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D. Myc-Tdh3/Afch3 Myc-Tdh3C150SC154SA/fdh3

D. Myc-Tdh3/Afch3 Myc-Tdh3C150SC154SA/fdh3

Fig. 13.5. 2D-gel electrophoresis of oxidized protein-thiols purified using biotin-HPDP, as described in Sects. 3.2.2 and 3.2.3. Typical profiles of oxidized protein-thiols purified from wild type cells that were: (A) left untreated, or (B) treated with 1 mM of H2O2 during 1 minute, or (C) purified from cells lacking both cytoplasmic thioredoxins. Arrows indicate proteins that show an increased oxidation upon H2O2 treatment or upon inactivation of the thioredoxin pathway. (D) A control experiment showing the thiol-specificity of the biotin-HPDP-based purification procedure (From ref. 14): Tdh3 cannot be purified when lacking its two cysteine-residues. A strain with inactivation of the TDH3 gene was transformed with plasmid expressing a Myc-tagged version of Tdh3 (left gel) or Myc-Tagged Tdh3 in which the two cysteines were replaced by serine residues (right gel). The region of gel were Tdh3 migrates is enlarged.

Fig. 13.5. 2D-gel electrophoresis of oxidized protein-thiols purified using biotin-HPDP, as described in Sects. 3.2.2 and 3.2.3. Typical profiles of oxidized protein-thiols purified from wild type cells that were: (A) left untreated, or (B) treated with 1 mM of H2O2 during 1 minute, or (C) purified from cells lacking both cytoplasmic thioredoxins. Arrows indicate proteins that show an increased oxidation upon H2O2 treatment or upon inactivation of the thioredoxin pathway. (D) A control experiment showing the thiol-specificity of the biotin-HPDP-based purification procedure (From ref. 14): Tdh3 cannot be purified when lacking its two cysteine-residues. A strain with inactivation of the TDH3 gene was transformed with plasmid expressing a Myc-tagged version of Tdh3 (left gel) or Myc-Tagged Tdh3 in which the two cysteines were replaced by serine residues (right gel). The region of gel were Tdh3 migrates is enlarged.

3.2. Basic Protocols for Proteome-Wide Oxidized Protein Thiols Identification

3.2.1. Acid Quenching of Protein Thiol by TCA Precipitation

The protocol detailed here is adapted for the proteome-wide identification of protein-thiol oxidation in the single-celled eukaryote Saccharomyces cerevisiae, using [s 4C]NEM as the thiol-labeling reagent. Modifications required when using other thiol-labeling reagents are indicated with reference to this protocol.

1. Cells are grown in CASA medium supplemented with adenine, uracil, and tryptophane (5 g/L) at 30°C (200 rpm shaking)

2. TCA is added to a 20% final concentration into a 20-mL cell culture at an O. D. 0.6, which has been treated or not with H2O2, at concentrations between 0.2 and 1 ml (see Notel)

3. The cell culture is centrifuged 5 min at 4,000g and at 4°C

4. The pellet is washed with 1 mL of a 20% TCA cold solution

5. The sample is centrifuged 5 min at 4,000g and at 4°C

6. The pellet is immediately frozen in dry ice

7. 0.2 mL of a 20% TCA cold solution is added to the TCA-precipitated pellet together with 0.1-mL glass beads

8. Cells in suspension in a 1.5-mL Eppendorf are broken by vigorous agitation on a vortex during 10 min. Agitation is repeated three or four times after samples are cooled down by incubation on ice 1 min

9. Glass beads are brought down by gravity during a few minutes. The supernatant is collected in a 1.5-mL Eppendorf tube. The beads are washed with 0.2 mL of 5% TCA cold solution, and the wash solution is added to the first supernatant

10. After centrifugation at 13,000g for 10 min at 4°C, the protein pellet is washed three times in 1 mL of cold acetone (see Note2) to remove TCA

11. The pellet is then air-dried in a speed-vacuum for 5 min

12. Steps 1-11 are identical when using fluorescent NEM or IAM instead of [s 4C]NEM. However, when using biotin-HPDP, the starting culture volume is 500 ml, and steps 1-11 should be adapted to this volume

3.2.2. Protein-Thiol 1. Protein pellets are dissolved in 200 pL of denaturing buffer

Labeiing (Figs. 13.3 containing 50 mMS IAM. Alkylation of free thiols is performed and 13.4) by a 45-min incubation at 30°C under constant shaking

2. To remove aggregates, samples are centrifuged 10 min at 13,000g at room temperature.

3. To remove excess of IAM, supernatants are precipitated with cold TCA at a 10% final concentration as in Section 3.2.1, steps 10 and 11.

4. Dried pellets are solubilized in 200 pL of denaturing buffer containing 20 mM of DTT. Samples are incubated 45 min at 30°C under shaking to allow full reduction of oxidized thiols.

5. Aggregated proteins are removed by centrifugation 10 min at 13,000g at room temperature.

6. To remove excess of DTT, steps 3, 4, and 5 are repeated once.

7. To label oxidized thiols that have been reduced by DTT in step 4, dried pellets are dissolved in 200 pL of denaturing buffer and incubated during 15 min at 30°C, after which 30 pL (15 pCi) of [14C]NEM stock solution (2 mM final concentration) is added. Samples are further incubated during 15 min at 30°C under shaking.

8. Protein samples are then centrifuged during 10 min at 13,000g at room temperature to remove aggregated proteins.

9. To remove unbound [14C]NEM, repeat steps 3 and 5.

10. The dried protein pellets are dissolved in 100 pL of denaturing buffer with shaking at 30°C.

11. After centrifugation, the concentration of the resulting supernatant is determined by the Micro BCA assay reagent kit (see Note4).

12. 100 pg of the [14C]NEM labeled proteins are then subjected to 2D-gel electrophoresis.

13. When using biotin-HPDP, buffer volumes are adapted to the starting culture volume; biotin-HPDP labeling (step 7) is performed by incubating samples during 45 min at 30°C, in the dark. Then, steps 8-10 are identical, except for the volume of denaturing buffer that should be such to optimize protein solubilization (between 500 pl and 3 ml).

14. For fluorescent NEM thiol labeling, dyes are used at a 0.1 mM final concentration in the presence of 200 mM IAM (step 1 and/or 7). The pH of the denaturing buffer is 9 in steps 1 and 4, and 7.5 in step 7. Labeling is performed upon an incubation of 15 min at 4°C in the dark (step 7). Steps 9 and 10 are omitted (see Notes 5 and 6).

3.2.3. Purification of 1. Three to five milligrams of biotin-HPDP-labeled extracts

Biotin-HPDP Labeled in denaturing buffer from step 11 (Sect. 3.2.2) are diluted

Proteins (Fig. 13.5) by addition of one volume of buffer containing 50 mM

Tris-HCl, pH 8.8; and 100 mM NaCl. Extracts are then precleared by incubation with 500 pL of sepharose CL-4B beads for 3 h at 4°C, with tumbling.

2. The sepharose beads are discarded and 500 pL of streptavi-din-sepharose beads are added to the extracts that are then incubated overnight at 4°C with tumbling.

3.2.4.2D-Gel First Dimension

3.2.5.2D-Gel Second Dimension

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