inhomogeneities in gel content, electric field applied, and thermal fluctuations (9). Due to these differences, proteins may migrate slightly differently between gels thus causing spot matching to become a difficult task.
In order to circumvent the need to run separate gels for various samples, 2D-DIGE was developed. This technique involves the same separation steps as 2D-PAGE, but also incorporates the use of fluorescent dyes (9). Through the use of these dyes, differentially stained samples can be run on the same gel to allow for direct comparison between samples (11). Differences in fluorescence spot intensity between samples leads to comparison of protein expression levels. 2D-DIGE is also advantageous in the study of low abundance proteins which may not exhibit high spot intensity in the absence of fluorescence dyes.
In order to gain an understanding of redox regulation as it occurs in mitochondria, a technique was developed known as redox-DIGE. Thiol-containing proteins are believed to be involved in mitochondrial redox regulation through modification by ROS/RNS. However, the specifically targeted thiol-containing proteins are difficult to detect by common methods of protein identification. Through the development of redox-DIGE, proteins containing redox-sensitive thiols could be identified (3). Thiol-containing proteins were labeled with two different fluorescent dyes to compare control samples with samples undergoing redox-challenging. Upon detection of changes in relative fluorescence between samples, proteins could be pinpointed and studied by mass spectrometry. This allowed for identification of redox-sensitive thiol-containing proteins found to be predominantly involved in cellular processes such as fatty acid oxidation.
E. Experimental Design
In order to study the effect of ROS/RNS on thiol-containing mitochondrial proteins, mitochondria were prepared from rat heart through homogenization. Following homogenization, differential centrifugation was used to obtain rat heart mitochondria specifically since different organelles have different sizes and shapes and can be pelleted through various centrifugation steps. Free mitochondrial protein thiols were measured through the use of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) which is commonly referred to as Ellman's reagent. The use of this reagent stems from a stoichiometric reaction involving the reaction between thiol groups and the reagent to produce the NTB anion. This anion appears yellow in color which allows the absorbance to be measured in order to determine the concentration/number of thiol groups in a sample (12) (See Figure 1 for the reaction).
Figure 1: Reaction of a thiol group with DTNB to form the NTB anion (12).
Figure 2: Redox-DIGE Schematic (3).Prior to the reaction with DTNB, glutathione was removed. Glutathione is an antioxidant protein with the responsibility as the main thiol antioxidant molecule in the cell (2). Removal ensures lack of defense against the ROS/RNS being tested in thiol modification. Protein concentration was determined using the BCA Assay.
Mitochondria were incubated for 5 minutes at 37oC in the presence of ROS/RNS (H2O2, SNAP) of varying concentration. Following this incubation, N-ethylmaleimide (NEM) was added to the mitochondrial suspension to block exposed thiol groups in a control sample and a sample exposed to ROS/RNS. NEM was then removed, the proteins were denatured, and any thiol groups existing in an oxidized form were reduced with dithiothreitol (DTT). The control and redox challenged samples were then labeled with Cy3 or Cy5 maleimide dyes respectively. Equal amounts of these samples were then pooled together and subjected to two-dimensional electrophoresis (2-DE). (Figure 2 (3)).
2-DE was carried out by IPG strips (pH 3-10) rehydrated with the samples for 10 hours. Following isoelectric focusing, the strips were loaded onto an acrylamide gel and overlaid with 1% agarose in SDS running buffer which contained bromophenol blue. The gels were run until the bromophenol blue dye front was seen to run off the bottom of the gels. Following 2-DE, gels were transferred to an imager which allowed fluorescent spots to be viewed using lasers differing in excitation wavelengths (532, 632nm lasers). Analysis was performed using DeCyder, software specifically designed for DIGE. Total protein abundance was measured by staining the gels using Deep Purple total protein stain following fluorescence detection.
Protein spots determined to correspond to modified thiol-containing proteins were excised using a spot picking software in conjunction with a list generated by DeCyder. These excised protein spots were subjected to in-gel tryptic digestion. The resulting peptides were extracted from the gel and matrix-assisted laser desorption ionization-time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry was used to identify modified proteins. This identification was accomplished through database searching using MASCOT which enabled peptide mass fingerprinting to determine protein identity.
F. Preliminary Results
Figure 3: A. Effect of SNAP B. Effect of H2O2 Prior to gel analysis, loss of reactive thiol groups was measured using the DTNB assay for determining total protein thiol content. After exposure to varying concentrations of ROS/RNS (H2O2 and SNAP), it was determined how the presence of ROS/RNS affected the redox state of thiol-containing proteins. No detectable loss of free protein thiols was measured upon exogenous ROS/RNS exposure (Figure 3 (3)). This led to the conclusion that ROS/RNS modification of protein thiols may be overlooked by the insensitivity of the technique. Redox-DIGE was developed to test this conclusion.
Upon examination of data produced through redox-DIGE, it was found that protein thiols undergoing oxidation upon exposure to ROS/RNS could be identified. Control samples (not exposed to ROS/RNS) were labeled with Cy3 maleimide leading to visualization of green color upon fluorescence excitation. Samples exposed to ROS/RNS were labeled with Cy5 maleimide leading to production of a red color. These samples were pooled and separated on the same gel. Gels were run to test the exposure of protein thiols to varying concentrations of ROS/RNS. Figure 4 (3) shows the effect of ROS/RNS on mitochondrial thiol-containing protein oxidation state. Green indicates presence of oxidized proteins in the control sample, red indicates modified thiols upon exposure to ROS/RNS, and yellow indicates where spots were equally labeled by both dyes. This situation occurred due to reactive thiol groups being folded to the inside of the protein such that exposure to NEM failed to block these thiol groups. Upon denaturation, these reactive thiols were labeled in both samples leading the appearance of a yellow spot when fluorescent images were superimposed.
Figure 4: Effect of oxidants on mitochondrial protein thiols determined by redox-DIGE
Protein spots identified as containing significant change in fluorescent intensity were excised and identified by MALDI-TOF-TOF mass spectrometry. Two of the proteins identified by mass spectrometric analysis (propionyl-CoA carboxylase (PCC) and pyruvate dehydrogenase kinase (PDHK)) were chosen as candidate proteins for redox regulation. Enzyme activities were measured for these proteins in order to determine whether or not protein function changed upon exposure to ROS/RNS. Enzyme activity for both species was found to be inhibited upon exposure to ROS/RNS.
G. Major Findings
Redox-DIGE is a sensitive technique that has the ability to identify oxidatively modified thiol-containing proteins. The identification of these modified proteins is difficult, but through the use of fluorescent dyes, identification is possible. Modified proteins were easily distinguished from NEM-blocked proteins. Although redox-DIGE appears to be highly sensitive in detection, the major downfall to the technique involves the achievable resolution. In this study, isoelectric focusing was performed in the pH range from 3-10. This results in the loss of separation for very acidic and basic proteins. These proteins remain unresolved and as such, may not be identified as possible modified proteins. Also, the advantage of redox-DIGE is the elimination of a need to run separate gels for comparison purposes. However, without running comparative gels of samples exposed to identical conditions, no insight into reproducibility can be gained. In order to accurately quantify proteins identified, results should be reproducible from experiment to experiment. Although multiple gels leads to variability, they should still be run in order to test reproducibility. In addition, including an internal standard labeled with an additional fluorescent dye would allow quantitation of expression changes between samples and on comparative gels (13). Finally, although redox-DIGE is a sensitive technique in detecting oxidatively modified proteins, it still uses conventional 2D-PAGE techniques. This leads to a long analysis time since isoelectric focusing takes hours, running the gel takes hours, and tryptic digestion also consumes a reasonable amount of time, and these are only three steps in the overall process. The development of a technique not based on gel electrophoresis could lead to a noticeable decrease in analysis time.
The authors were able to identify a small subset of proteins that contained thiols particularly sensitive to redox modification. This conclusion was based on the results of the experiment testing ROS/RNS levels and the effect on thiol redox state. Redox-DIGE allowed the determination that thiol-containing proteins exposed to low levels of ROS/RNS showed a significant increase in thiol oxidation. When the DTNB test was used, no change was observed in total thiol content of mitochondrial proteins. Again, this illustrates the sensitivity achievable with the redox-DIGE technique.
The existence of green fluorescence indicated that proteins not exposed to exogenous ROS/RNS were also oxidized. No tests were conducted to identify proteins oxidatively modified in the control samples. Therefore, further tests should be performed to identify those proteins containing oxidized thiol groups in the absence of ROS/RNS exposure. This identification could then lead to the determination of whether or not these oxidized thiol-containing proteins exhibit the same cellular functions as those proteins oxidized upon ROS/RNS exposure.
H. Conclusions/Future Outlook
Redox-DIGE has been identified as a sensitive technique in the study of oxidatively modified thiol-containing proteins. The technique, however, cannot be applied to a variety of systems. DIGE can be used to study differences in protein expression between samples, with redox-DIGE specifically allowing the examination of oxidatively modified proteins. At the present time only two sets of fluorescent dyes exist: one set targets lysine residues while the other targets cysteine residues (10). If additional fluorescent dyes could be synthesized, more amino acids could be targeted for tagging. Also, it would be helpful to conjugate the dyes to molecules other than maleimides in order to target different types of oxidative modification. Since oxidative stress is known to play an important role in aging and age-related functional decline, the application of redox-DIGE to studying oxidative modification would allow for direct analysis between samples across a span of ages. This would enable analysis of accumulated oxidative damage which could lead to determination of important biomarker proteins. In addition, further studies could be conducted to allow more insight to be gained into possible oxidative modifications which allow specific proteins to function as signaling molecules. Nevertheless, redox-DIGE has emerged as a technique that allows expression of oxidatively modified proteins to be studied and compared between samples without having to use multiple gels. Elimination of the requirement to run multiple gels for comparison has allowed the direct comparison of samples separated under identical conditions. Relative changes in the fluorescence detected provide a means of studying changing protein expression between samples.
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