TITLE: Thiol-Redox Proteomics to Study Reversible Protein Thiol Oxidations in Bacteria
AUTHORS: Martina Rossius, Falko Hochgräfe, Haike Antelmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Thiol-redox proteomics methods are rapidly developing tools in redox biology. These are applied to identify and quantify proteins with reversible thiol oxidations that are formed under normal growth and oxidative stress conditions inside cells. The proteins with reversible thiol oxidations are usually prepared by alkylation of reduced thiols, subsequent reduction of disulfide bonds followed by a second differential alkylation of newly released thiols. Here, we describe two methods for detection of protein </span><span style = "font-style:italic;">S</span><span>-thiolations in Gram-positive bacteria using the direct shotgun approach and the fluorescent-label thiol-redox proteomics method that have been successfully applied in our previous work.</span></div></div>

[STEPS]
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Inoculate an  culture of bacterial cells into two flasks each containing  to an optical density at 500 nm (OD500) of 0.05.
[defined minimal medium]
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Grow bacterial cells until the mid-exponential phase (e.g., OD500 of 0.4). Flask 1 is used as control and flask 2 is exposed to sublethal NaOCl concentration which allows the cells to recover from growth arrest. The sublethal NaOCl concentration has to be determined for the specific bacterium before the stress experiment.
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Harvest bacterial cultures in flask 1 before the stress (control) and that in flask 2 after  of NaOCl stress exposure in 2 × 50 mL Falcon-like tubes  by rapid centrifugation (ca ).
on ice
Centrifuge: 9500 34, 5 min, 4 10
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Wash cell pellets immediately in – to remove the oxidant, transfer the cells to 2 mL Eppendorf-like tubes, repeat centrifugation using a microfuge ().
[TE-IAM buffer]
[TE-IAM buffer]
Centrifuge: 19400 34, 5 min, 4 10
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Resuspend washed cell pellet  immediately in . Do not vortex to avoid air bubbles and resuspend the cell pellet slowly using a pipette tip (see Note 2).
on ice
[UCE buffer with 100 mM IAM]
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Break cells in UCE-IAM buffer immediately using a homogenizer in the presence of glass beads and remove glass beads by short centrifugation in a microfuge (). Thiol alkylation is performed for  in the dark (see Note 2).
Centrifuge: 19400 34, 5 min, 4 10
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Add four parts pure acetone to one part of protein extract in 2 mL Eppendorf-like tubes (e.g., add 0.4 mL protein extract to 1.6 mL acetone), precipitate proteins for  and centrifuge protein pellet in a microfuge ().
on ice
Centrifuge: 19400 34, 20 min, 4 32
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Wash protein pellet with  by mixing up the pellet mechanically with a pipette tip to ensure the proteins are suspended and any trace of IAM is removed. Remove supernatant after centrifugation using a microfuge  (). Repeat washing and centrifugation with  four times:
[acetone ]
Centrifuge: 19400 34, 20 min, 20 10
[acetone]
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Wash 1/4: Wash protein pellet with  by mixing up the pellet mechanically with a pipette tip to ensure the proteins are suspended and any trace of IAM is removed. Remove supernatant after centrifugation using a microfuge  ().
[acetone ]
Centrifuge: 19400 34, 20 min, 20 10
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Wash 2/4: Wash protein pellet with  by mixing up the pellet mechanically with a pipette tip to ensure the proteins are suspended and any trace of IAM is removed. Remove supernatant after centrifugation using a microfuge  ().
[acetone ]
Centrifuge: 19400 34, 20 min, 20 10
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Wash 3/4: Wash protein pellet with  by mixing up the pellet mechanically with a pipette tip to ensure the proteins are suspended and any trace of IAM is removed. Remove supernatant after centrifugation using a microfuge  ().
[acetone ]
Centrifuge: 19400 34, 20 min, 20 10
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Wash 4/4: Wash protein pellet with  by mixing up the pellet mechanically with a pipette tip to ensure the proteins are suspended and any trace of IAM is removed. Remove supernatant after centrifugation using a microfuge  ().
[acetone ]
Centrifuge: 19400 34, 20 min, 20 10
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Dry the washed protein pellet in a vacuum centrifuge and store proteins frozen at  until reduction and BODIPY labeling (see Note 3).
-20 °C
?. [3.2 Fluorescent Labeling of Reversibly Oxidized Proteins by BODIPY-IAM and Isoelectric Focusing of the Labeled Protein Extracts]
Dissolve dried protein pellet from a 50 mL cell culture in  by shaking for –. Remove cell waste by centrifugation in a microfuge (), transfer the supernatant into a new Eppendorf tube and determine protein concentration using Bradford reagent according to manufacturer instructions.
[UCE buffer (without IAM)]
Centrifuge: 17900 34, 20 min
?. [3.2 Fluorescent Labeling of Reversibly Oxidized Proteins by BODIPY-IAM and Isoelectric Focusing of the Labeled Protein Extracts]
Transfer  into a new Eppendorf tube and fill up to . Reduce protein disulfides with  for  at  in the dark (add 5 μL 10 mM TCEP to 50 μL protein extract). Perform fluorescence labeling of the protein extract by addition of  and incubate for  at  in the dark (see Note 4).
[ protein extract]
[UCE buffer]
[TCEP]
0 Room temperature
[BODIPY-IAM stock solution]
0 Room temperature
?. [3.2 Fluorescent Labeling of Reversibly Oxidized Proteins by BODIPY-IAM and Isoelectric Focusing of the Labeled Protein Extracts]
Equilibrate spin columns with , centrifuge twice () and discard the flow-through. Add BODIPY-IAM-labeled protein extract to the spin columns, centrifuge () and collect the protein containing flow-through into a new Eppendorf-like tube. The excess BODIPY-IAM will be retained in the spin columns. Add  to the BODIPY-IAM-labeled proteins, centrifuge () and use the supernatant for rehydration of IPG strips in the dark  (see Note 5).
[RH solution]
Centrifuge: 1020 34, 1 min
Centrifuge: 1020 34, 4 min
[RH solution]
Centrifuge: 17900 34, 15 min
?. [3.2 Fluorescent Labeling of Reversibly Oxidized Proteins by BODIPY-IAM and Isoelectric Focusing of the Labeled Protein Extracts]
For rehydration of IPG strips use SERVA IPG Blue Strips pH 4–7 NL /18 cm. Add  containing the BODIPY-IAM-labeled proteins to the rehydration chamber. Place IPG strips into the rehydration solution within the chamber and remove air bubbles below the IPG strips. Cover the rehydration chamber with a parafilm to avoid drying out of IPG strips. Protect rehydration chamber from light. Reswelling of the IPG strips is performed  at  (see Note 6).
[RH solution]
0 Room temperature
?. [3.2 Fluorescent Labeling of Reversibly Oxidized Proteins by BODIPY-IAM and Isoelectric Focusing of the Labeled Protein Extracts]
Place the IPG strips in the isoelectric focusing chamber. On top at both ends of the IPG strips place electrode strips that are soaked with distilled water. Place the electrodes on top of these electrode strips and connect these with the cathode and anode. Overlay the IPG strips with mineral oil, start running the IEF using the power supply EPS3500-XL according to the IEF following parameters (see Note 7):  ABCDEF11.Step: 150 V1 mA5 W150 Vh1 h22.Step: 300 V1 mA5 W300 Vh1 h33.Step: 600 V1 mA5 W600 Vh1 h44.Step: 1500 V1 mA5 W1500 Vh1 h55.Step: 3000 V1 mA5 W57,000 Vh19 h
ABCDEF11.Step: 150 V1 mA5 W150 Vh1 h22.Step: 300 V1 mA5 W300 Vh1 h33.Step: 600 V1 mA5 W600 Vh1 h44.Step: 1500 V1 mA5 W1500 Vh1 h55.Step: 3000 V1 mA5 W57,000 Vh19 h
?. [3.2 Fluorescent Labeling of Reversibly Oxidized Proteins by BODIPY-IAM and Isoelectric Focusing of the Labeled Protein Extracts]
Remove IPG strips from the chamber and soak the mineral oil from the surface of the strips by a filter paper. Store IPG strips at  until separation by SDS-PAGE.
-20 °C
?. [3.3 Second Dimension of 2D SDS-PAGE of Fluorescent-Labeled Proteins]
For 12.5% acrylamide–bisacrylamide 2D gels prepare the separating gel solution as described in Subheading 2.1.2 item 19 "Components for 2D-PAGE and Staining Used in Fluorescent-Label Thiol-Redox Proteomics" in the Materials Tab. Immediately after initiating the polymerization process cast the separating gel solution into the funnel of the multicasting chamber. Remove bubbles by pressing the flexible tube which connects the funnel and the multicasting chamber. Open the valve to cast slowly the gel solution through the flexible tube into the multicasting chamber. Make sure to avoid air bubbles in the chamber. Cover the separating gels with butanol to ensure a homogeneous polymerization of the gel. Acrylamide polymerization is finished within .
?. [3.3 Second Dimension of 2D SDS-PAGE of Fluorescent-Labeled Proteins]
Prepare 100 mL stacking gel solution as described in Section 2.1.2 item 20 "Components for 2D-PAGE and Staining Used in Fluorescent-Label Thiol-Redox Proteomics" in Materials Tab. Keep  for embedding the IPG strips at . Rinse the surface of the gels with distilled water to remove the butanol. Add  and  to the stacking gel solution to initiate the polymerization process and mix gently. Load stacking gel solution with APS and TEMED immediately on top of the separating gel using a pipette. Cover the stacking gel with butanol to ensure a homogeneous polymerization of the gel. Polymerization is finished within .
[stacking gel solution]
4 °C
[10% (w/v) APS]
[TEMED ]
?. [3.3 Second Dimension of 2D SDS-PAGE of Fluorescent-Labeled Proteins]
After stacking gel polymerization, remove the 2D gels from the multicasting chamber and insert it into the running gel chamber. Fill the running gel chamber with 1× SDS–Tris–glycine running buffer. Equilibrate each IPG strip with  for . Remove solution (A) and equilibrate each IPG strip with  for . Remove solution (B), and load the IPG strips onto the 2D gels. Mix 30 mL stacking gel solution (from step 18 in this section) with  and  using a magnetic stirrer and cover IPG with this stacking gel solution. Finally, fill the upper gel chamber with 1× SDS–Tris–glycine running buffer. Perform the SDS-PAGE run according to the following parameters: 300 V and 300 mA; 10 min 20 W per gel and overnight 2 W per gel. Stop the run when the bromphenol blue front has reached the bottom of the gel. After the run is finished, remove the gels from the glass plates. Scan the gels to observe fluorescence images immediately using a Typhoon scanner according to the following parameters: Fluorescence setup: 520 nm, BP40, 488 nm; Filter: Blue FAM; Mode: platen.
[equilibration solution (A)]
[solution (B)]
[10% (w/v) APS]
[TEMED ]
?. [3.3 Second Dimension of 2D SDS-PAGE of Fluorescent-Labeled Proteins]
Fix 2D-gels in gel fixation solution for at least . Mix  with  and use this solution for staining of the gels . Next day, rinse the gels with distilled water twice and scan the Coomassie image. 2D gel images are quantified using the Decodon Delta 2D software by calculating ratios of fluorescence/protein amounts (see Note 8) (Figs. 2b and 3).
[Colloidal Coomassie stock solution]
[methanol ]
?. [3.4 Bacterial Cultivation, Harvesting of Cells, and Preparation of Protein Extract for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
Inoculate three flasks each containing  with overnight cultures to an optical density of OD500 of 0.07 and grow cells until the mid-exponential growth phase (OD500 of 0.5–1.0). Flask 1 is used as untreated control. Flask 2 is exposed to diamide, and flask 3 is treated with NaOCl at sublethal concentrations each for . These sublethal NaOCl and diamide concentrations should reduce the growth rate and have to be determined before in detailed growth analyses.
[minimal medium]
?. [3.4 Bacterial Cultivation, Harvesting of Cells, and Preparation of Protein Extract for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
Harvest the three bacterial cultures from untreated cells (control) and cells exposed for 30 min to diamide and NaOCl stress in 50 mL Falcon tubes  by rapid centrifugation ().
on ice
Centrifuge: 9500 34, 10 min, 4 10
?. [3.4 Bacterial Cultivation, Harvesting of Cells, and Preparation of Protein Extract for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
Wash bacterial cell pellets in TE-IAM buffer, transfer to Eppendorf tubes, centrifuge again () and resuspend the cell pellets in .
Centrifuge: 17900 34, 5 min, 4 10
[UCE-IAM buffer]
on ice
?. [3.4 Bacterial Cultivation, Harvesting of Cells, and Preparation of Protein Extract for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
Disrupt cells using the homogenizer  and centrifuge cell extracts twice for  at  to remove the cell debris. Alkylate the proteins containing free thiol-groups by incubation of the protein extract for  at  in the dark.
on ice
4 °C
0 Room temperature
?. [3.4 Bacterial Cultivation, Harvesting of Cells, and Preparation of Protein Extract for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
Determine the protein concentration by using Bradford reagent according to manufacturer instructions. Concentrate aliquots of 200 μg protein extract in the vacuum centrifuge to  and dissolve proteins in . Continue with protein separation using SDS gels.
15 µl
[non-reducing SDS-sample buffer (without DTT)]
?. [3.5 Protein Fractionation by Separation on 15% SDS-PAGE for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
For preparation of two 15% non-reducing SDS-PAGE separating gels, prepare 1D separating gel solution freshly (Subheading 2.2.2, item 7 "Components for SDS-PAGE" in Materials Tab) and immediately cast the separating gels between two glass plates, cover the gel surface with  and allow polymerization for –.
[butanol ]
?. [3.5 Protein Fractionation by Separation on 15% SDS-PAGE for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
For preparation of 1D SDS-PAGE stacking gel, first remove butanol and rinse the gel surface with Aqua dest. Prepare 1 D stacking gel solution (Subheading 2.2.2, item 8 "Components for SDS-PAGE" in Materials Tab) and cast the stacking gel solution on top of the separation gel. Place the comb into the stacking gels and allow polymerization for .
?. [3.5 Protein Fractionation by Separation on 15% SDS-PAGE for Identification of Protein S-Thiolation Using ESI-LC-MS/MS Analysis]
Place the gels into the running chamber and fill up with running buffer. For protein fractionation, load 2 wells each with  in non-reducing SDS-sample buffer for control, diamide, and NaOCl extract, respectively. Separate proteins in 15% non-reducing SDS-PAGE according to manufacturer’s description. Fix the SDS gels in gel fixation solution for . Mix  with  and stain the gels . Next day, rinse the gels with distilled water twice. At this point gel can be stored at  covered in for example transparent film to avoid drying till in-gel digestion. Each gel lane is cut into ten slices and used for in-gel tryptic digestion and subsequent LC-MS/MS-analysis for identification of protein S-thiolations (see Note 9).
[protein extract]
[Colloidal Coomassie stock]
[methanol ]
4 °C
?. [3.6 In-Gel Tryptic Digestion and Peptide Preparation for Mass Spectrometry]
Destain the gel pieces using Digester-A solution under vigorous agitation for  at . Discard the supernatant and repeat destaining several times until the gel pieces are completely destained.
37 °C
?. [3.6 In-Gel Tryptic Digestion and Peptide Preparation for Mass Spectrometry]
Shrink the destained gel pieces in Digester-B solution under vigorous agitation for  at . Discard the supernatant and dry the gel pieces in a vacuum centrifuge for –.
0 Room temperature
?. [3.6 In-Gel Tryptic Digestion and Peptide Preparation for Mass Spectrometry]
For the tryptic in-gel digestion add – to the dried gel pieces until the gel is completely reswollen. Perform tryptic digestion  at .
[trypsin solution]
[trypsin solution]
37 °C
?. [3.6 In-Gel Tryptic Digestion and Peptide Preparation for Mass Spectrometry]
Extract tryptic peptides with  under vigorous agitation for  at . Concentrate the peptides in the vacuum centrifuge and store at  until LC-MS/MS analysis.
[spotter solution]
0 Room temperature
-20 °C
?. [3 Methods]
Fluorescent-label thiol-redox proteomics to visualize reversibly oxidized proteins in bacteria in response to oxidative stress.
?. [3.1 Fluorescent-Label Thiol-Redox Proteomics: Growth, Harvesting of Cells, and Blocking of Reduced Thiols by IAM]
Fluorescent-label thiol-redox proteomics is carried out to visualize reversibly oxidized proteins in bacteria in response to oxidative stress.