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    A Proteomics Approach to Identify Redox-Sensitive Proteins

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    实验试剂

     

    1. Arabidopsis suspension culture: T87 suspension culture (RIKEN BioResource Center).

    2. Liquid medium for T87 cells: 1/2 MS (Murashige and Skoog) salts, 3% sucrose, 0.29 g/L KH2 PO4 , 0.2 mg/L 2, 4-D, and B5 vitamins.

    3. 0.5 M H2 O2 stock solution (prepare just before use).

    4. Glass vacuum filter (VWR).

    5. Homogenization buffer: 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 5 mM EGTA, 50 mM NaCl, 1 mM PMSF, and 1/100 volume of plant protease inhibitor cocktail (Sigma).

    6. 10 mM stock of IAF in dimethylformamide (DMF).

    7. 1 M stock of NEM in methanol.

    8. Phenol (Tris buffered, pH 6.4–6.8).

    9. Rehydration buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT, and 0.5% IPG buffer (Bio-Rad).

    10. CB-X Protein Assay kit (G-Biosciences).

    11. Reduction buffer: 50 mM Tris–HCl, pH 8.0, 7 M urea, 2 M thiourea, 1% CHAPS, and 1 mM DTT.

    12. 11 cm ReadyStrip IPG strips (pH 4–7) (Bio-Rad).

    13. SDS-PAGE running buffer (10×): 250 mM Tris, 1,920 mM glycine, and 1% (w/v) SDS.

    14. 8–16% Criterion Precast Gels (Bio-Rad).

    15. Sypro Ruby (Bio-Rad).

    16. Destaining solution: 10% methanol and 7% acetic acid.

    17. Multiprobe II Plus (PerkinElmer).

    18. Solvent A: 0.1% formic acid in MilliQ water.

    19. Solvent B: 0.1% formic acid in ACN.

    实验步骤

     

    1. Cell Culture and H2 O2 Treatment

       1) The T87 cell culture is maintained in 50 ml of liquid medium in 250-ml flasks by gentle agitation (50 rpm) in dark at 22℃. Cells are subcultured weekly by transferring 3 ml of culture to a new flask with 50 ml of the culture medium (see Notes 1 and 2).

       2) For the oxidant treatment, a 0.5 M H2 O2 stock solution is added to T87 cells 3 days after subculturing to a final concentration of 5 mM. For control samples, the same amount of H2 O is added to the culture cells. Ten minutes after the H2 O2 addition, cells are harvested by filtering through a glass vacuum filter and immediately frozen in liquid nitrogen for further analysis (see Notes 3 and 4).

    2. Protein Extraction and Labeling: For Direct Labeling Method

       1) Five hundredmicroliters of cells are broken in 1.5-ml Eppendorf tubes with plastic micro-pestle on ice in 500 ml homogenization buffer containing 20 mM IAF from a 10 mM stock in DMF and the protein homogenate is incubated on ice for 30 min (see Note 5).

       2) The homogenate is then centrifuged for 45 min at 20,000 × g under 4℃.

       3) The supernatant is mixed with an equal volume of ice-cold phenol and centrifuged at 20,000 × g for 15 min at 4℃ to separate phenol and aqueous phases.

       4) The upper aqueous phase is removed leaving the interface intact, and the phenol phase is extracted twice with 50 mM Tris–HCl, pH 8.0, then mixed with five volumes of cold 0.1M ammonium acetate in methanol, and left at 20℃ overnight to precipitate proteins.

       5) After centrifugation at 20,000 × g for 15 min, the protein pellet is washed five times with 1 ml of methanol and air dried for 10 min in a fume hood.

       6) The pellet is resuspended in the rehydration buffer. After centrifugation at 15,000 × g for 3 min, the supernatant is transferred to a new tube, and the protein concentration is determined with the CB-X Protein Assay kit.

    3. Protein Extraction and Labeling: For Blocking Method

       1) Five hundred microliters of cells are broken in 1.5-ml Eppendorf tubes with plastic micro-pestle on ice in 500 ml of homogenization buffer containing 10 mM NEM from a 1 M stock in methanol and the protein homogenate is incubated on ice for 30 min (see Note 5).

       2) The homogenate is then centrifuged for 45 min at 20,000 × g at 4℃.

       3) The supernatant is mixed with an equal volume of ice-cold phenol and centrifuged at 20,000 × g for 15 min at 4℃ to separate phenol and aqueous phases.

       4) The upper aqueous phase is removed leaving the interface intact, and the phenol phase is extracted twice with 50 mM Tris–HCl, pH 8.0, then mixed with five volumes of cold 0.1M ammonium acetate in methanol, and left at -20℃ overnight to precipitate proteins.

       5) After centrifugation at 20,000 × g for 15 min, the protein pellet is washed five times with 1 ml of methanol and air dried for 10 min in a fume hood.

       6) The pellet is resuspended in the reduction buffer.

       7) After sitting at room temperature for 30 min, 10 mM IAF is added to a final concentration of 40 mM and the mixture is incubated at room temperature for 30 min.

       8) The reaction is stopped by adding five volumes of methanol and free IAF is removed by precipitation with centrifugation.

       9) The pellet is resuspended in the rehydration buffer (make sure that the concentration of protein exceeds 1.4 mg/ml) and protein concentration is determined with the CB-X Protein Assay kit.

    4. Two-Dimensional Gel Electrophoresis

       1) Two hundred and fifty micrograms of protein are mixed with more rehydration buffer to bring the volume to 180 ml.

       2) Samples are then applied to 11 cm ReadyStrip IPG strips and the strips are rehydrated overnight at room temperature (see Note 6).

       3) IEF is carried out on a Bio-Rad PROTEAN IEF cell at 20℃ with maximum 50 mA/strip and the following setting: 250 V for 30 min, 500 V for 1 h, a gradient increase to 8,000 V in 2.5 h, and remaining at 8,000 V until reaching 35,000 V h.

       4) After IEF, IPG strips are washed five times by dipping into the SDS-PAGE running buffer.

       5) The strips are then transferred to 8–16% Criterion Precast Gels for the second-dimension electrophoresis using Criterion Cell System (Bio-Rad). SDS-PAGE is run at 60 V for 15 min and then at 200 V until the bromphenol blue dye front reaches the gel end (see Note 7).

       6) Gels are stained with Sypro Ruby according to the manufacturer’s instructions and destained with destaining solution.

    5. Two-Dimensional Gels’ Image Scanning and Analysis

       1) The gels are scanned using a Typhoon 9410 scanner (GE Healthcare). Signal of IAF-labeled protein is detected using 488-nm laser and a 520-nm band-pass emission filter. Signal of Sypro Ruby fluorescent dyes is detected using 532-nm laser and 610-nm band-pass emission filter. All gels are scanned at 100-mm pixel size (see Note 8).

       2) The 2-D gel images are analyzed using Progenesis SameSpots software version 2.0 as described in the user’s instruction (Nonlinear Dynamics). Briefly, a sample is chosen as reference image, and alignment vectors are manually added between the reference image and other samples. After alignment, the images of three replicate samples for the same treatment are grouped together and the aligned images are analyzed for spot volume quantification and volume ratio normalization of different samples in the same treatment group. Statistical, quantitative, and qualitative analysis sets are created between the control group and treated group. Protein spots with more than twofold increase or decrease in the normalized spot volume (with p-value <0.05) between the H2 O2 -treated samples and the control samples are picked for identification.

    6. Spot Picking and Mass Spectrometry

       1) A spot-picking list generated from Phoretix 2D Evolution gel analysis software (Nonlinear Dynamics Ltd.) is exported to Gelpix (Genetix Inc.). The excised spots are then digested with Multiprobe II Plus.

       2) Protein digests are subjected to nano-LC-ESI–MS/MS analysis. Nano-LC is performed with a nanoLC-2D (Eksigent) equipped with a capillary trap LC Packings PepMap (DIONEX) and LC Packings C18 Pep Map 100 (75 mm, 15 cm) connected to the MS. Peptides (5 ml of injections) are desalted for 10 min with a flow rate 5 ml/min of 90.5% solvent A. Peptides are then resolved on a gradient from 9.5 to 35% solvent B for 4 min, from 35 to 45% solvent B for 31 min, and from 45 to 90.5% solvent B over the final 6 min at 200 nl/min flow rate.

       3) The MS analysis is performed on an ABI QSTAR XL (Applied Biosystems) hybrid QTOF MS/MS mass spectrometer equipped with a nanoelectrospray source (Protana XYZ manipulator). Positive-mode nanoelectrospray is generated from fused-silica PicoTip emitters with a 10 mm aperture (New Objective) at 2.5 kV. The m/z response of the instrument is calibrated daily with manufacturer standards. TOF mass and product ion spectra are acquired using information-dependent data acquisition (IDA) in Analyst QS v1.1 with the following parameters: mass ranges for TOF MS and MS/MS are m/z 300–2,000 and 70–2,000, respectively. Every second, a TOF MS precursor ion spectrum is accumulated, followed by three product ion spectra, each for 3 s. The switching from TOF MS to MS/MS is triggered by the mass range of peptides (m/z 300–2,000), precursor charge state (2–4), and ion intensity (>50 counts). The DP, DP2, and FP settings are 60, 10, and 230, respectively, and rolling collision energy is used.

    7. Protein Database Search

       1) The peptide tandem MS (mass spectra) are processed using Analyst QS software v1.1 (Applied Biosystems) and searched against the NCBI Protein database (5,162,317 sequences, June 2007) using an in-house version of MASCOT v2.20 (Matrix Science Inc).

       2) The following parameters are selected: tryptic peptides with &le;1 missed cleavage site; precursor and MS/MS fragment ion mass tolerance of 0.8 and 0.8 Da, respectively; fixed carbamidomethylation of cysteine; and variable oxidation of methionine. Positive identification is determined based on the following criteria: 2 peptide sequences; protein sequence coverage; total MASCOT and individual ion scores (http://www.matrixscience.com/help/scoring_help.html); and MS/MS spectral quality judged by a full-length y-ion series of peptides comprising at least six consecutive amino acid sequence tags with no missed cleavages.

    注意事项

     

    1. We used Arabidopsis suspension cells rather than whole plant tissues since cells can be exposed to the oxidant more uniformly, which increases the sensitivity for detecting oxidatively modified proteins.

    2. Avoid using cell culture older than 3 days for H2 O2 treatment. Cultures in which cells growing exponentially are better than cultures in the plateau phase which might contain many cells that are undergoing cell death and producing a high level of ROS.

    3. We used 0.5–5 mM H2 O2 to induce oxidative stress of Arabidopsis suspension cells and detected similar protein oxidation patterns based on the 2-DE images of IAF-labeled proteins. However, we have not done experiments for direct and extensive comparison on identities of proteins that become oxidatively modified following treatments with different concentrations of the oxidant.

    4. H2 O2 is not very stable and degrades easily. Purchase new H2O2 to replace old one every 2 months for reproducible results.

    5. All steps of protein extraction, blocking of free thiols, and fluorescence labeling were carried out under reduced light.

    6. Choose ready-made strips with appropriate pH ranges according to the isoelectric points of proteins of interest.

    7. The size of the gel for the second dimension should be chosen according to the complexity of the protein samples analyzed.

    8. When gels were scanned, the photomultiplier tube (PMT) was set to ensure maximum pixel intensity between 40,000 and 80,000 to avoid saturation.

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