Detection of Disulfides in Protein Extracts of Arabidopsis thaliana Using Monobromobimane (mBB)

Materials and Reagents

1. Finntip Pipette Tips (Finntip Flex 10, 200, and 1,000)
2. 1.5 ml microcentrifuge tube (VIOLAMO, F-1.5, catalog number: 1-1600-01)
3. Arabidopsis thaliana wild-type plants (Ecotype: Col-0)
4. Arabidopsis thaliana mutant plant of interest (Ecotype: Col-0)
5. Liquid nitrogen
6. Tris(hydroxymethyl)aminomethane (Tris) (Nacalai Tesque, catalog number: GR35406-91)
7. cOmplete^TM, Mini, Protease Inhibitor Cocktail (Roche, catalog number: 4693159001)
8. Bio-Rad Protein Assay Kit (Bio-Rad, catalog number: 5000001ja)
9. FOCUS^TM Protein Alkylation (TaKaRa, catalog number: GA503)
10. Iodoacetamide (IAA) (Sigma-Aldrich, catalog number: I1149-5G)
11. Trichloroacetic acid solution (TCA), 100% (w/v) (Nacalai Tesque, catalog number: SP06275-24)
12. Acetone (Nacalai Tesque, catalog number: SP09888-85) 
13. FOCUS^TM Protein Reductant (TaKaRa, catalog number: GA501)
14. Tris(2-carboxyethyl)phosphine Hydrochloride (TCEP) (Nacalai Tesque, catalog number: SP06342-21)
15. Monobromobimane (mBB) (Tokyo Chemical Industry, catalog number: B4220)
16. Glycerol (Nacalai Tesque, catalog number: SP17045-94)
17. Bromophenol Blue (BPB) (Nacalai Tesque, catalog number: B-2609)
18. Glycine (Nacalai Tesque, catalog number: SP17141-24)
19. Sodium lauryl sulfate (SDS) (Nacalai Tesque, catalog number: SP08933-34)
20. Protein extraction buffer (see Recipes)
21. IAA solution (see Recipes)
22. SDS-PAGE sample buffer (see Recipes)
23. SDS-PAGE buffer (see Recipes)
    

Equipment

1. Pipettes (Thermo Fisher Scientific, Finnpipette^TM F2)
2. Cork borer (4.0 mm, inside diameter of the hole)
3. Conventional Dewar’s vessel
4. Vortexer (Scientific Industries, Vortex-Genie 2)
5. Homogenizer pestle (VIOLAMO, F-1.5, catalog number:1-2955-02)
6. Refrigerated microcentrifuge (TOMY, MX305)
7. Absorption spectrometer (Thermo Fisher Scientific, Multiskan^TM GO)
8. Mini-PROTEAN Tetra System (Bio-Rad, catalog number: 1658004ja)
9. Mini-PROTEAN TGX Gels (Bio-Rad, catalog number: 456-1095)
10. Power supply (ATTO, catalog number: AE-8135)
11. UV Transilluminator (Maestrogen, model: UV-01)
12. Yellow filter (Kenko, Black & White Filter, Y2 Professional, λ_cut-on 470-480 nm)
13. Conventional digital camera with hood (RICHO, WG-4)
    

Software

1. ImageJ (https://imagej.nih.gov/ij//)

Procedure

1. Protein extraction
   1. Place freshly prepared leaf discs (10-50 mg) punched-out with a cork borer in a 1.5 ml microcentrifuge tube and immediately freeze in liquid nitrogen in a Dewar’s vessel. The estimated average weight of five leaf discs should be approximately 10 mg. 
   2. Grind the sample in the microcentrifuge tube with a homogenizer pestle and add 200 µl of the ice-cold protein extraction buffer (prepare immediately prior to use, Recipe 1) per 50 mg sample on ice before sample lysis.
      Note: Make sure not to dissolve the sample. We recommend grinding the sample in the presence of liquid nitrogen rather than on ice. In the case of Arabidopsis leaf discs, it is easy to obtain fully homogenized sample in a couple of seconds. 
   3. Centrifuge at 14,000 x g, 4 °C for 10 min.
   4. Transfer the supernatant to a new 1.5 ml microcentrifuge tube for removing sample debris.
2. Protein measurement
   1. Prepare the dye for Bio-Rad Protein Assay by diluting 1 volume of the dye reagent concentrate with 4 volumes of distilled, deionized (DDI) water.
   2. Add 2 and 5 µl protein extract into 198 and 195 µl diluted dye reagent, respectively.
   3. After 5 min, measure absorbance at 595 nm and estimate the protein concentration with protein standard II (BSA) in Bio-Rad Protein Assay Kit (0.2 to 2.0 mg/ml).
3. Thiol alkylation
   1. Adjust the protein concentration to 1.0 mg/ml with the extraction buffer and combine several samples to prepare 1.5 ml of protein extract in a single tube.
   2. Divide the solution into three 1.5 ml microcentrifuge tubes. Each tube contains 500 µl of the extract, one each for the determination of free thiols (tube A), disulfide bonds (tube B), and total thiols (tube C) in the protein extracts. All the tubes should be kept on ice until use.
      Note: For all experiments, we strongly recommend starting from a single protein extract to discriminately detect free thiols, disulfide bonds, and total thiols in proteins to minimize the sample variations.
   3. Add 2.5 µl FOCUS^TM alkylation buffer to tubes B and C, and vortex for 10 s.
   4. Add 25 µl 0.4 M IAA (prepare immediately prior to use, Recipe 2) only in tube B for detection of disulfide bonds. 
   5. Incubate tubes B and C without shaking at room temperature (approximately 23 °C), under dark for 30 min.
4. Protein purification
   1. Add an equal volume of cold 20% (w/v) TCA solution to tubes B and C.
   2. Centrifuge the tubes at 14,000 x g, 4 °C for 30 min.
   3. Remove the supernatant, leaving the protein pellet intact.
   4. Remove TCA by washing pellet twice with 500 µl cold acetone and retain the pellet.
5. Disulfide reduction
   1. Adjust the protein reduction solution by adding 2.5 µl FOCUS^TM Reductant buffer and 10 µl FOCUS^TM Protein Reductant (or 250 mM TCEP in DDI water) to 487.5 µl DDI water.
   2. Add 50 µl reduction solution to tubes B and C, and then suspend the pellet by gently pipetting up and down. Here, the pellet is not completely dissolved.
   3. Incubate the tubes at 55 °C for 1 h. Vortex for 10 s every 10 min and confirm the complete dissolution of pellet after incubation.
6. mBB labeling
   1. Add 25 µl of 60 mM mBB solution to tubes A, B, and C.
      Note: TCEP can be involved in dehalogenation of mBB and might weaken the efficiency of fluorescent labeling in solution (Graham et al., 2003). Be sure to keep the mBB concentration higher than that of TCEP in the labeling solution.
   2. Incubate at room temperature (approximately 23 °C) for 15 min.
   3. To remove excess unreacted mBB, add an equal volume of cold 20% (w/v) TCA solution to tubes A, B, and C.
   4. Centrifuge the tubes at 14,000 x g, 4 °C for 30 min.
   5. Remove the supernatant, leaving the protein pellet intact.
   6. Remove TCA by washing pellet twice each with 500 µl cold acetone and retain the pellet.
7. Separation of the labeled proteins
   1. Suspend the pellets in 10-25 µl SDS-PAGE sample buffer (can be stored at room temperature for 1 week, Recipe 3) and boil for 3 min.
   2. Assemble the Mini-PROTEAN TGX gel in Mini-PROTEAN Tetra system and pour SDS-PAGE buffer (can be stored at room temperature for 1 month, Recipe 4).
   3. Load the boiled samples, equivalent to 20-40 µg of proteins, to the sample well.
      Note: The initial amount of protein per tube is approximately 50 µg. Adjust and determine the loading volume based on some pilot experiments in the laboratory. We recommend setting an experimental replicate for SDS-PAGE with approximately 20 µg protein, for example, a replicate with 10 µl of the solution obtained by resuspending the pellet in 25 µl SDS-PAGE sample buffer. 
   4. Start the electrophoretic run at 100 V and stop after the samples have traversed the desired distance.
8. Protein visualization
   1. Place the gel on UV transilluminator and irradiate the gel.
   2. Use yellow filter to obtain fluorescence derived from mBB-labeled protein.
      Note: mBB has an excitation maximum at 394 nm and an emission maximum at 490 nm.

Data analysis

1. Interpretation of the difference between wild type (Col-0) and mutant is based on densitometric analysis of bands with conventional monochrome photographs or fluorescence images using any conventional software, such as ImageJ (https://imagej.nih.gov/ij//).
2. The representative data indicate comparable band intensity in R-SH staining between Col-0 and the mutant (Figure 2). However, only on the basis of R-SH data, we cannot assess the redox state of thiol-containing proteins. Here, the R-S-S-R data show a slight increase in band intensity in the mutant and the “Total” data show a slight decrease in band intensity (Figure 2), suggesting that the mutant has an oxidizing atmosphere for thiol-containing proteins. The combined interpretation clearly raises the possibility that the mutant has an oxidizing atmosphere in thiol-containing proteins and the redox state may contribute to the decrease in the amount of thiol-containing proteins.
3. Figure 3 exemplifies several different cases to be assumed. Case A indicates comparable band intensities in R-SH staining between Col-0 and the mutant. Case B indicates the R-SH data, showing a decrease in band intensity in the mutant and Case C indicates the R-SH data, showing an increase in band intensity in the mutant.
4. In all these cases, interpretations about the redox state can be changed by the R-S-S-R data and the “Total” data (compare cases A1 to A2, B1 to B2, and C1 to C2).
5. For example, in Case B1, the R-S-S-R data demonstrating an increase in band intensity in the mutant support the interpretation based on the R-SH data, suggesting an oxidizing atmosphere in the mutant. On the other hand, in Case B2, the R-S-S-H data demonstrating a decrease in band intensity in the mutant is contrary to the hypothesis based on the R-SH data. The total (R-SH and R-S-S-R) data represent that the mutant has lesser variety of thiol-containing proteins. The combined interpretation raises the possibility that the mutant has a comparable redox state in thiol-containing proteins as Col-0 and, thereby, the free thiol might decrease in Case B2.
6. Many Arabidopsis mutants have a variety of proteome, resulting from comprehensive proteomic reorganization in response to mutations in various regulatory networks (Figure 3, Case B2 and C1). To minimize possible misinterpretations regarding the redox state of thiol-containing proteins, it is desirable to obtain not only the R-SH data, but also the R-S-S-R and total thiol data.
   
   
   Figure 2. Representative data for monobromobimane (mBB) staining of thiol-containing proteins. Free thiols (R-SH) in proteins were detected by mBB staining without alkylation or reduction (IAA: −, TCEP: −). Disulfide bonds (R-S-S-R) were stained by mBB followed by alkylation and reduction (IAA: +, TCEP: +). Total thiols (R-SH and R-S-S-R) were visualized with mBB only after reduction (IAA: −, TCEP: +). Asterisks indicate different band intensities in Col-0 and the mutant.
   
   
   Figure 3. Representative interpretation for the mutant of interest. Representative images of monobromobimane (mBB) stained gels. In all the cases, the left lane is of wild type (Col-0) and the right lane is of the mutant. R-SH and R-S-S-R indicate free thiols and disulfide bonds, respectively, in the proteins.
   

Recipes

1. Protein extraction buffer
   30 mM Tris/HCl, pH 7.0, with one tablet of cOmplete^TM, Mini Protease Inhibitor Cocktail per 10 ml
2. IAA solution
   0.4 M IAA 
3. SDS-PAGE sample buffer
   50 mM Tris/HCl, pH 7.0
   2% (w/v) SDS
   5% (w/v) glycerol
   0.01% (w/v) BPB
4. SDS-PAGE buffer
   250 mM Tris
   1.92 M glycine
   1% (w/v) SDS
   

Acknowledgments

This protocol was adapted and modified from previous work by Hashida et al. (2018). This work was supported by the JSPS KAKENHI Grant Number 17H05714 to M.K.Y.

Competing interests

The authors declare that there are no conflicts of interest.

References

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