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. Author manuscript; available in PMC: 2024 Jan 1.
Published in final edited form as: Methods Mol Biol. 2023;2591:219–236. doi: 10.1007/978-1-0716-2803-4_13

Identification of Deubiquitinase Substrates in Xenopus Egg Extract

Valentina Rossio 1, Joao A Paulo 1, Randall W King 1,*
PMCID: PMC9662951  NIHMSID: NIHMS1784093  PMID: 36350551

Abstract

Deubiquitinases (DUBs) antagonize protein ubiquitination by removing ubiquitin from substrates. Identifying the physiological substrates of each DUB is critical for understanding DUB function and the principles that govern the specificity of this class of enzymes. Since multiple DUBs can act on the same substrate, it can be challenging to identify substrates using inactivating a single enzyme. Here, we outline a method that enables the identification of proteins whose stability depends on DUB activity and an approach to profile DUBs specificity in Xenopus egg extract. By coupling broad DUB inhibition with quantitative proteomics, we circumvent DUB redundancy to identify DUB substrates. By adding back recombinant DUBs individually to the extract, we pinpoint DUBs sufficient to counteract proteasomal degradation of these newly identified substrates. We apply this method to Xenopus egg extract but suggest that it can also be adapted to other cell lysates.

Keywords: Deubiquitinases, substrates, UPS, TMT-proteomics, ubiquitin

1. Introduction

Reversible post-translational modifications of proteins regulate virtually all essential functions of the cell. An example is a covalent attachment of ubiquitin, mediated by a cascade of enzymes in the ubiquitin-proteasome system (UPS), in which ubiquitin is initially activated by E1 and then attached to substrates by specific E2 and E3 enzymes [1, 2]. Ubiquitination targets some proteins to proteasomal degradation but can also regulate protein function independently of degradation [1, 3]. Deubiquitinases (DUBs) catalyze ubiquitin removal from proteins and process ubiquitin precursors, ensuring the availability of a free ubiquitin pool [4, 5].

DUBs comprise a large group of enzymes that fall into two main families: the zinc metalloproteases and the cysteine-proteases [6, 7]. Although a number of DUBs have been well characterized, most DUBs still do not have any known substrates, making it difficult to understand their functional roles or the molecular basis of substrate specificity. A major challenge in identifying DUB substrates is that multiple DUBs may function redundantly on the same substrates [8, 9]. Thus, the inactivation of a single DUB may not be sufficient to produce changes in substrate ubiquitination or stability.

Here, we describe a method that circumvents DUB redundancy to enable substrate identification in Xenopus egg extract. By chemically inhibiting many cysteine-protease DUBs simultaneously, with the highly specific DUB inhibitor ubiquitin vinyl sulfone (UbVS) [10], we applied tandem mass tag (TMT)-based quantitative proteomics to identify proteins whose stability depends on DUB activity. By restoring the activity of specific DUBs in UbVS-treated extract, we illustrate how these substrates can be used to define enzyme-substrate relationships, overcoming hurdles associated with redundant enzyme function.

2. Materials

2.1. Xenopus egg extract preparation

  1. Extract buffer (XB): 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM potassium HEPES (pH 7.7 with KOH), 50 mM sucrose

  2. MMR (1X): 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES (pH 7.8 with NaOH)

  3. Energy mix (20X): 150 mM creatine phosphate, 20 mM ATP, 2 mM EGTA, 20 mM MgCl2 (pH 7.7)

  4. Dejellying solution (prepare fresh within one hour of use): dissolve 5% w/v cysteine HCl in water (pH 7.8 with NaOH)

  5. Protease inhibitors (1000X): mixture of leupeptin, chymostatin, pepstatin (final concentration of 10 mg/ml each) in DMSO

  6. Cytochalasin B (1000X): 10 mg/ml in DMSO

  7. Calcium ionophore A23187 (5000X): 10 mg/ml in DMSO

  8. Cycloheximide (100X): 10 mg/ml in water

  9. Liquid nitrogen

  10. Equipment: Sorvall centrifuge with HB-6 rotor, vortex, pH meter

2.2. Reagents for extract treatment

  1. UbVS (stock concentration can range from 150–250 mM) in 50 mM MES, pH 6

  2. MES buffer: 50 mM MES, pH 6

  3. Recombinant human ubiquitin, dissolved to 1–1.5 mM in XB buffer

  4. TnT-quick coupled transcription/translation system, rabbit reticulocyte lysate (Promega)

  5. Standard PCR reagents and PCR purification kit

  6. 35S-methionine 500 mCi, stabilized aqueous solution (PerkinElmer)

  7. Human recombinant DUBs

  8. Equipment: thermomixer, nanodrop, PCR thermocycler

2.3. SDS-polyacrylamide gel electrophoresis and immunoblotting

  1. MES SDS running buffer (20X): 50 mM MES, 50 mM Tris base, 0.1% SDS, 1 mM EDTA (pH 7.3)

  2. Tris-acetate SDS-running buffer (20X): 50 mM Tricine, 50 mM Tris base, 0.1% SDS (pH 8.24)

  3. 4X LDS sample buffer: 988 mM Tris, 2.04 mM EDTA, 8% LDS, 40% glycerol, 0.88% Comassie Brilliant Blue G250, 0.7 mM Phenol red

  4. Polyacrylamide protein gels 4–12%, 3–8%, and PVDF transfer membrane (0.45 micron pore size)

  5. Transfer buffer: 25 mM Tris base, 192 mM glycine, 20% methanol

  6. Tris-buffered saline (TBS-T; 10X): 1.5 M NaCl, 500 mM Tris–HCl, pH 7.5, 1% Tween

  7. Blocking solution: 5% dry milk in TBS-T

  8. Primary antibody: anti-Ube2C antibodies (R & D systems)

  9. Secondary antibody: anti-rabbit IgG conjugated with horseradish peroxidase (HRP)

  10. SuperSignal West Femto Sensitivity Substrate

  11. Equipment: heat block, Novex mini-cell gel running apparatus, power supply, Mini PROTEAN TetraCell transfer apparatus, Amersham Imager 600 (Amersham)

2.4. Sample preparation for TMT-proteomic analysis

All the following reagents used to prepare proteomic samples should be prepared with HPLC grade solvents.

  1. Pierce BCA protein assay

  2. Quenching buffer: 50 mM Tris pH 8.8, 1% SDS, 5 mM DTT, protease inhibitor tablet

  3. 200 mM 3-[4-(2-Hydroxyethyl)-1-piperazinyl] propanesulfonic acid (EPPS or HEPPS) buffer, pH 8.5 (NaOH)

  4. Lysyl endopeptidase (LysC), trypsin, TMT-10plex Isobaric Label Reagent Set (ThermoFisher)

  5. C18-based SepPak prepacked Cartridge 100 mg (Waters)

  6. SepPak washing buffer: 5% acetonitrile (ACN), 1% trifluoroacetic acid (TFA)

  7. SepPak elution buffer: 70% ACN, 1% TFA

  8. Peptide resuspension buffer (prior to HPLC fractionation): 10 mM ammonium bicarbonate, 5% ACN, pH 8

  9. 300 Extend C18 column, 2.1 × 150 mm, 3.5 micron for peptide fractionation (Agilent)

  10. C18 solid phase extraction disks (SPE) for creating homemade StageTips or commercially available StageTips

  11. StageTip washing buffer: 5% ACN, 1% formic acid

  12. StageTip elution buffer: 70% ACN, 1% formic acid

  13. Resuspension buffer: 5% ACN, 5% formic acid

  14. Equipment: speed vacuum concentrator, benchtop centrifuge, vortex, Agilent 1260 HPLC system, Orbitrap Fusion mass spectrometer coupled to a Proxeon EASY-nLC 1000 liquid chromatography (LC) (ThermoFisher)

2.5. Coomassie staining and drying of SDS-polyacrylamide protein gels

  1. Coomassie blue solution: 0.2% Coomassie blue, 7.5 % acetic acid, 50% methanol

  2. Destaining solution: 40% methanol, 10% acetic acid

  3. Drying gel solution: 50% methanol, 10% acetic acid

  4. Phosphor screen (Fuji)

  5. Equipment: gel dryer (Bio-Rad), Typhoon Biomolecular Imager (Amersham)

3. Methods

3.1. Preparation of interphase Xenopus egg extract

Interphase Xenopus egg extract is prepared based on Murray’s protocol [11] and further adapted with a few modifications from Salic and King [12]. Extracts are prepared from eggs laid overnight by frogs placed separately in each container with 2 L of MMR solution (18°C). Each single batch of eggs is examined and if they contain a considerable number of abnormal eggs (such as white color, abnormal morphology, or connected) they are omitted. All the other eggs are pooled together and processed to prepare the egg extract. All the steps are carried out at room temperature, if not otherwise indicated.

  1. Wash the collected eggs in three volumes of MMR (prechilled to 16°C) and allow them to settle. Repeat two additional times with fresh MMR.

  2. Incubate the eggs with three volumes of 5% cysteine to remove the egg jelly coat (dejellying). This procedure is complete when all the eggs are tightly packed together (5–10 minutes).

  3. Wash the eggs gently with MMR, as you did in step 1, until the buffer is transparent (typically 3–5 times).

  4. Activate the eggs with two volumes of MMR containing 2 μg/mL calcium ionophore to drive the eggs into interphase. Incubate until the pigmented area of the eggs contracts, indicating that egg activation has occurred, typically about 5 minutes.

  5. Wash eggs three times with two volumes of Extract Buffer (XB) (prechilled at 16°C). Wash one last time with one volume of XB containing 10 μg/ml protease inhibitors. Incubate eggs in this solution for 20 minutes. Remove the XB.

  6. Carefully pour the eggs into a centrifuge tube (see Note 1) containing a small volume of XB with 100 μg/ml cytochalasin B and 10 mg/ml protease inhibitors (see Note 2). Cytochalasin B addition is important to prevent actin polymerization. For a 50 ml centrifuge tube, we use 2 ml of XB buffer. Spin tubes at low speed (Sorvall HB-6 rotor at 860 rpm (127 g) at 4°C) for 1 min to pack the eggs and aspirate excess buffer (see Note 3).

  7. Crush the eggs by spinning for 15 min at 4°C in an HB-6 rotor at 12,000 rpm (23,000 g). The cytoplasmic layer (the middle tan layer between the yellow lipid layer on the top and the dark yolk at the bottom) is removed by needle puncture (see Note 4) and collected in a clean falcon tube placed in ice.

  8. Add to the extract cytochalasin B and protease inhibitors at final concentrations of 10 μg/ml and cycloheximide at 100 μg/ml. Mix well by pipetting. Repeat step 7 once again.

  9. Supplement the extracts with 1X energy mix and 4% glycerol. Mix well by pipetting to dissolve the glycerol (see Note 5). The final extract should be clear and of a tan-yellowish color. Aliquot the extract, snap freeze in liquid nitrogen, and store at −80°C (see Note 6).

3.2. Evaluation of egg extract quality

After extract preparation, we suggest verifying the activity of the extract by checking the degradation of known proteasome substrates. Typically, degradation of the known APC substrates, Cyclin B1 or Securin, is measured after driving the extract into mitosis by adding non-degradable Cyclin B1 [13].

3.3. Monitoring level of ubiquitin depletion by analysis of E2 charging

Since protein synthesis is inhibited in the extract by cycloheximide, the only source of free ubiquitin is generated by the action of DUBs on their substrates. Inhibiting multiple DUBs simultaneously with UbVS depletes free monomeric ubiquitin [13, 14]. Indeed, treatment of extract with UbVS must be coupled with the addition of exogenous ubiquitin to the extract to sustain UPS activity. As a readout of ubiquitin availability, the charging status of the E2s enzymes is monitored by using non-reducing gels to access the fraction of Ube2C that is present in the form of a ubiquitin thioester (charged Ube2C). Ube2C is fully charged in unperturbed extracts but is rapidly discharged in UbVS-treated extract in a manner that can be fully rescued with exogenous ubiquitin [13, 14]. Therefore, monitoring Ube2C charging is a convenient measure of functional ubiquitin availability in the extract (see Note 7). We observed that 10 μM UbVS is sufficient to inhibit multiple DUBs (n=35) and rapidly deplete free ubiquitin [14]. At this concentration of UbVS, ubiquitin depletion can be fully restored by the addition of 50 μM exogenous ubiquitin [14]. Although higher UbVS concentrations may inhibit more DUBs, we have found that it is also more challenging to sustain Ube2C charging with exogenously added ubiquitin. We observed that 50 μM ubiquitin is not sufficient to restore ubiquitin availability in extract treated with 30 μM UbVS [14]. Since ubiquitin dynamics can vary in different batches of extract, we suggest determining the UbVS concentration sufficient to induce ubiquitin depletion while allowing for the rescue of ubiquitin availability after adding exogenous ubiquitin.

  1. Thaw the Xenopus egg extract rapidly in hand and then incubate on ice.

  2. Split the extract into four tubes (see Note 8). Add to each tube MES buffer (control) or UbVS at the final concentrations of 5, 10 and 20 μM, respectively (see Note 9 and Note 10). Mix by pipetting gently multiple times to be sure that the UbVS is well mixed. Collect time 0 from all the conditions by pipetting 1 mL of extract into 10 mL of non-reducing LDS sample buffer and briefly vortex it. Keep the sample on ice until the end of the experiment. Incubate the control- and UBVS-treated extracts at 24°C with gentle shaking in a thermomixer (1250 rpm) for 10 minutes. After 10 minutes, collect samples from the different conditions as described for the time 0 sample.

  3. Perform non-reducing SDS-PAGE electrophoresis (see Note 11) followed by immunoblotting with antibodies against Ube2C (see Note 12). After 10 minutes, 10 μM UbVS should be sufficient to induce rapid ubiquitin depletion, visualized as a complete loss of Ube2C-thioester.

  4. Next, check if ubiquitin availability can be rescued in UbVS-treated extract by adding exogenous ubiquitin to the extract. Thaw an aliquot of the Xenopus egg extract, as before. Treat the extract with 10 μM UbVS for 10 minutes, as previously. Split the extract into four clean tubes. Add XB buffer (control) or human recombinant ubiquitin to each tube at the final concentrations of 10, 20, and 50 μM, respectively. Mix by pipetting a couple of times gently. Collect time 0 and treat it as in step 2. Incubate at 24°C with gentle shaking, as before. Collect time points at 5, 30, and 60 minutes and quench them with non-reducing LDS sample buffer.

  5. Analyze samples by non-reducing SDS PAGE and immunoblotting with Ube2C antibodies.

  6. In our experimental conditions, 50 μM ubiquitin is typically sufficient to sustain Ube2C charging for at least one hour in extract pre-treated with 10 μM UbVS (see Note 13).

3.4. Identification of proteins protected from degradation by DUBs with TMT-proteomics

To identify proteins protected from degradation by DUBs, TMT-based quantitative proteomics can be used [15] to compare protein levels in different conditions in a quantitative manner. Proteins that decrease specifically in the presence of UbVS or UbVS/ubiquitin but are stable in extract treated with XB buffer or ubiquitin represent potential DUB substrates. In this experiment, E2 charging is monitored in parallel with performing the proteomic analysis in the same experiment as a control for DUB inhibition by UbVS and restoration of ubiquitin charging by exogenous ubiquitin. At each time point of the experiment described below, you will collect samples for proteomic analysis (see Note 14) and one sample for analysis by non-reducing SDS-PAGE, followed by immunoblotting to measure E2 charging. The logic and expected findings from this experiment are illustrated in Figure 1.

  1. Determine the protein concentration of the extract. We use a BCA assay kit according to the manufacturer’s instructions. Each sample collected and processed for TMT-proteomics should contain at least 100 μg of protein. The Xenopus egg extract that we prepare typically has a concentration of ~50 mg/mL, and thus we usually use 2 μL of extract for each sample.

  2. Depending on the number of samples you want to collect, calculate the amount of extract required, based on 2 μL per sample, and add a further 5–10 μL. Distribute this volume of extract to each of two tubes. Treat one sample with UbVS to achieve a final concentration of 10 μM UbVS and the other with an equivalent volume of MES buffer (control). Mix by pipetting gently multiple times to ensure that the UbVS is well mixed.

  3. Incubate samples at 24°C with gentle shaking (1,250 rpm).

  4. After 10 minutes, split both samples in half. For each pair of tubes, add ubiquitin to achieve a final concentration of 50 μM to one tube and add an equivalent volume of XB buffer to the other. Mix by pipetting gently multiple times.

  5. Collect 2 μL for proteomic analysis as time 0 from the extract treated with only XB buffer (control) by pipetting it in a clean 1.5 mL tube and adding 100 μL of quenching buffer for mass spectrometry. After buffer addition, mix by pipetting, vortex for at least 30 seconds, and flash freeze the samples and store at −80°C. Samples can be stored for years. In parallel, collect time 0 from all the conditions by pipetting 1 μL of extract into 10 μL of non-reducing LDS sample buffer and vortex briefly.

  6. Incubate the remaining extract at 24°C (1250 rpm) for one hour. Collect samples for proteomic analysis and E2 charging as described above.

  7. Before treating the samples for mass spectrometry analysis, analyze the samples by non-reducing SDS-PAGE and immunoblotting for Ube2C. UbVS treatment should induce ubiquitin depletion visualized as the discharge of Ube2C. The addition of ubiquitin should be sufficient to re-charge Ube2C for the experiment duration.

Figure 1. Identifying proteins protected from degradation by DUBs with TMT-proteomics.

Figure 1.

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(A) Top: Experimental scheme. Treat the extract with 10 μM UbVS or buffer for 10 minutes. After 10 minutes, split both samples in two and add 50 μM ubiquitin to one sample and buffer to the other. Collect samples for proteomic analysis at time 0 from extract treated with only buffer. Collect time 0 from all the conditions for Ube2C immunoblot analysis and quench with non-reducing LDS sample buffer. Incubate the remaining extract at 24°C and collect samples for proteomic analysis after one hour and samples for E2 charging analysis after 30 minutes and one hour. Bottom: Immunoblot analysis of Ube2C is shown. (B) Summary of treatment and analysis of the samples for TMT-mass spectrometry. R.A.: relative amount. The graph represents the expected behavior of potential DUB substrates, whose abundance should decrease specifically in the presence of UbVS/ubiquitin but remain stable in all the other conditions.

3.5. Processing samples for TMT-proteomic analysis

3.5.1. Methanol-chloroform precipitation, protein digestion, and TMT isobaric labeling

  1. Thaw frozen samples rapidly. Add 400 μL of 100% methanol to each sample and vortex for 5–10 seconds. Add 100 μL of 100% chloroform and vortex for 5–10 seconds. Lastly, add 300 μL of water and vortex for 5–10 seconds (see Note 15).

  2. Centrifuge the samples for 5 minutes at top speed (23,000g). After centrifugation, you will see the formation of 2 layers (one aqueous and one organic). A thin white disk will form at the interface between the two layers, the proteins. Aspirate both layers using a P1000 pipette, starting from the top layer, leaving the disk intact (see Note 16).

  3. Wash the protein pellet twice by adding 800 μL methanol. Vortex briefly and centrifuge at 23,000g for 2 minutes. Remove the supernatant without drying the pellet completely (usually, around 10 μL of methanol remain).

  4. Resuspend the samples in 100 μL of 200 mM EPPS (pH 8.5). Add LysC at a 1:50 enzyme/protein ratio and incubate samples overnight at room temperature with gentle agitation (see Note 17). Add trypsin at 1:100 protease/protein ratio and digest the samples for 6 hours at 37°C (see Note 18).

  5. Halt digestion by adding 25 μL anhydrous acetonitrile (ACN) to each sample.

  6. Add 10 μL of TMT labeling reagent (the stock is at 20 μg/μL) to the digested peptides. Mix with a gentle vortex (2 sec). Incubate for 1 hour at room temperature, briefly vortexing the samples every 10 minutes.

3.5.2. Quality control analysis

In TMT-based quantitative proteomics, each sample is separately labeled with a different isobaric TMT tag. The samples are subsequently combined, fractionated, and analyzed by mass spectrometry to identify and quantify each sample’s relative abundance of peptides. Prior to proceeding with the final pooling of the samples and their fractionation, it is important to perform a quality control step in which a small amount of each sample is pooled together and analyzed by mass spectrometry to confirm that peptide digestion is successful, whether each of the labeled samples contains roughly the same amount of total peptides and if the degree of labeling is sufficient [16]. The labeling efficiency should be greater than 95% before the analysis. If the labeling efficiency is <95%, we suggest repeating the labeling (step 6 of section 3.4.1). For example, if you have a labeling efficiency of 80%, we suggest adding 20% more labels and incubating for another hour. We suggest repeating the label check after adding more label. If the labeling efficiency is sufficient, we pool the final sample. We sum the TMT signal for each channel for all peptides and adjust the amount to mix for each sample using the sample with the lowest TMT signal as the baseline. Samples should vary by less than 1.5-fold. If the ratio between samples is greater than 1.5-fold, it is better to perform the experiment again and collect new samples. Some possible causes of large disparaties include inaccuracy in concentration estimate with the BCA assay, improper digestion conditions (e.g., pH or protein:protease ratio), and/or inconsistent protein precipitation.

  1. Pool together 2 μL of each labeled sample and add 100 μL of 1% formic acid to the combined samples. Freeze the remaining samples for analysis without pooling them by placing at −80°C. These samples will be processed following the quality control analysis described in this section.

  2. Desalt and clean up the combined sample by solid-phase extraction (SPE), using commercially available StageTips (see materials) or a homemade equivalent. C18 disks 0.4 mm in diameter and 0.5 mm in length have a binding capacity of 2–4 μg of digested proteins [17]. We use the equivalent of six disks per StageTip. StageTips are made manually by inserting these small disks of C18 beads embedded in a soft mesh of PTFE (polytetrafluoroethylene) into P200 pipette tips [17].

  3. Place a StageTip adaptor in a 2 mL microcentrifuge tube and insert the StageTip in the adaptor. Add 100 μL methanol, centrifuge for 3 minutes at 1,500g, and discard the eluate. The time is an estimate as it is important that all of the added buffer flows out of the tip before the new buffer is added and that the tip does not dry entirely from excessive centrifugation time.

  4. Add 100 μL of 1% formic acid and 70% ACN, centrifuge as before, and discard the eluate.

  5. Equilibrate the StageTip with 100 μL of 1% formic acid and 5% ACN, centrifuge, and discard the eluate.

  6. Load the sample from Step 1 on the StageTip, centrifuge, and discard the eluate.

  7. Wash StageTip with 100 μL of 1% formic acid and 5% ACN, centrifuge, and discard the eluate.

  8. Elute with 100 μL of 1% formic acid and 70% ACN, centrifuge, and dry the sample by vacuum centrifugation.

  9. Add 100 μL of 5% ACN and 5% formic acid. The sample is now ready for mass spectrometry analysis.

  10. Perform the ratio and label checks as described [16].

3.5.3. Peptide desalting and fractionation via basic-pH HPLC

  1. If the results show an acceptable ratio and the sample is adequately labeled, thaw the labeled samples from Step 1 on ice. Add hydroxylamine at a final concentration of ~0.3% and incubate for 15 minutes at room temperature to quench the labeling reaction (see Note 19).

  2. Combine the samples based on the results of the ratio check. If all the samples contain the same amounts of peptides, you can combine them 1:1. If there are slight differences among the samples (less than 1.5 fold), adjust the relative amounts. Freeze the sample in dry ice and evaporate all the ACN by vacuum centrifugation (see Note 20).

  3. To clean up and desalt the sample, use one C18-based SepPak cartridge under vacuum (pull through at about 1mL/min). SepPak units are solid-phase extraction (SPE) cartridges. The principle of operation is identical to that of StageTips, but SepPaks can accommodate a larger capacity of peptides. The binding capacity of the column is estimated to be 1% of the column matrix. In this case, for 1 mg of peptides (10 samples, 100 μg each a 100 mg SepPak cartridge (column volume 3 mL) can be used.

  4. Activate the SepPak with 1 column volume (CV) of methanol. Wash with 1 CV of a solution containing 70% ACN, 1% trifluoroacetic acid (TFA), and equilibrate with 1 CV of 1% TFA, 5% ACN.

  5. Acidify the sample by adding a few μL of TFA 100% (pH 2–3) and load the sample (typically 1–1.5 mL) to the column.

  6. Wash with 1CV of 1% formic acid (FA), 5% ACN and elute with 500–750 μL of 70% ACN, 1% formic acid.

  7. Freeze the sample and evaporate off the ACN by vacuum centrifugation.

  8. Resuspend peptides in 500 μL of 10 mM ammonium bicarbonate and 5% ACN at pH 8.

  9. Fractionate up to 300 mg of the peptide mixture (with a 60 min linear gradient from 13% to 42% ACN in 10 mM ammonium bicarbonate pH 8) into a total of 96 fractions of 200 μL each and collect them in a 96-well plate. Use an Agilent 300 Extend C18 column (3.5 μm particles, 4.6 mm ID and 220 mm long) on an Agilent 1260 HPLC (flow rate: 0.6 mL/min) or equivalent column and LC.

  10. Next, we suggest concatenating these fractions to improve proteome coverage and reduce mass spectrometry acquisition time [18]. We combine and consolidate 96 fractions into 24. Specifically, we combine four alternating wells vertically into one fraction (i.e., wells A1, C1, E1, G1 will constitute fraction 1; B1, D1, F1, H1, fraction 2, etc.), resulting in 24 super-fractions. Note that each super fraction has on average 12.5 mg of peptides. Because peptides elute horizontally in the 96 wells plate during HPLC fractionation, these four wells combined have minimal overlapping peptides. On the other hand, because adjacent wells in the plate contain overlapping peptides, we analyze only 12 super-fractions from non-adjacent columns [18]. We usually analyze the columns that have even numbers. The remaining 12 super-fractions (collected from the columns with odd numbers) can be stored at −80°C as a backup if needed or if specific peptides of interest are sought.

  11. Desalt each fraction via StageTip as described previously (step 2, paragraph 4.2). Lyophilize to dryness by vacuum centrifugation, and reconstitute in 10 μL of 5% ACN and 5% formic acid for LC-MS/MS.

  12. Inject 4 μL onto the mass spectrometer. This volume should represent 3–4 μg of peptides. We analyzed our samples on an Orbitrap Fusion mass spectrometer coupled with Proxeon EASY-nLC 1000 liquid chromatography (LC). For peptide detection, identification and quantification, see Rossio et al. [14].

3.6. Validation of candidate substrates identified by TMT-based proteomic analysis

We suggest independently validating the findings of the proteomics experiment. We usually in vitro translate and label the candidate DUB substrates with 35S-methionine [14]. Substrates are added to Xenopus egg extract, and stability is monitored by autoradiography. The addition of UbVS together with ubiquitin should induce their specific degradation. As an alternative to in vitro transcription/translation, and immunoblotting experiment with specific antibodies against the candidate substrates can be performed.

  1. Amplify the cDNAs of potential DUB substrates by PCR, using a primer that includes a T7 promoter, to allow T7-dependent transcription. Clean up the reaction using a PCR purification kit.

  2. Translate the PCR product using a coupled transcription/translation TNT system in the presence of 35S-methionine. Use the amount of DNA and methionine suggested by the manufacturer’s instructions of the coupled transcription/translation TNT system.

  3. Incubate the reaction at 30°C for 90 minutes.

  4. Add cycloheximide to a final 100 μg/mL concentration to the translation mix and incubate for 10 min at room temperature (see Note 21).

  5. Add the translation mix to the Xenopus extract (at 8 % final volume) pre-treated with XB buffer, ubiquitin, UbVS, or UbVS/ubiquitin, as in the proteomic experiment. Mix well, collect a sample immediately after substrate addition (time 0) and quench it with sample buffer. Incubate the extract at 24°C in the orbital mixer (1250 rpm) for one hour. Collect samples at 30 and 60 minutes for all the conditions and quench with sample buffer.

  6. Boil the samples for at least two minutes, spin at 23000g (30 seconds) in a bench centrifuge and analyze by reducing SDS-PAGE followed by autoradiography on film or phosphorimaging.

  7. The radioactive signal of the translated substrates should decrease over time, specifically in the extract treated with UbVS or UbVS/ubiquitin [14]

3.7. Identification of DUBs sufficient to rescue substrate degradation in UbVS/ubiquitin-treated extract

To identify DUBs that are sufficient to counteract proteasomal degradation of candidate substrates, we tested a panel of recombinant DUBs for the ability to stabilize 35S-methionine-labeled substrates in extracts treated with UbVS/ubiquitin. In these conditions, the UbVS is fully consumed by reaction with endogenous DUBs, so that the activity of the added DUB will be not affected. This approach circumvents DUB redundancy to reveal the activity and specificity profile of single DUBs. The presence of the endogenous DUBs in Xenopus extract are in the concentration range from 5 nM to more than 2 mM [19]. We suggest starting with a high concentration of enzyme (500 nM-1 μM), followed by a dose-response analysis of active DUBs.

  1. Before testing whether DUBs can rescue protein degradation, it is essential to confirm the activity of each recombinant, purified DUB. For cysteine protease DUBs, we do this by confirming that the DUB is UbVS-reactive.

  2. To verify DUB enzyme activity, incubate every single DUB with a saturating amount of UbVS for one hour at 37°C. We suggest incubating 1 μM DUB with 10 μM of UbVS (see Note 22). After one hour, add sample buffer to quench the reaction, boil for 2 minutes at 100°C and analyze by SDS-gel electrophoresis (see Note 23). If the DUB is active, it will covalently react with the UbVS and be visible as a gel shift of around 9 kDa.

  3. Stain the gel with Coomassie blue solution for one hour. Incubate the gel in the destaining solution as long as needed.

  4. After the activity of the DUB is verified, it can be added to the extract. It is important to demonstrate that the DUB added to the extract is not getting inactivated by unreacted UbVS present in the extract (see Note 24). Incubate the extract 30 minutes with UbVS or XB buffer (control), and then add one of the recombinant DUBs of interest to the extract (see Note 25). Mix well, take a timepoint (time 0) and quench it with sample buffer. Continue to incubate the extract and take time points at 15 and 30 minutes and quench them with sample buffer.

  5. Analyze the samples by reducing SDS-PAGE followed by immunoblotting with specific antibodies against the recombinant DUB. If the DUB is inactivated by residual UbVS present in the extract, it will be present as a gel shift of around 9 kDa, specifically in UbVS-treated extract (see Note 26).

  6. After the DUBs are verified not to be inactivated after their addition to the UbVS-treated extract, you can proceed to identify the DUBs that can rescue degradation of the protein substrates revealed by the previous proteomic experiment (see Note 27).

  7. Translate the PCR product of your selected substrates using the coupled transcription/translation system in the presence of 35S-methionine, as done previously. Pre-treat the extract with UbVS for 30 minutes or with MES buffer as a control. Add the in vitro labeled substrates to both the reactions (see Note 28) and in addition ubiquitin to the UbVS-treated extract. Keep the reactions on ice.

  8. Split the extract treated with UbVS/ubiquitin and contain the labeled substrates into clean tubes kept on ice. The number of tubes will depend on the number of DUBs tested in the experiment, as each DUB should be tested in an independent reaction (see Note 29).

  9. Add XB buffer (control) and the recombinant DUBs to the extract kept on ice and mix very well. As previously, collect time 0 from each reaction and incubate the rest at 24°C, as previously. Take samples at 30 and 60 minutes.

  10. Process and analyze the samples by reducing SDS-PAGE as before, followed by autoradiography on film or phosphorimaging.

  11. Determine the fraction of substrate remaining at 60 minutes relative to time zero for each condition by quantifying the radioactive signal. We use the Quantity One software (Bio-Rad). If a DUB is sufficient to antagonize substrate degradation, you will see an increase in the fraction of substrate remaining at 60 minutes compared to the reaction lacking the DUB. Since multiple DUBs can act on the same substrates, more than one DUB may rescue substrate degradation.

Acknowledgments

We acknowledge W. Harper for the gift of plasmids (ORFeome collection). This work was supported by the Cell Biology Education and Fellowship Fund (V.R.), NIH grants R01 GM132129 (J.A.P.) and R35 GM127032 (R.W.K.).

Footnotes

1.

The transfer of the eggs into the tube containing the XB buffer must be done gently because the eggs are easily broken after they have been dejellied.

2.

When preparing this buffer, vortex immediately after you add cytochalasin as it tends to precipitate.

3.

For small preparations, a microcentrifuge can be used. In this case, pack the eggs with a brief spin at 160 g, remove the excess buffer, and then crush the eggs at 14000 rpm (21,000g).

4.

Heating the needle with a flame helps in puncturing the tube.

5.

Undissolved glycerol is visible as a transparent gel at the bottom of the falcon tube.

6.

The extract can be defrosted once. Thawing and refreezing the extract are not recommended due to potential activity loss.

7.

Since ubiquitin antibodies cannot distinguish between free monomeric ubiquitin and unreacted UbVS in extract, this method is less helpful in monitoring free ubiquitin availability in UbVS-treated extract.

8.

The extract is very viscous. Cutting the pipette tip helps to pipet the correct volume of extract.

9.

We started the UbVS dose-response at 5 μM UbVS because cysteine protease DUBs in Xenopus egg extract have an estimated total concentration of around 7 μM [19].

10.

The extract should constitute at least 75% of the reaction volume in all experiments since excessive dilution can decrease extract activity.

11.

Non-reducing conditions are essential to preserving the thioester bond of Ube2C with ubiquitin. Because the thioester is sensitive to heat, it is important to keep collected samples on ice and not boil them. Performing gel electrophoresis in a cold room is preferable to avoid excessive gel heating.

12.

We usually load 50 μg of protein per lane of the gel, as this amount is sufficient to detect most proteins by immunoblotting.

13.

We observed that the UbVS concentration required to induce ubiquitin depletion decreases at longer incubation times. For example, 5 μM UbVS can induce the full discharge of Ube2C by 30 minutes (data not shown). This time- and concentration-dependence likely reflects the ongoing conjugation of ubiquitin to extract proteins by E2 and E3 enzymes in the extract.

14.

All samples for mass spectrometry analysis should be collected at least in duplicate, with triplicate or quadruplicate being preferable for obtaining lower p-values during statistical analysis.

15.

Both volumes and tubes can be scaled up, if necessary.

16.

By gently tilting the tube, the protein pellet attaches to the side of the tube, making it easy to aspirate both layers.

17.

The solution may turn cloudy due to the precipitation of undigested proteins. This precipitation is not a problem as the cloudiness disappears with digestion by trypsin.

18.

We observed that the number of identified peptides increased significantly with Lys-C/trypsin sequential digestion compared to single enzyme digestion.

19.

We usually quench the labeling reaction after the label check. If the check reveals incomplete labeling, we add more labeling reagents. This check will not be possible if we have already quenched the reaction.

20.

The samples do not need to be completely dry as overdrying pellets often makes them challenging to resuspend, and proteases remain active in up to 20% methanol.

21.

The addition of cycloheximide to the translation mix ensures that there is no protein synthesis after adding the reticulocyte mix to the Xenopus egg extract. The extract has already been treated with cycloheximide (see section 3.1).

22.

Most DUBs should completely react with the UbVS [14]. However, some DUBs that require accessory proteins for activation may not react with UbVS when the individual enzyme is treated with the inhibitor, as is the case for USP14, which requires the proteasome [10].

23.

Since most DUBs have a high molecular weight (more than 100 kDa), we suggest performing the SDS-gel electrophoresis with a gel containing a low polyacrylamide concentration (3–8%) in Tris-acetate SDS Running buffer. This concentration of polyacrylamide facilitates the detection of the mobility shift induced by the covalent addition of UbVS.

24.

We observed that increasing the duration of UbVS incubation in an extract from 10 to 30 minutes is essential to consume all the unreacted UbVS present in the extract.

25.

To establish that there is no residual unreacted UbVS in the extract, we usually add 800 nM USP7 because it is highly sensitive to UbVS.

26.

If the DUB is inactivated by residual UbVS present in the extract, we suggest increasing the incubation time of the extract with UbVS or decreasing the concentration of UbVS.

27.

We suggest starting with substrates that have been independently validated (see paragraph 3.4).

28.

Multiple substrates can be examined together in the same experiment if the substrates have different molecular weights. In this way, you can test the effect of one DUB on the stability of multiple substrates simultaneously.

29.

We suggest testing the DUB at a concentration of 800 nM to 1 μM.

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