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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Free Radic Biol Med. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2727357
NIHMSID: NIHMS118079

Methods for imaging and detecting modification of proteins by reactive lipid species

Abstract

Products of lipid peroxidation are generated in a wide range of pathologies characterized by those associated with oxidative stress and inflammation. Many oxidized lipids contain reactive functional groups that can modify proteins, change their structure and function, and affect cell signaling. However, intracellular localization and protein adducts of reactive lipids have been difficult to detect and rely largely on antibodies raised against specific lipid-protein adducts. As an alternative approach for monitoring oxidized lipids in cultured cells, we have tagged the lipid peroxidation substrate arachidonic acid, and an electrophilic lipid, 15-deoxy- Δ12,14-prostaglandin-J2 (15d-PGJ2) with either biotin or the fluorophore BODIPY. Tagged arachidonic acid can be used in combination with conditions of oxidant stress or inflammation to assess the subcellular localization and protein modification by oxidized lipids generated in situ. Furthermore, we show that reactive lipid oxidation products such as 15d-PGJ2 can also be labeled and used in fluorescent and Western blotting applications. This article describes the synthesis, purification, and selected application of these tagged lipids in vitro.

Keywords: lipid peroxidation, cyclopentenone, electrophile, nucleophile, electrophilic lipid, oxidative stress, redox signaling

INTRODUCTION

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid, are primary components of the diet, biological membranes, and lipoproteins and are substrates for lipid peroxidation (LPO) [13]. They readily undergo non-enzymatic peroxidation during oxidative stress or are oxidized through reactions involving enzymes such as cyclooxygenase (COX), cytochrome P450, or the lipoxygenases [48]. A role for oxidized lipids in pathophysiology is supported by the increased levels of LPO apparent in several diseases including Alzheimer’s disease [9, 10], renal failure [11, 12], and atherosclerosis [13, 14].

Many LPO products contain functional groups capable of covalently modifying proteins [1518]. For example, both non-enzymatic and enzymatic oxidation of PUFAs result in the formation of reactive lipid species that have electrophilic carbons, usually in the β-position of an α,β-unsaturated carbonyl (Figure 1), which are reactive with protein nucleophiles. Electrophilic products of non-enzymatic lipid oxidation include aldehydes such as 4-hydroxynonenal (HNE), malondialdehyde, and acrolein, and the J-and A-series isoprostanes (Figure 1). Other products include the isoketals, which occur through non-enzymatic LPO by rearrangement of endoperoxide intermediates of the isoprostane pathway [19]. Isoketals are highly electrophilic γ-ketoaldehydes that rapidly and covalently modify lysine residues, forming a variety of adducts including lactam, hydroxylactam, imine, and pyrrole species. The enzymatic oxidation of arachidonic acid also results in the formation of reactive LPO products. However, in this case the variety and reactivity of these species is constrained by the active site of enzymes such as lipoxygenase which limits the number of stereoisomers generated during the enzymatic reaction.

Figure 1
Examples of reactive lipid species produced by enzymatic or non-enzymatic oxidation of arachidonic acid

Studying the complex mixture of reactive lipids formed in vivo during oxidative stress or inflammation has been limited by existing techniques, which have focused primarily on following the reactions of a defined lipid in conjunction with a candidate target protein approach. The covalent modification of proteins by oxidized lipids is an important mechanism by which LPO may initiate cell signaling as well as contribute to tissue injury [15, 2022]. Although many studies have shown that the formation of oxidized lipids are increased in disease, it is unclear whether they represent the footprints of oxidative stress or are mediators of pathology. A major obstacle to a complete understanding of the biological roles of oxidized lipids is the unambiguous detection and quantification of biologically relevant protein modifications. Hence, improved technologies that allow a more comprehensive definition of the reactive lipid proteome are needed to determine the mechanism, the extent, and under what circumstances oxidized lipids affect cell signaling and physiology.

Due to the immunogenic potential of some lipid peroxidation products such as isoketals, levuglandins, and aldehydes, antibodies against specific protein-lipid adducts have been raised that allow for their detection by immunological methods [2327]. This approach has been used to identify specific lipid modifications using proteomics and mass spectrometry techniques [2830]. However, many antibodies to protein-lipid adducts are susceptible to epitope bias thus protein modifications detected using antibodies may represent a small subset of the reactive lipid proteome. Another disadvantage of this approach is that since complex lipid mixtures are typically generated in cells and tissues during oxidative stress, an antibody to a specific lipid protein adduct will necessarily give only a partial picture of the reactive lipid proteome.

Detection of the carbonyl moiety present on proteins conjugated to α,β-unsaturated aldehydes and ketones has been a useful marker of lipid peroxidation and oxidative stress [31, 32]. Aldehydes and ketones can react with protein nucleophiles such as histidine, cysteine, and lysine by a Michael addition reaction to form stable adducts [18]. This type of adduct can be detected by derivatizing the carbonyl group using hydrazine or hydrazide chemistry to form a stable hydrazone product [31, 33, 34]. In particular, the use of avidin or streptavidin detection techniques in conjunction with biotin hydrazide increases the sensitivity for detecting low abundance proteins. One important caveat, however, is that protein carbonyls can be introduced by reaction with oxidants other than reactive lipids, decreasing the specificity of this approach for the unique detection of the oxidized lipid proteomes.

Other methods of detection, such as the radioactive labeling of precursor lipids, have been used to measure protein-lipid adducts [3537]. Nevertheless, the intracellular targets for reactive lipid oxidation products remain largely undefined due to a lack of suitable detection reagents and protocols. Herein, we describe a non-radioactive method for the labeling, purification, and utilization of substrates for LPO (i.e., arachidonic acid) and of a specific oxidized lipid, 15-deoxy-prostaglandin J2 (15d-PGJ2). In previous studies, we and others have shown that conjugation of 15d-PGJ2 to biotin or fluorescent tags can be used to monitor both the subcellular localization of the lipid and to identify specific protein targets [16, 17, 3842]. Using this approach, signaling proteins such as Keap-1 were shown to form covalent adducts with 15d-PGJ2, which mediated the induction of antioxidant defenses including glutathione and heme oxygenase-1 [16, 17, 38]. The insertion of the tags on the lipid via the carboxyl group has little or no impact on the potency of the lipid electrophiles to induce either antioxidant defenses at low concentrations or apoptosis at higher levels. The BODIPY analog of 15d-PGJ2 has been particularly interesting, revealing mitochondrial targeting of the lipid, and biotin-tagged 15d-PGJ2 has been used to identify the proteome modified by reactive prostaglandins in mitochondria and cells [41, 42]. This paper describes the methods for detecting protein-lipid adducts using fluorescent and Western blotting approaches.

PRINCIPLES

To follow the formation of lipid adducts with proteins, we describe a protocol to conjugate biotin or BODIPY to the carboxylic acid group of the unsaturated lipid arachidonic acid or the lipid electrophile. This method was adapted from two previously published methods which describe the biotinylation of prostaglandin A2 [43] and 15d-PGJ2 [44]. Tagging arachidonic acid offers the important advantage of detecting proteins that are reactive with multiple LPO species. As shown in Figure 2, tagged arachidonic acid can be oxidized to lipid peroxidation products in the presence of catalytic metals, oxidants, or in response to an inflammatory stimulus. Many of these tagged products contain functional groups such as reactive aldehydes, ketones, and electrophilic carbons capable of modifying protein nucleophiles. Hence, this technique enables the detection of protein-reactive lipid products containing the BODIPY or biotin tag. Incorporation of the fluorescent BODIPY tag also allows the detection of protein modification by oxidized lipids using in-gel fluorescence, thereby eliminating the need for protein transfer and allowing a more high-throughput proteomics approach.

Figure 2
Proteins modified by reactive lipids can be detected or enriched using tagged arachidonic acid

MATERIALS

  1. Arachidonic Acid (Calbiochem, Product # 181198)
  2. EZ-Link 5-(Biotinamido) pentylamine (Pierce Biotechnology, Product # 21345)
  3. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC; Pierce Biotechnology, Product # 22980)
  4. 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (BODIPY FL-EDA; Invitrogen, Product # D2390)
  5. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2; Cayman Chemical, Product # 18570)
  6. Ethanol, HPLC grade
  7. Acetonitrile, HPLC grade
  8. Methanol, HPLC grade
  9. Chloroform, HPLC grade, preserved with 0.75% ethanol
  10. Acetic Acid, glacial, certified ACS plus grade
  11. Chambered coverglass, 4-well (Nunc; Product # 155383)
  12. Hoechst 33258 (Invitrogen, Product # H-3569)
  13. Protein assay reagent (Biorad, Product # 500–0006)
  14. 30% Acrylamide/Bis Solution (Biorad, Product # 161–0158)
  15. Tris base (Fisher, Product # BP152–1)
  16. Triton X-100 (Sigma-Aldrich, Product # T-9284)
  17. 1-Butanol (Fisher, Product # A399–500)
  18. TEMED (Acros Organics, Product # 420580050)
  19. Sodium dodecyl sulfate (SDS; Fisher Scientific, Product # BP166–500)
  20. Nitrocellulose (Biorad, Product # 162–0112)
  21. Supersignal West Dura(Pierce Biotechnology, Product # 34076)
  22. Amber glass vials, 1.5 ml (SUN SRi, Product # 200 252)
  23. Caps with rubber septa (SUN SRi, Product # 500 062)
  24. Borosilicate glass tubes, 16 × 100 mm (Fisher, Product # 14–961–29)
  25. Hemin chloride (MP biomedical, Product # 15489–47–1)
  26. DMSO (Sigma-Aldrich, Product # D2438)
  27. Hybond-LFP, low fluorescence PVDF (GE Healthcare, Product # RPN303LFP)
  28. PVDF (Millipore, Product # IPVH00010)

INSTRUMENTS

  1. Mass balance (sensitive to 0.1 mg)
  2. Pipettes
  3. Rocker (tilting shaker)
  4. Vortex mixer
  5. HPLC system: BioCad Perfusion Chromatography system (PerSeptive Biosystems Incorporated) or other HPLC system equipped with ultraviolet/visible spectrophotometer and fraction collector.
  6. Preparative Column: Gemini C18 reversed phase column with 10 μm particle size and dimensions of 250 × 21.2 mm. (Phenomenex, Product # 00G-4436-P0)
  7. Semipreparative Column: Luna C18 reversed phase column with 5 μm particle size and dimensions of 250 × 10 mm. (Phenomenex, Product # 00G-4041-N0)
  8. Microscope: Leica DMIRBE Microscope with TCS SP Laser Scanning Confocal system or other confocal microscopy system equipped with lasers suitable for fluorescein and DAPI excitation and detection (Leica; Wetzlar, Germany).
  9. Gel Imaging System with CCD Camera (Alpha Innotech)
  10. Fluorescent Imager: Typhoon or other fluorescence imaging system suitable for gel scanning and equipped with a filter and laser suitable for BODIPY excitation and detection (GE Healthcare).

PROTOCOL

Biotinylation of arachidonic acid

The structures for unlabeled and biotinylated arachidonic acid are shown in Fig. 3A. The biotin moiety is covalently attached to the fatty acid at the carboxyl group using the activating compound 1-ethyl-3-(3-dimethyl) aminopropylcarbodiimide (EDC) causing it to be reactive with the amino group of 5-(Biotinamido) pentylamine. This condensation reaction results in the formation of a stable amide bond between the fatty acid and the biotin tag. To minimize oxidation, exposure to room air and light must be avoided and all solvents should be purged with an inert gas such as argon or nitrogen prior to use. In addition, new glassware that has been pre-rinsed with ethanol and dried should be used to prevent contamination and loss of lipid. The reaction should be performed in an amber or foil-covered glass vial to further limit light-dependent oxidation of the lipid.

Figure 3
Structures of lipids and tagged versions
  1. Prepare 10 mg/ml EDC in 100% acetonitrile. Vortex until in solution.
  2. Make a solution of 10 mg/ml 5-(Biotinamido) pentylamine in 44% ethanol/44% acetonitrile/12 % water (v/v).
  3. Add components to the glass vial in the following order:
    1. 6 μl arachidonic acid (neat oil), approximately 5 mg
    2. 500 μl EDC solution (10 mg/ml in acetonitrile)
    3. 400 μl 5-(Biotinamido) pentylamine solution (10 mg/ml in ethanol, acetonitrile, and water)
    4. Add 94 μl acetonitrile to bring volume up to final 1 ml reaction volume.
  4. Blow argon over the headspace in the vial and cap tightly.
  5. Incubate covered in foil for 16–18 h at room temperature with gentle rocking.
  6. Store at −80 °C, if necessary. Otherwise purify immediately by HPLC.

Synthesis of BODIPY-arachidonic acid

This synthesis of BODIPY-labeled arachidonic acid is also performed using a carbodiimide-mediated conjugation reaction. The structure of BODIPY-arachidonic acid is shown in Fig. 3A. The reaction should be performed in a new, pre-rinsed amber or foil-covered glass vial to prevent light-dependent oxidation of the unsaturated lipid.

  1. Prepare EDC (10 mg/ml) in acetonitrile. Vortex until in solution.
  2. Resuspend BODIPY FL-EDA (5 mg vial) in 150 μl of ethanol.
  3. Add components to a clean glass vial in the following order:
    1. 6 μl arachidonic acid (neat oil), approximately 5 mg
    2. 500 μl EDC
    3. 150 μl BODIPY FL-EDA dye
  4. Rinse the original BODIPY FL-EDA vial with 344 μl acetonitrile. Pipet suspension into the light-protected glass vial to bring the total volume to 1 ml.
  5. Blow argon over the headspace in the vial and cap tightly.
  6. Incubate the vial for 16–18 h at room temperature with gentle rocking.
  7. Purify immediately by HPLC or store at −80 °C until purification.

Synthesis of a Biotinylated Electrophilic Lipid

The electrophilic prostaglandin, 15-deoxy-prostaglandin-J2, can also be conjugated to biotin; the structure of biotinylated 15-deoxy-prostaglandin-J2 is shown in Fig. 3B. The protocol for this conjugation reaction is as follows:

  1. Evaporate solvent from a 1 mg vial of 15d-PGJ2 under nitrogen gas. Resuspend the neat oil immediately in 600 μl acetonitrile. Note: It is recommended that a pure preparation of 15d-PGJ2 which does not contain mixed isomers is used (Product information is given in the Materials section).
  2. Prepare 4 mg/ml of 5-(Biotinamido)pentylamine in 77% aqueous acetonitrile
  3. Make a 5 mg/ml stock solution of EDC in acetonitrile.
  4. In an amber glass vial combine the following:
    1. 600 μl 15d-PGJ2 (1 mg)
    2. 200 μl of EDC in acetonitrile (1 mg)
    3. 200 μl 5-(Biotinamido) pentylamine (0.8 mg)
  5. Fill the headspace with argon, and close the vial with a Teflon-lined cap.
  6. Incubate the vial for 18 h at room temperature in the dark with constant, gentle agitation.
  7. Purify the reaction mixture by HPLC or store at −80°C until purification.

Synthesis of a BODIPY-tagged Electrophilic Lipid

The protocol used for this conjugation reaction is as follows:

  1. Evaporate solvent from five 1 mg vials of 15d-PGJ2 under nitrogen gas. Resuspend each vial immediately in 340 μl acetonitrile.
  2. Add 1ml acetonitrile to a 5 mg vial of BODIPY FL-EDA, mix, and add 300 μl of ethanol to facilitate solubility.
  3. Make a 5 mg/ml stock solution of EDC in acetonitrile.
  4. In five clean amber glass vials combine the following. (The total volume in each vial should equal 1 ml to allow for adequate mixing):
    • D) 340 μl 15d-PGJ2 (1 mg)
    • E) 400 μl of EDC in acetonitrile (2 mg)
    • F) 260 μl BODIPY FL-EDA dye (1 mg)
  5. Fill headspaces with argon, and close vials with Teflon-lined caps.
  6. Incubate the reaction mixture for 18 h at room temperature in the dark with constant, gentle agitation.
  7. Purify the reaction mixture by HPLC or store at −80°C until purification.

Purification of Tagged Lipids by HPLC

After synthesis, the reaction preparations have a mixture of the original unreacted lipid, the tag, the expected product, and the priming compound, EDC. High performance liquid chromatography (HPLC) with UV-Vis detectors is needed to purify the tagged lipid. Following chromatographic separation, product purity is verified using electrospray ionization mass spectrometry (ESI-MS).

The mobile phases used for HPLC separations are prepared as follows:

  • Solvent [A] (10% acetonitrile/0.24% acetic acid/90% H2O (v/v))
  • Solvent [B] (100% acetonitrile)
  • Solvent [C] (100% ultrapure water)

A summary of all HPLC parameters used for each lipid is given in Table 2. For all purifications, the column is equilibrated in the initial mobile phase which varies depending on which tagged lipid is being separated (Table 1). The mass of each lipid, typical retention times, and optimum ionization mode for mass spectrometry are given in Table 1.

Table 1
Parameters for tagged lipids
Table 2
HPLC gradient profiles for purification of tagged lipids

(I) HPLC separation of arachidonic acid conjugates

For purification of tagged arachidonic acid, reversed-phase HPLC should be performed on a C18 semi-preparative column as indicated in the Instruments section. Typically, an aliquot of the reaction mix containing approximately 1 mg of starting material is eluted at a flow rate of 4.5 ml/min using a linear gradient of 50–95% B after equilibration in 50% solvent A/50% solvent B (Table 2, Gradient I). Fractions containing the peak of interest are collected and verified using electrospray ionization mass spectrometry (ESI-MS).

Figure 4A represents an example separation of the Bt-AA reaction mixture by HPLC, with the product typically eluting at a retention time of 13 min (peak b), arachidonic acid eluting late (27 min, peak c), and oxidized products of arachidonic acid eluting early (peak a). To verify separation of Bt-AA, a wavelength scan from 200–300 nm (Figure 4B) should be performed using a spectrophotometer. Figure 4C is a characteristic spectrum obtained from ESI-MS in the positive ionization mode with the major peak at 616 (M+H)+, the m/z value for Bt-AA.

Figure 4
HPLC purification, UV spectra, and mass spectrometric verification of biotinylated arachidonic acid

BD-AA can also be separated to high purity by HPLC (Figure 5A) using the same HPLC gradient profile as Bt-AA. As with Bt-AA, oxidized products containing the tag elute before the product (peak a). BD-AA elutes at approximately 29 min, and purity can be verified by UV/Vis spectrophotometry (Figure 5B) at 200–600 nm. The maximum absorbance is observed at 504 nm (for BODIPY) with the second largest peak at 205 nm (for arachidonic acid). Other components in the spectra are contributions of the BODIPY tag. Product purity must be verified using ESI-MS in the negative ionization mode as shown in Figure 5C. The m/z value for BD-AA is 619 (M+H)+.

Figure 5
HPLC purification, UV spectra, and mass spectrometric verification of BODIPY-arachidonic acid

(II) HPLC separation of tagged electrophilic lipids

For purification of biotinylated and BODIPY-tagged 15d-PGJ2, HPLC separation should be carried out on a C18 preparative column as indicated in the Instruments section. Approximately 2 mg of starting material can be separated using Gradient II, at a flow rate of 20 ml/min. The absorbance of the eluant should be measured at 306 nm (λmax for 15d-PGJ2). Fractions containing the peak of interest are collected and verified using ESI-MS.

BD-15d-PGJ2 is purified using HPLC Gradient profile II, which begins at a concentration of 100% A/0% B and after 13 min increases over a linear gradient to 5% A/95% B. BD-15d-PGJ2 elutes during the linear gradient as Peak c with a retention time of 26 min (Figure 6A). A characteristic UV/Vis wavelength scan from 200–600 nm is shown in Figure 6B, with major peaks observed at 504 nm (BODIPY) and 306 nm (15d-PGJ2). Purity of BD-15d-PGJ2 should be verified using ESI-MS in the negative ionization mode (Figure 6C). BD-15d-PGJ2 is apparent at 632 (M-H). A formic acid adduct formed during the mass spectrometry can also be seen in the spectra (m/z = 678).

Figure 6
HPLC purification, UV spectra, and mass spectrometric verification of BODIPY-15d-PGJ2

Extraction of tagged lipids after HPLC purification

Following HPLC purification, the tagged lipids are in a solution containing acetic acid, acetonitrile, and water. To eliminate water and exchange solvents, it is necessary to extract the product using the following protocol:

  1. Combine fractions of interest in a glass separatory funnel. Record the volume.
  2. Mix with a 2:1 volume of 1:1 methanol:chloroform.
  3. Add one drop of 1 M acetic acid per 5 ml fraction. Shake until a uniform layer has formed and vent.
  4. Add one volume of 1 M sodium chloride and one volume of chloroform.
  5. Shake well and vent.
  6. Allow phases to equilibrate for 10 min.
  7. Discard the aqueous (top) layer, wash the bottom lipid-containing layer with water, and repeat the extraction. Collect the bottom layer containing the lipid product in glass tubes.
  8. Evaporate the solvent (from bottom layer) in a chemical hood under a steady stream of nitrogen gas.
  9. Reconstitute the purified product in ethanol, and measure the concentration with a spectrophotometer using the absorbance and extinction coefficient given in Table 1.
  10. Store tagged lipids at −80°C under inert gas. Verify quality by spectrophotometry and mass spectrometry before use. In our experience, if care is taken to exclude exposure to both light and air the compounds are stable for several months.

Mass Spectrometry Verification

Once synthesized, the tagged lipids are purified and fully characterized by mass spectrometry as shown for Bt-AA in Figure 4C. Typically, derivatives of arachidonic acid are diluted to 1 μM in ethanol and injected in an infusion mobile phase of 50% acetonitrile containing 10 mM ammonium acetate for ESI-MS. Tagged 15d-PGJ2 can be diluted to 1 μM in ethanol and injected in an infusion mobile phase of 50% acetonitrile containing 0.1% formic acid. The mass for each lipid and optimal ionization mode for each is reported in Table 1.

General tips on using tagged lipids

Since tagged lipids are typically used for in vivo or in vitro detection of lipid products, lipid derivatives should be resuspended and stored at −80°C in a vehicle suitable for cell culture, such as ethanol or DMSO. As with any new experimental treatment, it must be verified that the vehicle used does not change the biological outcome observed in your particular model.

Due to evaporation of the storage solvent, the concentration of tagged lipids may change over time and should be measured prior to each experiment using a spectrophotometer. For this reason, it is also not recommended to make small aliquots of the stock, since they evaporate more quickly. The absorbance and extinction coefficient for calculating the concentration of each lipid in ethanol is given in Table 1. In addition, a wavelength scan of arachidonic acid derivatives can be used to detect oxidation of the lipid which will be apparent through the presence of a peak at 233 nm due to conjugated dienes [45, 46].

Fluorescent microscopy with tagged lipids

The BODIPY-FL EDA moiety allows for visualization of lipid products in situ. This can be combined with tracker dyes to determine the subcellular localization of the lipids during experimentation. To detect BODIPY-tagged lipids using microscopy, the following protocol is used:

  1. Typically, cells are grown in chambered coverglasses to allow live-cell visualization of fluorescence. In the example shown in Figure 7, bovine aortic endothelial cells (BAEC) were used as previously described [47].
    Figure 7
    Confocal microscopy of bovine aortic endothelial cells (BAEC) demonstrating live-cell visualization of fluorescently tagged lipids
  2. Serum deprive cells for 16 h prior to the experiment by changing media to DMEM containing 0.5% FBS.
  3. Treat cells with 10 μM BD-AA or 5 μM BD-15d-PGJ2.
  4. If using AA, treat BAEC with a pro-oxidant to induce lipid peroxidation (in this case hemin).
  5. Incubate for 2 h at 37°C, 5 % CO2.
  6. Stain nuclei with 1 μg/ml Hoechst 33258 for 20 min at room temperature.
  7. Wash cells twice with warm 0.5% FBS-DMEM (500 μl per well).
  8. Detect BODIPY-FL fluorescence using an argon laser with a 488 nm laser line.
    Emission should be detected between 500–570 nm.
  9. Detect Hoechst nuclear stain using an ultraviolet laser. Emission should be detected between 350–450 nm.
  10. Single channel images can be merged using Adobe Photoshop or Image J.

SDS-PAGE and Western Blotting

The detection of covalent protein adducts by oxidized lipids using tagged lipids is facilitated by the biotin or BODIPY moiety present on the lipid. Both types of tagged lipids offer unique advantages and should be considered depending on the research goal, available laboratory equipment, and the experimental conditions. Biotinylated lipids allow for sensitive detection as well an accurate quantification of protein modification using Western blot analysis [39]. BODIPY-labeled lipids can also be detected using Western blot analysis; however, an advantage of using fluorescent tags is that the gel can be imaged immediately after electrophoresis using “in-gel” imaging, eliminating the need for transfer to a membrane. For both biotin as well as BODIPY-tagged lipids, high resolution digital imaging techniques are advantageous In our experience, the use of film is rarely optimal, primarily due to the fact that, even at low exposures the film easily saturates. The result is that quantification obtained from a film image is often nonlinear and may underestimate experimental changes. The dynamic range of digital cameras and fluorescent imagers (e.g., Typhoon imagers) are far superior and are therefore recommended for Western blotting applications using biotin tags and/or in-gel fluorescent imaging. Protocols specific for each tagged technology (biotin or BODIPY) are detailed below:

(I) Detecting protein modification by biotinylated lipids

One advantage of using biotinylated lipids is that the amount of lipid-protein adducts may also be calculated using a standard, such as biotinylated cytochrome c (bt-cyt c). It is recommended to include such a standard on the same gel to quantitate the biotin content in experimental samples [39]. Since each mole of lipid is tagged with one mole of biotin, the quantification of biotin on a Streptavidin blot serves as a quantitative marker for the lipid adduct, therefore allowing the calculation of the mol lipid/mol protein in proteomics format. The following protocol is used to detect proteins modified by biotinylated lipids:

  1. After experimentation using Bt-AA or Bt-15d-PGJ2, the protein samples are separated by 1D- or 2D-SDS-PAGE.
  2. To blot proteins, transfer to a nitrocellulose or PVDF membrane at 100 V for 2 h with cooling or at 25 V overnight at 4°C. Note: If chemifluorescent imaging will be used, a low fluorescence PVDF membrane such as Hybond-LFP, is recommended. See Materials section.
  3. Block the membranes with 5% milk in Tris-buffered saline (25 mM Tris, 137
    mM sodium chloride, 2.6 mM potassium chloride, 0.05% Tween-20) for 1 h.
  4. Wash membranes thoroughly to remove all milk. Note: Some milk blotting formulations contain biotin. Since this could interfere with streptavidin binding to biotinylated proteins, milk should not be included after the blocking step.
  5. Incubate blots with Streptavidin-HRP (1:10,000 dilution in TBS-T) for 1 h at room temperature to ensure even distribution of Strep-HRP.
  6. Wash Western blots with TBS-T 3 × 10 min at room temperature.
  7. Add the chemiluminescent or chemifluorescent substrate evenly to the blot, ensuring coverage of the entire surface.
  8. If imaging using chemiluminescence, acquire a series of images using a CCD camera imager (AlphaInnotech). For chemifluorescence imaging, use a fluorescence scanner such as the Typhoon imager (GE Healthcare).
  9. An image that is not saturated and is within the linear range for band intensity should be chosen for absolute quantitation with the standard.
  10. Refer to [39] for the complete method on using bt-cyt c as a standard. Briefly, a range of bt-cyt c concentrations should be used in order to construct a standard curve. The concentrations chosen will depend on the exact experimental protocol and amount of biotin labeling; however, in our experience 0.01–10 pmol is the ideal range. Quantitate the band intensity per lane, and plot this versus the known amount of biotin for each lane of bt-cyt c to obtain a standard curve. A representative standard curve and quantitated blot are shown in Figure 8A.
    Figure 8
    Detection of proteins modified by tagged lipids by Western blotting and in-gel fluorescence techniques

(II) Detecting protein modification by BODIPY-tagged lipids with Western blotting

The following protocol is used to detect proteins modified by BODIPY-tagged lipids by Western blotting. Alternatively, if access to a fluorescent imager is available, in-gel fluorescence imaging may be used. Refer to [(III) Visualizing BODIPY-tagged adducts using in-gel fluorescence].

  1. Protein samples are separated by 1D- or 2D-SDS-PAGE.
  2. To blot proteins, transfer to nitrocellulose or PVDF membrane at 100 V for 2 h with cooling or at 25 V overnight at 4°C
  3. Block the membranes with 5% milk in TBS-T for 1 h at room temperature.
  4. Incubate with anti-BODIPY antibody (1:1,000 dilution in 5% milk) for 3 h at room temperature to ensure even distribution of the BODIPY antibody.
  5. Wash Western blots with TBS-T 3 × 10 min at room temperature.
  6. Incubate with anti-rabbit antibody (1:3,000 dilution in 5% milk) for 1 h at room temperature.
  7. Wash Western blots with TBS-T 3 × 10 min to decrease non-specific binding.
  8. Add the chemiluminescent or chemifluorescent substrate evenly to the blot, ensuring coverage of the entire surface.
  9. For chemiluminescent imaging, acquire a series of images using a CCD camera such as the Alpha Imager (Alpha Innotech).
  10. For chemifluorescent imaging, dry membranes after incubating with ECL Plus reagent. In our experience, less background is observed when the membrane is dried completely before imaging. After exposure to ECL Plus reagent, transfer the membrane to a stacked layer of lab tissue and gently blot the membrane dry. Afterwards, place the membrane on a piece of filter paper and allow to dry for at least 15 min prior to imaging. Failure to allow the blot to dry completely will result in uneven background and image artifacts. Lay the blot face-down on the Typhoon plate and overlay with a piece of non-fluorescent plastic such as 3M Dual Purpose transparency film. Scan image using the fluorescence acquisition mode, the 520 BP 40 emission filter and the blue (488 nm) laser (note: if using a Typhoon Trio + imager, use the ECL + excitation setting). Set the PMT voltage to 450–500 V initially. Do not press the sample; the plastic transparency film will adequately press the membrane against the plate. Set the pixel size to 500–1000 μm initially. Adjust the PMT voltage to obtain an image that is intense but not saturated. Take the final image using a pixel size of 100 μm or less.

(III) Visualizing BODIPY-tagged adducts using in-gel fluorescence

Since Bromophenol blue is a fluorescence quenching agent [48, 49], a dye-free SDS sample loading buffer is recommended if using fluorescence imaging. Our laboratory uses a 5× sample buffer containing 250 mM Tris HCl (pH 6.8), 25% glycerol, and 10% sodium dodecyl sulfate. If another loading buffer is used, it should be adapted so that components that interfere with BODIPY fluorescence are removed or substituted with a similar but non-interfering reagent. The Typhoon imaging system or another analogous system may be used for imaging BODIPY-labeled proteins. In addition, we recommend staining the gels with Coomassie blue or another stain such as Deep Purple (GE Healthcare) after acquiring the BODIPY image to verify even protein loading. This allows for simultaneous or post-visualization of the total protein amount as well as the modifications.

  1. Due to potential photobleaching, the BD-labeled protein samples should be separated by 1D- or 2D-SDS-PAGE under low light conditions. It is important to run the dye front completely off of the gel prior to imaging because the unreacted BODIPY-labeled lipid agent can interfere with imaging and result in poor image quality in the lower half of the gel.
  2. After separation, the gel can be left in the glass plates and imaged or removed from the plates and stained with Coomassie, Deep Purple, or another stain with excitation/emission properties distinct of BODIPY.
    1. For gel imaging directly in the glass plates using a Typhoon or similar imager, wash the outside of the glass plates well with 70% ethanol, followed by distilled water. Allow plates to dry completely.
    2. Place the dry glass plates containing the gel on 1.5 mm spacers, and adjust the focal plane accordingly (e.g., to 2–4 mm to account for the width of the glass plate).
    3. If using a Typhoon imager, use the 520 BP40 or 526SP emission filter and the 488 nm blue laser. Use similar settings if using another imager, or capture image using UV light and a camera.
  3. Alternatively, the gel can be removed from the plates and stained immediately with a total protein stain. The gel can then be imaged using the laser and emission filter described above for BODIPY. Subsequently, a fluorescent Coomassie image can be acquired on the Typhoon, using the 670BP30 emission filter and 633 nm laser. Note: in our experience, we have found that imaging the BODIPY label while the gel is in the glass plates used for electrophoreses provides the best results.

CALCULATIONS AND EXPECTED RESULTS

Confocal microscopy with fluorescently labeled lipids

As shown in Figure 7, BD-15d-PGJ2 and BD-AA are capable of entering bovine aortic endothelial cells (BAEC) after 2 h. In this experiment, BD-15d-PGJ2 fluorescence showed a reticular pattern, consistent with previous studies demonstrating its ability to concentrate in mitochondria [41, 42]. After entering cells, the fluorescent tag of BD-AA retains its excitation/emission properties and does not appear to be oxidized due to hemin treatment.

Protein modification by tagged lipids

Both the BODIPY and biotin moieties allow the detection of protein modification by endogenously produced lipid peroxidation products in lysates prepared from in vitro and in vivo samples. Hemin has been shown to increase lipid peroxidation resulting in the formation of protein reactive lipid species; however, the formation of protein-lipid adducts has not been previously reported. As shown in Figure 8A, Bt-AA can be used to detect protein modifications by lipid peroxidation products in cells. In this experiment, RAW 264.7 macrophages were treated with the ethanol vehicle (control), Bt-AA, or hemin and Bt-AA. In control lysates, a small number of proteins can be detected on the Strep-HRP blot corresponding to endogenous biotin-containing carboxylases. Bt-AA only slightly increases the reactivity with Strep-HRP, suggesting that arachidonic acid itself is not reactive with proteins, as expected. However, addition of hemin, which catalyzes lipid peroxidation, results in an increase in Strep-HRP signal. BD-AA can be used in a similar manner and also facilitates the detection of protein reactive lipid species. In addition, other controls, such as the addition of the chain-breaking antioxidants butylated hydroxytoluene (BHT) and α-tocopherol, can be used to decrease LPO and protein adduct formation (data not shown).

The electrophilic lipid, 15d-PGJ2, has been shown to modify proteins in several experimental models [17, 20, 5054] and is capable of modifying the mitochondrial proteome [40]. The mitochondrial proteome modified by BD-15d-PGJ2 can be investigated using proteomic techniques. Figure 8C shows the results from such an experiment. Briefly, isolated cardiac mitochondria were treated with BD-15d-PGJ2, and the proteins were separated by 2D Blue Native PAGE. This method uses the protein-binding and charge-conferring characteristics of Coomassie dye to facilitate protein separation in the 1st dimension [5557]. After separation in the 2nd dimension, the proteins remain bound to much of the Coomassie dye. Hence, both the BODIPY and Coomassie fluorescence can be detected using the guidelines in (III) Visualizing BODIPY-tagged adducts using in-gel fluorescence. As shown in Figure 8C, equal protein loading was verified on 2D Blue Native PAGE gels of isolated heart mitochondria using the intrinsic Coomassie fluorescence. BODIPY fluorescence could be detected with a Typhoon imager in samples treated with BD-15d-PGJ2. No interfering fluorescence of proteins was detected at this wavelength in control mitochondria. The BN-PAGE gels shown in Figure 8C demonstrate the utility of using tagged electrophilic lipids to detect reactive subproteomes. Similar experiments with these tagged lipids can be performed using other 1D and 2D gel-based separation techniques.

Quantification of protein modification by tagged lipids

Protein modification by biotin tagged lipids can be quantified using standards such as bt-cyt c. The development of this method is described in [39]. Briefly, addition of bt-cyt c allows for the absolute determination of the moles of biotin per milligram of protein in a biological sample. Due to the addition of the biotin tag, this increase in biotin is assumed to be due to protein modifications by the biotin-containing oxidized lipid. Therefore, each biotin molecule is equivalent to one lipid covalently adducted to a protein. Since covalent modification of proteins is an important regulatory mechanism in cell signaling, reliable measurement of these modifications is advantageous for these studies [20, 40, 52, 53]. As shown in Figure 8A, proteins modified by oxidized products of Bt-AA were detected on Strep-HRP western blot. Quantification of adducts (nmol biotin/mg protein) was facilitated using the internal standard, bt-cyt c and is shown in Figure 8B.

CAVEATS

Do tagged lipids have biological effects different from untagged derivatives?

Tagged derivatives should be compared with the untagged version of the same lipid for all new experimental models to ensure that the biological effects are not changed by the tag. For example, induction of antioxidant defenses, and the cytoprotection elicited by the electrophilic lipid, 15d-PGJ2, are not significantly different for the tagged derivatives of this electrophile [17]. Importantly, the covalent addition of BODIPY and biotin occurs at the carboxylic acid distant from the electrophilic carbons on the molecule, and will not affect the electrophilicity of the lipid. However, modification of the carboxyl group may change other properties of the lipid [58]. For example, the biotinylated derivative of 15d-PGJ2 is not an effective ligand for PPARγ.

While the use of tagged unsaturated fatty acids allows the identification of lipid protein adducts derived from lipid peroxidation there are a number of important limitations. For example, modifications of the fatty acid other than oxidation which lead to covalent modification of proteins could also result in increased protein adduct formation. As a control for this the attenuation of modification by chain breaking antioxidants, which should inhibit lipid peroxidation but not other modifications of the fatty acid such as acetylation can be used. In interpreting the accumulation of lipid-protein adducts a number of mechanisms should be considered. For example, it is known that electrophilic lipids can inhibit the proteasome and thus the turnover of adducted proteins may be inhibited [30, 51].

Identification of lipid-protein adducts

Use of the tagged lipids described herein enable the discovery and identification of protein targets of reactive LPO products. A limitation of this approach is the products of LPO in which the tag is lost or oxidized will not be detected. For example, 4-hydroxy-2-nonenal generated from the oxidation of the PUFA, will not contain a tag, which is attached to the carboxyl group. In addition, it should be noted that using the tagged substrate arachidonic acid and the oxidation systems described (i.e. hemin) will not enable the investigator to know the specific oxidized lipid species that modified a particular protein without coupling this technology to mass spectrometry analysis. However, use of the tagged substrate does allow a more comprehensive and thorough approach to identifying the proteins modified by LPO in response to oxidative stress or inflammation.

Acknowledgments

The authors would like to thank Ray Moore for mass spectrometric data acquired at the Comprehensive Cancer Center Mass Spectrometry Shared Facility, University of Alabama at Birmingham.

This research was supported by NIH grants ES10167, DK 75865, DK 079337 (VDU), T32 HL007457 (BGH) T32 HL007918 (BPD), and American Heart Association funding through the Scientist Development Grant 0635361N (AL), and Predoctoral Fellowship 0815177E (ANH).

Abbreviations

AA
arachidonic acid
BAEC
bovine aortic endothelial cells
BD-AA
BODIPY arachidonic acid
BODIPY FL-EDA
4, 4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride
Bt-AA
biotinylated arachidonic acid
DMSO
dimethyl sulfoxide
EDC
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
ERP
electrophile responsive proteome
ESI-MS
electrospray ionization mass spectrometry
LPO
lipid peroxidation
PUFA
polyunsaturated fatty acid
ROS
reactive oxygen species
RLS
reactive lipid species
15d-PGJ2
15-deoxy- Δ12,14-prostaglandin-J2

Footnotes

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References

1. Porter NA. Chemistry of lipid peroxidation. Methods Enzymol. 1984;105:273–282. [PubMed]
2. Porter NA, Caldwell SE, Mills KA. Mechanisms of free radical oxidation of unsaturated lipids. Lipids. 1995;30:277–290. [PubMed]
3. Pacifici EH, McLeod LL, Sevanian A. Lipid hydroperoxide-induced peroxidation and turnover of endothelial cell phospholipids. Free Radic Biol Med. 1994;17:297–309. [PubMed]
4. Kuehl FA, Jr, Egan RW. Prostaglandins, arachidonic acid, and inflammation. Science. 1980;210:978–984. [PubMed]
5. Patrignani P, Panara M, Greco A, Fusco O, Natoli C, Iacobelli S, Cipollone F, Ganci A, Créminon C, Maclouf J. Characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases. Adv Prostaglandin Thromboxane Leukot Res. 1995;23:129–131. [PubMed]
6. Capdevila JH, Falck JR, Estabrook RW. Cytochrome P450 and the arachidonate cascade. FASEB J. 1992;6:731–736. [PubMed]
7. O’Donnell VB, Maskrey B, Taylor GW. Eicosanoids: generation and detection in mammalian cells. Methods Mol Biol. 2009;462:5–23. [PubMed]
8. Yamamoto S. Enzymatic lipid peroxidation: reactions of mammalian lipoxygenases. Free Radic Biol Med. 1991;10:149–159. [PubMed]
9. Sultana R, Boyd-Kimball D, Poon HF, Cai J, Pierce WM, Klein JB, Merchant M, Markesbery WR, Butterfield DA. Redox proteomics identification of oxidized proteins in Alzheimer’s disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD. Neurobiol Aging. 2006;27:1564–1576. [PubMed]
10. Montine TJ, Morrow JD. Fatty acid oxidation in the pathogenesis of Alzheimer’s disease. Am J Pathol. 2005;166:1283–1289. [PubMed]
11. Baud L, Ardaillou R. Reactive oxygen species: production and role in the kidney. Am J Physiol. 1986;251:F765–776. [PubMed]
12. Suzuki D, Miyata T, Saotome N, Horie K, Inagi R, Yasuda Y, Uchida K, Izuhara Y, Yagame M, Sakai H, Kurokawa K. Immunohistochemical evidence for an increased oxidative stress and carbonyl modification of proteins in diabetic glomerular lesions. J Am Soc Nephrol. 1999;10:822–832. [PubMed]
13. Polidori M, Praticó D, Parente B, Mariani E, Cecchetti R, Yao Y, Sies H, Cao P, Mecocci P, Stahl W. Elevated lipid peroxidation biomarkers and low antioxidant status in atherosclerotic patients with increased carotid or iliofemoral intima media thickness. J Investig Med. 2007;55:163–167. [PubMed]
14. Chisolm G, Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic Biol Med. 2000;28:1815–1826. [PubMed]
15. Go Y, Halvey P, Hansen J, Reed M, Pohl J, Jones D. Reactive aldehyde modification of thioredoxin-1 activates early steps of inflammation and cell adhesion. Am J Pathol. 2007;171:1670–1681. [PubMed]
16. Ceaser E, Moellering D, Shiva S, Ramachandran A, Landar A, Venkartraman A, Crawford J, Patel R, Dickinson D, Ulasova E, Ji S, Darley-Usmar V. Mechanisms of signal transduction mediated by oxidized lipids: the role of the electrophile-responsive proteome. Biochem Soc Trans. 2004;32:151–155. [PubMed]
17. Levonen A, Landar A, Ramachandran A, Ceaser E, Dickinson D, Zanoni G, Morrow J, Darley-Usmar V. Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products. Biochem J. 2004;378:373–382. [PubMed]
18. Shonsey E, Eliuk S, Johnson M, Barnes S, Falany C, Darley-Usmar V, Renfrow M. Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal. J Lipid Res. 2007 [PubMed]
19. Davies S, Amarnath V, Roberts Ln. Isoketals: highly reactive gamma-ketoaldehydes formed from the H2-isoprostane pathway. Chem Phys Lipids. 2004;128:85–99. [PubMed]
20. Cernuda-Morollón E, Pineda-Molina E, Cañada F, Pérez-Sala D. 15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit. J Biol Chem. 2001;276:35530–35536. [PubMed]
21. Gutierrez J, Ballinger S, Darley-Usmar V, Landar A. Free radicals, mitochondria, and oxidized lipids: the emerging role in signal transduction in vascular cells. Circ Res. 2006;99:924–932. [PubMed]
22. Watanabe N, Zmijewski J, Takabe W, Umezu-Goto M, Le Goffe C, Sekine A, Landar A, Watanabe A, Aoki J, Arai H, Kodama T, Murphy M, Kalyanaraman R, Darley-Usmar V, Noguchi N. Activation of mitogen-activated protein kinases by lysophosphatidylcholine-induced mitochondrial reactive oxygen species generation in endothelial cells. Am J Pathol. 2006;168:1737–1748. [PubMed]
23. Davies S, Talati M, Wang X, Mernaugh R, Amarnath V, Fessel J, Meyrick B, Sheller J, Roberts Ln. Localization of isoketal adducts in vivo using a single-chain antibody. Free Radic Biol Med. 2004;36:1163–1174. [PubMed]
24. DiFranco E, Subbanagounder G, Kim S, Murthi K, Taneda S, Monnier VM, Salomon RG. Formation and stability of pyrrole adducts in the reaction of levuglandin E2 with proteins. Chem Res Toxicol. 1995;8:61–67. [PubMed]
25. Salomon RG, Subbanagounder G, O’Neil J, Kaur K, Smith MA, Hoff HF, Perry G, Monnier VM. Levuglandin E2-protein adducts in human plasma and vasculature. Chem Res Toxicol. 1997;10:536–545. [PubMed]
26. Palinski W, Horkko S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest. 1996;98:800–814. [PMC free article] [PubMed]
27. Hashimoto M, Sibata T, Wasada H, Toyokuni S, Uchida K. Structural basis of protein-bound endogenous aldehydes. Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct. J Biol Chem. 2003;278:5044–5051. [PubMed]
28. Bennaars-Eiden A, Higgins L, Hertzel AV, Kapphahn RJ, Ferrington DA, Bernlohr DA. Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro in vivo. Evidence for a role in antioxidant biology. J Biol Chem. 2002;277:50693–50702. [PubMed]
29. Kapphahn RJ, Giwa BM, Berg KM, Roehrich H, Feng X, Olsen TW, Ferrington DA. Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets. Exp Eye Res. 2006;83:165–175. [PubMed]
30. Ferrington DA, Kapphahn RJ. Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal. FEBS Lett. 2004;578:217–223. [PubMed]
31. Han B, Stevens JF, Maier CS. Design, synthesis, and application of a hydrazide-functionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates. Anal Chem. 2007;79:3342–3354. [PubMed]
32. Comporti M. Lipid peroxidation and biogenic aldehydes: from the identification of 4-hydroxynonenal to further achievements in biopathology. Free Radic Res. 1998;28:623–635. [PubMed]
33. Pompella A, Comporti M. The use of 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B for the histochemical detection of lipid peroxidation in animal tissues--a microphotometric study. Histochemistry. 1991;95:255–262. [PubMed]
34. Ahn B, Rhee SG, Stadtman ER. Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels. Anal Biochem. 1987;161:245–257. [PubMed]
35. Waller RL, Recknagel RO. Determination of lipid conjugated dienes with tetracyanoethylene-14C: significance for study of the pathology of lipid peroxidation. Lipids. 1977;12:914–921. [PubMed]
36. Valentovic MA, Gairola C, Lubawy WC. Cigarette smoke exposure alters [14C]arachidonic acid metabolism in aortas and platelets of rats fed various levels of selenium and vitamin E. J Toxicol Environ Health. 1985;15:493–502. [PubMed]
37. Lubin BH, Shohet SB, Nathan DG. Changes in fatty acid metabolism after erythrocyte peroxidation: stimulation of a membrane repair process. J Clin Invest. 1972;51:338–344. [PMC free article] [PubMed]
38. Dickinson D, Levonen A, Moellering D, Arnold E, Zhang H, Darley-Usmar V, Forman H. Human glutamate cysteine ligase gene regulation through the electrophile response element. Free Radic Biol Med. 2004;37:1152–1159. [PubMed]
39. Landar A, Oh J, Giles N, Isom A, Kirk M, Barnes S, Darley-Usmar V. A sensitive method for the quantitative measurement of protein thiol modification in response to oxidative stress. Free Radic Biol Med. 2006;40:459–468. [PubMed]
40. Landar A, Shiva S, Levonen A, Oh J, Zaragoza C, Johnson M, Darley-Usmar V. Induction of the permeability transition and cytochrome c release by 15-deoxy-Delta12,14-prostaglandin J2 in mitochondria. Biochem J. 2006;394:185–195. [PubMed]
41. Landar A, Zmijewski J, Dickinson D, Le Goffe C, Johnson M, Milne G, Zanoni G, Vidari G, Morrow J, Darley-Usmar V. Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species. Am J Physiol Heart Circ Physiol. 2006;290:H1777–1787. [PubMed]
42. Zmijewski J, Landar A, Watanabe N, Dickinson D, Noguchi N, Darley-Usmar V. Cell signalling by oxidized lipids and the role of reactive oxygen species in the endothelium. Biochem Soc Trans. 2005;33:1385–1389. [PMC free article] [PubMed]
43. Parker J. Prostaglandin A2 protein interactions and inhibition of cellular proliferation. Prostaglandins. 1995;50:359–375. [PubMed]
44. Cernuda-Morollon E, Pineda-Molina E, Canada FJ, Perez-Sala D. 15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit. J Biol Chem. 2001;276:35530–35536. [PubMed]
45. Corongiu FP, Milia A. An improved and simple method for determining diene conjugation in autoxidized polyunsaturated fatty acids. Chem Biol Interact. 1983;44:289–297. [PubMed]
46. Corongiu FP, Banni S, Dessi MA. Conjugated dienes detected in tissue lipid extracts by second derivative spectrophotometry. Free Radic Biol Med. 1989;7:183–186. [PubMed]
47. Go YM, Levonen AL, Moellering D, Ramachandran A, Patel RP, Jo H, Darley-Usmar VM. Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein. Am J Physiol Heart Circ Physiol. 2001;281:H2705–2713. [PubMed]
48. Bertsch M, Mayburd AL, Kassner RJ. The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue. Anal Biochem. 2003;313:187–195. [PubMed]
49. Yue Q, Niu L, Li X, Shao X, Xie X, Song Z. Study on the interaction mechanism of lysozyme and bromophenol blue by fluorescence spectroscopy. J Fluoresc. 2008;18:11–15. [PubMed]
50. Aldini G, Carini M, Vistoli G, Shibata T, Kusano Y, Gamberoni L, Dalle-Donne I, Milzani A, Uchida K. Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement. Biochemistry. 2007;46:2707–2718. [PubMed]
51. Ishii T, Sakurai T, Usami H, Uchida K. Oxidative modification of proteasome: identification of an oxidation-sensitive subunit in 26 S proteasome. Biochemistry. 2005;44:13893–13901. [PubMed]
52. Pérez-Sala D, Cernuda-Morollón E, Pineda-Molina E, Cañada F. Contribution of covalent protein modification to the antiinflammatory effects of cyclopentenone prostaglandins. Ann N Y Acad Sci. 2002;973:533–536. [PubMed]
53. Sánchez-Gómez F, Cernuda-Morollón E, Stamatakis K, Pérez-Sala D. Protein thiol modification by 15-deoxy-Delta12,14-prostaglandin J2 addition in mesangial cells: role in the inhibition of pro-inflammatory genes. Mol Pharmacol. 2004;66:1349–1358. [PubMed]
54. Shibata T, Yamada T, Ishii T, Kumazawa S, Nakamura H, Masutani H, Yodoi J, Uchida K. Thioredoxin as a molecular target of cyclopentenone prostaglandins. J Biol Chem. 2003;278:26046–26054. [PubMed]
55. Grandier-Vazeille X, Guerin M. Separation by blue native and colorless native polyacrylamide gel electrophoresis of the oxidative phosphorylation complexes of yeast mitochondria solubilized by different detergents: specific staining of the different complexes. Anal Biochem. 1996;242:248–254. [PubMed]
56. Brookes P, Pinner A, Ramachandran A, Coward L, Barnes S, Kim H, Darley-Usmar V. High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes. Proteomics. 2002;2:969–977. [PubMed]
57. Schagger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem. 1991;199:223–231. [PubMed]
58. Johnson TE, Holloway MK, Vogel R, Rutledge SJ, Perkins JJ, Rodan GA, Schmidt A. Structural requirements and cell-type specificity for ligand activation of peroxisome proliferator-activated receptors. J Steroid Biochem Mol Biol. 1997;63:1–8. [PubMed]