Protocol of Preparation of Ubiquitinated Protein Samples
Ubiquitination refers to the process by which one or more ubiquitin molecules are covalently bound to other protein molecules by a series of enzymes such as ubiquitin activating enzyme (El), ubiquitin binding enzyme (E2), and ubiquitin ligase (E3). Proteins with polyubiquitin are recognized and degraded by proteasomes containing a variety of proteases, thus affecting important physiological processes such as biological signaling pathways, protein activity and their intracellular localization. From yeast to mammals, ubiquitination is a very important regulatory mechanism in the regulation of cellular protein degradation and function. Enrichment of ubiquitinated proteins is mainly based on labeling methods, which can be performed by affinity labeling of ubiquitin (commonly 6×His), combined with nickel chelate chromatography affinity or combined with SCX, RP-LC, and also dual affinity strategies to enrich ubiquitinated peptides/proteins.
Enrichment methods for ubiquitination-modified proteins
Enrichment of ubiquitinated proteins is mainly based on labeling method, i.e., affinity labeling of ubiquitin (commonly 6×His), followed by affinity extraction of ubiquitinated proteins using nickel chelate chromatography. The principle is that since the C-terminus of ubiquitin is an Arg-Gly-Gly structure, Gly-Gly stays on the peptide chain of the modified protein after trypsin hydrolysis, which increases the peptide mass by 114 and serves as a mass tagging ubiquitination site, and then the ubiquitination site is identified by tandem mass spectrometry.
To obtain more accurate ubiquitinated proteins, a strategy combining multiple protein enrichment methods, such as affinity tagging combined with SCX and RP-LC to separate ubiquitinated peptides, can also be used. This approach has been extended to ubiquitination studies in mammalian systems.
Dual affinity strategy enrichment methods have also been applied to the enrichment of ubiquitinated proteins. A polyubiquitin-binding column was combined with nickel chelate chromatography to label the ubiquitin moiety with a 6-histidine tag and a biotin tag, and then the ubiquitinated proteins were extracted using nickel integrated chromatography and streptavidin antibiotin protein affinity in sequence, while a two-step enzymatic digestion (Lys-C Trypsin) was used to elute and enzymatically digest the streptavidin antibiotin protein from the column for the tightly folded nature of the enrichment method. The ubiquitinated proteins were separated by SCX and RP-LC chromatography in combination.
1. Main Instruments and Equipment
High-speed centrifuge, ultracentrifuge, ultrasonic crusher, vertical shaker, probe micro-ultrasonic crusher, metal baths.
2. Experimental Materials
Protein sample of interest, usually a mixture of multiple proteins that have not been isolated.
3. Main reagents
(1) Some reagents refer to Ubiquitin Enrichment Kit (PIERCE Inc. products).
(2) Polyubiquitin affinity resin (PIERCE), Ni2+-NTA-agarose beads (Qiagen, Clon-tech), disposable columns (Bio-Rad poly-prep)*1*2, acid-washed glass beads (Sigma).
(3) Tris, TBS, PBS, 1 mol/L imidazole, 10% (V/V) trichloroacetic acid.
(4) Ammonium bicarbonate buffer NH4HCO3 (pH 7. 8).
(5) Washing buffer is a mixture of lysate and TBS, volume ratio 1:9.
(6) Guanidine hydrochloride wash buffer (add imidazole*3 according to the experiment)
| 6 mol/L | Guanidine hydrochloride |
| 50 mmol/L | Sodium phosphate buffer (pH 8.0) |
| 10 mmol/L | Tris-Cl (pH 8.0) |
| 300 mmol/L | NaCl |
| 5 mmol/L | N-Ethylmaleimide (NEM) protease inhibitor |
(7) Guanidine hydrochloride lysis solution
| 6 mol/L | Guanidine hydrochloride |
| 100 mmol/L | Sodium phosphate buffer (pH 8.0) |
| 5 mmol/L | Imidazole |
(8) Protease inhibitors
In the process of tissue cell fragmentation, the addition of diisopropyl fluorophosphate (DFP) can inhibit or slow down autolysis. The addition of iodoacetic acid can inhibit the activity of proteolytic enzymes that require sulfhydryl groups in active centers. The activity of proteolytic enzyme can also be eliminated by adding benzene sulfonyl fluoride (PMSF), which can be added according to the actual conditions in the experiment (Table 1-3-6). Use protease inhibitor complex tablets (Roche molecular Biochernicals), 5 mg/mL peptide inhibitor*4 prepared with 10 g/mL of DMSO as solvent.
Table 1-3-6 Broad-spectrum Protease Inhibitor Mixture
| Component | Final concentration |
|---|---|
| PMSF | 35 μg/mL (1 mmol/L) |
| EDTA | 0.3 mg/mL (1 mmol/L) |
| Pepstatin | 0.7 μg/mL |
| Leupeptin | 0.5 μg/mL |
Protein buffer (add imidazole*5 according to the experiment)
| 50 mmol/L | Sodium phosphate buffer (pH 8.0) |
| 100 mmol/L | KCl |
| 20 % (volume fraction) | Glycerin |
| 0.2 % (volume fraction) | NP-40 |
SDS buffer*6
| 1 % | SDS |
| 45 mmol/L | HEPES (sodium salt) (pH7.5) |
SDS-PAGE loading buffer*7
| 20 % (volume fraction) | Glycerin |
| 4 % | SDS |
| 0.125 mol/L | Tris-Cl (pH 6.8) |
| 0.2 mol/L | DTT |
| 0.01 % | Bromophenol Blue |
Urea sample buffer*8
| 8 mol/L | Urea |
| 4 % | SDS |
| 0.125 mol/L | Tris-Cl (pH 6.8) |
| 0.2 mol/L | DTT |
| 0.01 % | Bromophenol Blue |
Urea washing buffer*9
| 8 mol/L | Urea |
| 50 mmol/L | Sodium phosphate buffer (pH 8.0) |
| 10 mmol/L | Tris-Cl (pH 8.0) |
| 300 mmol/L | NaCl |
| 5 mmol/L | N-Ethylmaleimide (NEM) protease inhibitor |
Urea wash buffer (pH 6.0): 50 mmol/L sodium phosphate buffer (pH 6.0), other components as urea wash buffer (pH 8.0).
Method 1. Enrichment of Polyubiquitinated Proteins (refer to Ubiquitin Enrichment Kit)*10
(1) Mix gently several times to obtain a homogeneous suspension.
(2) Add samples to the centrifuge column and add 20 μL of suspension resin to each sample using a wide-mouth or cut-over tip.
(3) Cover the centrifuge column tightly and incubate on a vertical shaker at 4°C for 2h or overnight*11.
(4) Remove the centrifuge column containing the protein samples and affinity resin into a 1.5 mL centrifuge tube and centrifuge at 5000g for 15s.
(5) Remove the centrifuge column from the centrifuge tube.
(6) Put the bottom cap on the centrifuge column and remove the top cap. Add 300 μL of washing buffer to the column, close the top cap, and turn the column over several times. Remove the bottom cap, place the column into a new tube and centrifuge at 5000g for 15s.
(7) Repeat (6) twice, and collect the washing solution from each time in separate tubes.
(8) Put the bottom cap on the centrifuge column and add 50 - 75 μL of SDS-PAGE loading buffer or IEF sample buffer.
(9) Cap the top cap tightly and vortex shake for 2-3s.
(10) Place the capped column into the centrifuge tube and loosen the top cap. Warm the sample in a metal bath or water bath according to the operation manual.
(11) Remove the bottom cap and centrifuge the column and tube at 5000 g for 15 s. The eluate collected is the enriched ubiquitinated fraction. The fractions collected by this method are suitable for western blot or isoelectric focusing.
Method 2. Affinity Purification of Ubiquitinated Proteins from Cells Expressing His6-Ub (Mammalian Cells)
(1) Collect cultured cells, one set of mammalian cells expressing the protein of interest and His6-Ub*12, and the other set of control cells*13.
(2) Cells were lysed in 2 mL of guanidine hydrochloride lysis solution. The lysate is collected, and to reduce the viscosity of the lysate, light sonication can be performed using a probe micro-ultrasonic disruptor*14.
(3) Centrifuge at 14000g for 15 min at 4°C. Clarified extracts were mixed with 75 μL Ni2+ -NTA-agarose and incubated for 4h at 4°C on a vertical shaker.
(4) The mixture was added to a disposable column (e.g., Bio-Rad poly-prep) and washed in the following sequence of procedures.
1 mL 6 mol/L guanidine hydrochloride/100 mmol/L sodium phosphate buffer (pH 8.0), without imidazole.
2 mL 6 mol/L guanidine hydrochloride/100 mmol/L sodium phosphate buffer (pH 5.8).
1mL 6mol/L guanidine hydrochloride/100mmol/L sodium phosphate buffer (pH 8.0) without imidazole.
2mL volume ratio 1:1 of 6mol/L guanidine hydrochloride/100mmol/L sodium phosphate buffer (pH 8.0) and protein buffer without imidazole.
2mL 6mol/L guanidine hydrochloride/100mmol/L sodium phosphate buffer (1:3 v/v) (pH 8.0) and protein buffer without imidazole.
2mL protein buffer without imidazole.
1mL of protein buffer containing 10mmol/L imidazole.
(5) Elution of bound protein with 1 mL of protein buffer containing 200 mmol/L imidazole.
(6) Precipitate the eluate with 10% (v/v) TCA, then resuspend the precipitate with an equal volume of 2× SDS-PAGE loading buffer and treat in a boiling water bath for 5 min.
(7) Samples prepared in parallel can be separated and analyzed on the same SDS-PAGE, or antibodies to the target protein can be used for direct immunohybridization, and mass spectrometry can also be performed to identify ubiquitination sites*15.
Method 3. Affinity Purification of Ubiquitinated Proteins from Cells Expressing His6-Ub (Yeast Cells)
(1) Cultured cells were collected, one group of yeast cells with His6-Ub*15 and the other group of control cells. The cells were lysed by adding 425-600 μm acid-washed glass beads per 300 μL of collection by vortexing with imidazole-free guanidine hydrochloride washing solution for 5 min.
(2) Centrifuge at 14,000g for 15min at 4°C. Clarify the total protein contained in the extract by mixing each 2.75mg with 50μL Ni2+-NTA-agarose and adding 1mol/L imidazole to a final concentration of 10mmol/L. Incubate for 4h at 4°C on a horizontal shaker.
(3) Wash the magnetic beads sequentially according to the following procedure.
1mL of guanidine hydrochloride wash solution containing 20mmol/L imidazole.
1mL of urea wash buffer (pH 8.0) containing 20mmol/L imidazole.
1mL urea wash buffer (pH 6.0) containing 20mmol/L imidazole.
1mL of urea wash buffer (pH 6.0) containing 20mmol/L imidazole*17.
(4) Boiling in urea sample buffer to elute the bound proteins from the beads. Samples prepared in parallel can be separated and analyzed on the same SDS-PAGE, or antibodies to the target protein can be used for direct immunohybridization.
*1 The maximum volume that can be accommodated in the centrifuge column is 500 μL. If the sample volume exceeds 500 μL, the resin is incubated in a new tube and transferred to the centrifuge column.
*2 For optimal enrichment, use a minimum of 0.1 mg of total protein sample. The sample added in each centrifugation column needs to be diluted with TBS in equal volume. The total volume of the sample needs to be ≥ 200 μL. Samples should be diluted between 1 and 10 times.
*3 The solution can be stored at 4°C for 6 months without the addition of NEM or protease inhibitors.
*4 Needs to be freshly prepared on the same day.
*5 Can be stored at 4°C for 6 months.
*6 Can be stored at room temperature for 12 months.
*7 Can be stored at -20°C for 6 months.
*8 Can be stored at -20°C for 6 months.
*9 Protease inhibitors need to be freshly prepared on the same day with storage solution.
*10 Positive and negative controls are required during the enrichment of ubiquitinated proteins.
*11 The incubation time ensures that the protein samples are fully bound to the affinity resin.
*12 Protein expression can be done by transient transfection of appropriate expression vectors.
*13 Cells in which the protein of interest is not expressed are used as control experiments, and the procedure needs to be performed in parallel. Untagged proteins are commonly used as affinity-tagged substrates. Cells lacking His 6-tagged Ub cells also need to be assayed in parallel.
*14 Ultrasonication, taking care not to stir vigorously to prevent foam generation.
*15 Since the C-terminus of ubiquitin is an Arg-Gly-Gly structure, Gly-Gly remains on the peptide chain of the modified protein after trypsin hydrolysis, increasing the peptide mass by 114, which serves as a mass marker for ubiquitination sites, which in turn are identified by tandem mass spectrometry.
*16 Protein expression can be achieved by transient transfection of the appropriate expression vector.
*17 Washing the beads with a urea wash rigorously removes guanidine, which precipitates when SDS is added.
