Thiol-Redox Proteomics to Study Reversible Protein Thiol Oxidations in Bacteria

Fluorescent-label thiol-redox proteomics to visualize reversibly oxidized proteins in bacteria in response to oxidative stress.

Fluorescent-label thiol-redox proteomics is carried out to visualize reversibly oxidized proteins in bacteria in response to oxidative stress.

Inoculate an culture of bacterial cells into two flasks each containing 100mL to an optical density at 500 nm (OD₅₀₀) of 0.05.

Grow bacterial cells until the mid-exponential phase (e.g., OD₅₀₀of 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.

Harvest bacterial cultures in flask 1 before the stress (control) and that in flask 2 after 0h 30m 0s of NaOCl stress exposure in 2 × 50 mL Falcon-like tubes On ice by rapid centrifugation (ca 9500x g,4°C).

Wash cell pellets immediately in 1mL–2mL to remove the oxidant, transfer the cells to 2 mL Eppendorf-like tubes, repeat centrifugation using a microfuge (19400x g,4°C).

Resuspend washed cell pellet On ice immediately in 400µL. Do not vortex to avoid air bubbles and resuspend the cell pellet slowly using a pipette tip ( see Note 2 ).

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 (19400x g,4°C). Thiol alkylation is performed for 0h 15m 0s in the dark ( see Note 2 ).

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 1h 0m 0s On ice and centrifuge protein pellet in a microfuge (19400x g,4Room temperature).

Wash protein pellet with 80% (v/v) 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 (19400x g,20°C). Repeat washing and centrifugation with 80% (v/v) four times:

Wash 1/4: Wash protein pellet with 80% (v/v) 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 (19400x g,20°C).

Wash 2/4: Wash protein pellet with 80% (v/v) 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 (19400x g,20°C).

Wash 3/4: Wash protein pellet with 80% (v/v) 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 (19400x g,20°C).

Wash 4/4: Wash protein pellet with 80% (v/v) 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 (19400x g,20°C).

Dry the washed protein pellet in a vacuum centrifuge and store proteins frozen at -20°C until reduction and BODIPY labeling ( see Note 3).

Dissolve dried protein pellet from a 50 mL cell culture in 150µL by shaking for 0h 30m 0s–1h 0m 0s. Remove cell waste by centrifugation in a microfuge (17900x g), transfer the supernatant into a new Eppendorf tube and determine protein concentration using Bradford reagent according to manufacturer instructions.

Transfer 250µg into a new Eppendorf tube and fill up to 50µL. Reduce protein disulfides with 1millimolar (mM) for 0h 30m 0s at Room temperature in the dark (add 5 μL 10 mM TCEP to 50 μL protein extract). Perform fluorescence labeling of the protein extract by addition of 6µL and incubate for 0h 15m 0s at Room temperature in the dark ( see Note 4 ).

Equilibrate spin columns with 500µL, centrifuge twice (1020x g) and discard the flow-through. Add BODIPY-IAM-labeled protein extract to the spin columns, centrifuge (1020x g) 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 280µL to the BODIPY-IAM-labeled proteins, centrifuge (17900x g) and use the supernatant for rehydration of IPG strips in the dark ( see Note 5 ).

For rehydration of IPG strips use SERVA IPG Blue Strips pH 4–7 NL /18 cm. Add 360µL 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 Room temperature ( see Note 6 ).

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 ):

A	B	C	D	E	F
1.	Step: 150 V	1 mA	5 W	150 Vh	1 h
2.	Step: 300 V	1 mA	5 W	300 Vh	1 h
3.	Step: 600 V	1 mA	5 W	600 Vh	1 h
4.	Step: 1500 V	1 mA	5 W	1500 Vh	1 h
5.	Step: 3000 V	1 mA	5 W	57,000 Vh	19 h

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 -20°C until separation by SDS-PAGE.

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 2h 0m 0s.

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 30mL for embedding the IPG strips at 4°C. Rinse the surface of the gels with distilled water to remove the butanol. Add 380µL and 62.5µL 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 1h 0m 0s.

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 4.5mL for 0h 15m 0s. Remove solution (A) and equilibrate each IPG strip with 4.5mL for 0h 15m 0s. 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 460µL and 75µL 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.

Fix 2D-gels in gel fixation solution for at least 1h 0m 0s. Mix 200mL with 50mL 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).

Inoculate three flasks each containing 100mL with overnight cultures to an optical density of OD₅₀₀of 0.07 and grow cells until the mid-exponential growth phase (OD₅₀₀of 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 0h 30m 0s. These sublethal NaOCl and diamide concentrations should reduce the growth rate and have to be determined before in detailed growth analyses.

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 On ice by rapid centrifugation (9500x g,4°C).

Wash bacterial cell pellets in TE-IAM buffer, transfer to Eppendorf tubes, centrifuge again (17900x g,4°C) and resuspend the cell pellets in 1mL On ice.

Disrupt cells using the homogenizer On ice and centrifuge cell extracts twice for 0h 30m 0s at 4°C to remove the cell debris. Alkylate the proteins containing free thiol-groups by incubation of the protein extract for 0h 15m 0s at Room temperature in the dark.

Determine the protein concentration by using Bradford reagent according to manufacturer instructions. Concentrate aliquots of 200 μg protein extract in the vacuum centrifuge to 15µL and dissolve proteins in 15µL. Continue with protein separation using SDS gels.

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 1mL and allow polymerization for 0h 30m 0s–0h 45m 0s.

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 0h 40m 0s.

Place the gels into the running chamber and fill up with running buffer. For protein fractionation, load 2 wells each with 200µg 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 1h 0m 0s. Mix 200mL with 50mL and stain the gels . Next day, rinse the gels with distilled water twice. At this point gel can be stored at 4°C 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 ).

Destain the gel pieces using Digester-A solution under vigorous agitation for 0h 20m 0s at 37°C. Discard the supernatant and repeat destaining several times until the gel pieces are completely destained.

Shrink the destained gel pieces in Digester-B solution under vigorous agitation for 0h 30m 0s at Room temperature. Discard the supernatant and dry the gel pieces in a vacuum centrifuge for 0h 10m 0s–0h 20m 0s.

For the tryptic in-gel digestion add 5µL–20µL to the dried gel pieces until the gel is completely reswollen. Perform tryptic digestion at 37°C.

Extract tryptic peptides with 100µL under vigorous agitation for 1h 0m 0s at Room temperature. Concentrate the peptides in the vacuum centrifuge and store at -20°C until LC-MS/MS analysis.