Expression and purification of Lbpro*
Note: This is a modified protocol from Kirchweger et al., 19945.
(A) Protein expression
1. Transform Lbpro or Lbpro* plasmid into BL21(DE3) pLysE or pLysS E. coli cells using standard methods. Plate the cells onto a LB plate (supplement LB plate with 100 µg/ml Ampicillin and 30 µg/ml Chloramphenicol) and incubate the plate overnight at 37 °C. Critical Step: Lbpro* expression in E. coli strains other than pLysE or pLysS may lead to premature lysis and therefore reduced yields.
2. Pick a single colony from the LB plate and inoculate 100 mL of LB medium (supplement LB medium with 100 µg/ml Ampicillin and 30 µg/ml Chloramphenicol).
3. Incubate the culture overnight at 37 °C while shaking at 180 r.p.m.
4. Use the overnight culture to inoculate 900 mL of 2x YT medium (supplement 2x YT medium with 100 µg/ml Ampicillin and 30 µg/ml Chloramphenicol) in a 2 L flask.
5. Incubate the culture at 30 °C while shaking at 150 r.p.m.
6. Once the culture has reached an OD600 between 0.5 and 0.6 immediately cool the culture for 30 min at 18 °C while shaking at 150 r.p.m.
7. Induce expression of Lbpro* with 0.4 mM IPTG (final concentration).
8. Incubate the culture for 16-20 h at 18 °C while shaking at 110 r.p.m.
9. Harvest cells by centrifugation at 5,000 x g for 15 min at 4 °C.
10. Re-suspend cell pellet in 30 mL Buffer A and store at -80 °C. Alternatively, proceed onto the cell lysis procedure.
(B) Cell lysis
1. Add 1 mg/ml lysozyme and 0.1 mg/ml DNAse I to the resuspended cells, mix thoroughly, and incubate the solution on ice for 10 min.
2. Sonicate the resuspended cells on ice (recommended sonication settings: 10 sec burst, 10 sec off, amplitude 60 W for 3 min).
3. Clear the cell lysate by centrifugation at 50,000 x g for 30 min at 4°C.
4. Remove the supernatant and, together with a magnetic stir bar, place into a 100 mL beaker on ice overtop a stir plate at 4 °C.
Ammonium sulfate precipitation
1. With gentle stirring, slowly add the 100% saturated ammonium sulfate solution to the cleared cell lysate to a final concentration of 30% (v/v).
2. Incubate solution for 2 h at 4 °C with gentle stirring.
3. Centrifuge solution at 50,000 x g for 30 min at 4 °C.
4. Keep the supernatant and discard the pellet.
5. Further precipitate proteins from the supernatant, including Lbpro*, by adding the 100% saturated ammonium sulfate solution as before to a final concentration of 60%.
6. Incubate solution for 2 h at 4 °C with gentle stirring.
7. Centrifuge solution at 50,000 x g for 30 min at 4 °C.
8. Discard the supernatant and keep the pellet.
9. Re-suspend the pellet in Buffer A (8 mL of Buffer A per 1 L of cell culture) on a roller mixer.
10. Dialyze the resuspended pellet into 1 L of Buffer A at 4 °C overnight to remove excess ammonium sulfate.
Anion exchange chromatography
1. Prior to loading the sample onto the Resource Q anion exchange column, we recommend further diluting the sample 1:10 in Buffer A.
2. Load the diluted sample onto the Resource Q anion exchange column using either a peristaltic pump, sample pump, or superloop.
3. For the Resource Q run, we recommend the following settings (Fig. 1a):
- Column pre-wash 3 CVs, 0% Buffer B
- Column pre-wash 5 CVs, 100% Buffer B
- Column pre-wash 3 CVs, 0% Buffer B
- Sample application Inject all of the sample
- Column wash 5 CVs 0% Buffer B
- Elution 20 CVs 0-100% Buffer B linear gradient
- Column wash 5 CVs 100% Buffer B
- Column wash 5 CVs, 0% Buffer B
Set the flow rate to 6 ml/min for all steps, except for the sample application step which should be set at a flow rate of 3 ml/min.
Note: The Resource Q column requires regular maintenance with 2M NaCl/NaOH as per manufacturer’s instructions to maintain good resolution.
4. Analyze the ion-exchange run on an SDS-PAGE gel (Fig. 1b). Note: At this point in the purification it is expected to have contaminating proteins in the Lbpro*-containing fractions. However, these will be removed in subsequent steps of the purification.
5. Pool the peak Lbpro*-containing fractions and concentrate to 1.5 mL using an Amicon Ultra-15 centrifugal filter (10K MWCO). Note: Multiple anion exchange runs can be pooled for size exclusion chromatography.
Size exclusion chromatography
1. Run the concentrated sample on a size exclusion column (HiLoad 26/60 Superdex 75 pg) equilibrated in Buffer A (Fig. 1c).
2. Analyze the size exclusion chromatography run on an SDS-PAGE gel (Fig. 1d).
3. Pool the peak Lbpro*-containing fractions and concentrate the sample using an Amicon Ultra-15 centrifugal filter (10K MWCO). Note: Lbpro* can be concentrated to 20-30 mg/ml.
4. Determine the protein concentration, aliquot and flash-freeze the sample in liquid nitrogen, and store at -80 °C. Note: Lbpro* can be stored for several years at -80 °C, but is unstable at 4 °C and should never be stored at this temperature. Purification routinely yields 5-10 mg per L and is sufficient to perform all steps of the protocol below multiple times.
Generation of substrates for Ub-clipping
Ub-clipping can be performed on any ubiquitinated substrate or sample. Here we describe the preparation and Lbpro* digestion of three different ubiquitin samples. This includes: I) total ubiquitin from whole cell lysate, II) purified ubiquitin chains from cells, and III) isolated mitochondria. Together, these protocols will not only facilitate the analysis of these substrates, but also serve as a reference point for Ub-clipping analysis of other ubiquitinated proteins.
I. Ub-clipping on whole cell lysate
(A) Culturing and harvesting cells
1. Culture cells in one 15 cm petri dish containing Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal calf serum and PenStrep (Penicillin and Streptomycin).
2. Grow cells to 90-95% confluence.
3. Place petri dish on ice and remove DMEM medium.
4. Wash cells 2x with 10 mL of ice-cold PBS.
5. Remove excess PBS.
6. Add 500 µL PBS to the petri dish.
7. Scrape off cells and transfer to a pre-chilled 1.5 mL tube.
8. Centrifuge at 300 x g for 2 min at 4 °C.
9. Remove and discard the supernatant.
10. Flash-freeze the cell pellet in liquid nitrogen or proceed with the protein extraction procedure described below.
(B) Protein extraction
1. Resuspend cell pellet in 300 µL of freshly prepared extraction buffer.
2. Sonicate the sample using a microtip (recommended sonication settings: 10 sec burst, 10 sec off, amplitude 5-10 W for 1 min). Note: cells can also be lysed by passing the sample through a 26.5 Gauge needle.
3. Incubate sample on ice for 1 min.
4. Repeat sonication step.
5. Centrifuge cell lysate at 21,000 x g for 10 min at 4 °C.
6. Remove the supernatant and add fresh DTT to a final concentration of 10 mM.
7. Determine the protein concentration with a Bradford assay.
8. Dilute the protein extract 1:4 (vol:vol) with Lbpro* digestion buffer. Note: This will reduce the final concentration of urea to 1 M, a concentration in which Lbpro* is active.
9. Proceed with Lbpro* digestion and purification of total ubiquitin.
(C) Lbpro* digestion and purification of ubiquitin
1. Add Lbpro* to the diluted cell lysate and incubate for 16 h at 37 °C. Note: Overnight digestion with 10 μM Lbpro* is sufficient to collapse the ubiquitin smear (Fig. 2). For WT Lbpro, we recommend using a concentration higher than 10 μM during digestion.
2. Add Lbpro-treated sample to a pre-soaked dialysis tubing or cassette (3.5K MWCO).
3. Dialyze sample in cold Milli-Q water overnight at 4 °C with gentle stirring.
4. Remove sample from dialysis and place into a 1.5 mL tube.
5. Centrifuge sample at 21,000 x g for 10 min at 4 °C.
6. Remove the supernatant and discard the pellet.
7. Run the supernatant on a Superdex 75 Increase 3.2/300 gel filtration column using an ÄKTA Micro system. Note: We recommend connecting a filter immediately before the column. This will help minimize unwanted damage to the column.
8. Identify ubiquitin-containing fractions with anti-ubiquitin Western blots.
9. Pool the ubiquitin-containing fractions and concentrate using an Amicon Ultra-15 centrifugal filter (3.5K MWCO). Note: Minimize contamination of unwanted proteins from the early fractions containing the void elution, as these proteins will interfere with downstream mass spectrometry analysis.
10. Flash-freeze protein in liquid nitrogen or aliquot and lyophilize. Store sample at -80 °C.
II. Purification of ubiquitin chains and digestion with Lbpro*
(A) TUBE pull-down and digestion
Note: The TUBE pull-down procedure is a modified protocol from Hjerpe et al., 20094.
1. Culture and harvest one 10 cm petri dish of cells as described above.
2. Resuspend the cell pellets in 300 µL TUBE lysis buffer.
3. Supplement the lysis buffer with 100 µg of GST 4x ubiquilin 1 TUBE4.
4. Incubate on ice for 20 min.
5. Clear lysate by centrifugation at 21,000 x g for 10 min at 4 °C.
6. Incubate sample with 25 µL pre-washed Glutathione Sepharose 4B resin
on a rotating wheel for 2 h at 4 °C.
7. Centrifuge sample at 300 x g for 2 min at 4 °C and discard supernatant.
8. Wash beads with 500 µL ice-cold PBS + 0.1% Tween20 (v/v).
9. Repeat steps 7-8 two more times.
10. Wash beads with 500 µL PBS.
11. Centrifuge sample at 300 x g for 2 min at 4 °C and discard supernatant.
12. Incubate TUBE-bound ubiquitin chains with 100 µL of 10 µM Lbpro* in TUBE digestion buffer for 16 h at 37°C. Note: This is an on-bead cleavage assay, where Lbpro* clips and releases ubiquitin into supernatant.
13. Centrifuge sample at 300 x g for 2 min at 4 °C.
14. Transfer supernatant to a fresh tube and perform perchloric acid extraction.
(B) Perchloric acid extraction of ubiquitin
1. Slowly add perchloric acid to the ubiquitin sample to a final concentration of 0.5% (v/v), mix thoroughly.
2. Incubate sample for 10 min on ice.
3. Centrifuge sample at 21,000 x g for 10 min at 4 °C. Note: This step retains the ubiquitin species in the supernatant. Discard the pelleted protein precipitate.
4. Place the ubiquitin-containing supernatant in a pre-soaked Slide-A-Lyzer MINI 3.5 K (MWCO).
5. Dialyze sample in cold 50 mM Tris (pH 7.4) for 4 h at 4 °C with gentle stirring.
6. Dialyze sample in cold Milli-Q water overnight at 4 °C with gentle stirring.
7. Lyophilize sample and store at -80 °C.
III. Isolation of damaged mitochondria and digestion with Lbpro*
(A) Mitochondrial damage
1. Culture cells in two 15 cm petri dishes containing Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal calf serum and PenStrep (Penicillin and Streptomycin).
2. Grow cells to 80% confluence.
3. Treat cells with DMEM medium containing OA (10 μM oligomycin and 4 μM Antimycin A) and incubate for 2 h back in the 37 °C incubator.
4. Place petri dish on ice and remove DMEM medium.
5. Wash cells 2x with 10 mL of ice-cold PBS. Note: PBS can be supplemented with 200 mM chloroacetamide to further inhibit deubiquitinase activity.
6. Scrape off cells and transfer to a pre-chilled 1.5 mL tube.
7. Centrifuge at 1,000 x g for 5 min at 4 °C.
8. Remove and discard the supernatant.
9. Wash the cells with PBS and centrifuge again.
10. Remove and discard the supernatant.
11. Store at -80 °C or proceed to the purification of mitochondria.
B) Purification of mitochondria
Note: This protocol was adapted from Lazarou et al., 2007 6. All steps should be performed on ice or at 4 °C.
1. Resuspend cell pellets in 2.5-5 mL of mitochondrial isolation buffer containing 1x cOmplete Protease Inhibitor Cocktail and 1x PhosSTOP Phosphatase Inhibitor Cocktail.
2. Incubate on ice for 30 min.
3. Transfer resuspended cells to a 5 mL dounce homogeniser and homogenise with 25-30 strokes with a drill-fitted pestle. Note: To ensure the cells are properly disrupted without damage to mitochondria, the number of strokes may be varied depending on the torque of the drill.
4. Transfer the sample to a 50 mL tube.
5. Centrifuge at 1,000 x g for 5 min at 4 °C.
6. Carefully split the supernatant across several 1.5 mL tubes.
7. Centrifuge at 10,000 x g for 10 min at 4 °C.
8. Remove the supernatant. Note: Steps 1-3 can be repeated with the 1,000 x g pellet to liberate more mitochondria from unbroken cells.
9. Thoroughly resuspend and pool the 10,000 x g pellets in 1 mL of mitochondrial isolation buffer.
10. Centrifuge at 1,000 x g for 5 min at 4 °C. Note: This spin will remove any contaminating nuclei. Critical Step: Failure to thoroughly resuspend the mitochondria will cause them to be lost at this step.
11. Transfer the supernatant to a fresh 1.5 mL tube.
12. Centrifuge at 10,000 x g for 10 min at 4 °C.
13. Remove the supernatant and resuspend the pellet in 1 mL mitochondrial isolation buffer excluding the inhibitors.
14. Centrifuge at 10,000 x g for 10 min at 4 °C.
15. Remove supernatant.
16. Resuspend the pellet in 1 mL of sucrose storage buffer.
17. Determine the protein concentration using a BCA assay. Note: Samples can be stored at -80 °C until needed.
(C) Sodium carbonate extraction and Lbpro* digestion of purified mitochondria
Note: The sodium carbonate extraction procedure was adapted from Fujiki et al., 1982a & 1982b7,8
1. If frozen, thaw the isolated mitochondria on ice.
2. Aliquot the desired amount of protein in a 1.5 mL tube. Note: The amount of mitochondria can range between 0.2-1 mg per sample and will depend on the downstream application as well as density of ubiquitination.
3. Extract the soluble and peripheral membrane proteins by resuspending the pellet in fresh sodium carbonate solution at a ratio of 1 μL per μg mitochondria.
4. Incubate the sample for 30-45 min on ice with occasional vortexing.
5. Centrifuge at 21,000 x g for 30 min at 4 °C.
6. Discard the supernatant and keep the pellet containing integral membrane proteins. Note: The degree of extraction depends on the pH of the Na2CO3, so this should be measured and kept constant across experiments.
7. Resuspend the pellet in mitochondria Lbpro digestion buffer at a ratio of 1 μL per 10 μg mitochondria.
8. Add an equal volume of mitochondria Lbpro digestion buffer containing 20 μM Lbpro*.
9. Incubate overnight at 37 °C. Critical Step: In addition to helping reduce the catalytic cysteine of Lbpro*, DTT inhibits a mitochondrial carboxypeptidase that cleaves Arg74 of ubiquitin after Lbpro* cleavage.
10. Centrifuge sample at 21,000 x g for 30 min at 4 °C.
11. Remove the supernatant and transfer to a fresh tube.
12. Protein can be immediately flash-frozen in liquid nitrogen or aliquoted and lyophilized. Store sample at -80 °C.
Mass spectrometry & Ub-clipping analysis
Mass spectrometry analysis will largely depend on the available equipment. As a result, in this section of the protocol we have minimized instrument-specific details and provided general guidelines for analysis and data processing of samples. For specific details on reagents and instrumentation, please see the method section of our paper.
(A) Mass spectrometry
1. Resuspend sample in reconstitution buffer. Note: The resuspension volume will depend on the sample and sensitivity of the mass spectrometer.
2. Desalt your sample using C4 reverse phase media before mass spectrometry analysis. Note: This can be performed before or during HPLC runs.
3. Prior to ionization, separate ubiquitin species with an analytical column packed with C4 reverse phase media.
4. Standard acetonitrile gradients can be used to elute ubiquitin from the analytical column.
5. We recommend performing mass analysis at the highest mass resolution setting possible.
(B) Data analysis
1. The analysis of ubiquitin modifications from mass spectrometry data files can be performed by peak integration or spectra deconvolution. Note: In many cases both approaches yield comparable results. However, spectra deconvolution is preferred when quantifying both phosphorylation and ubiquitination sites.
2. For peak integration, quantitation should be performed on the ubiquitin species with the highest intensity (e.g. ubiquitin with a charge state of 12) and the nominal mass. After peak integration, export the area under the curve for each species and quantify their relative abundance.
3. For spectra deconvolution, all spectra corresponding to ubiquitin should be averaged and deconvoluted. After spectra deconvolution, export the intensities of each ubiquitin species and quantify their relative abundance.