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Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE
ARTICLE IN PRESS
Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE Yukiko Yoshida, Yasushi Saeki, Hikaru Tsuchiya, Keiji Tanaka* Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan *Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Detection of substrate ubiquitination by a given E3 using TR-TUBE 2.1 Comparison of TR-TUBE and ubiquitin overexpression methods 3. Identification of substrates for a given E3 ligase using TR-TUBE 3.1 Equipment 3.2 Buffers and reagents 3.3 Procedure 3.4 Notes 4. Conclusion References
2 3 3 7 7 7 8 11 12 12
Abstract Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).
Methods in Enzymology ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2018.12.032
#
2019 Elsevier Inc. All rights reserved.
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1. Introduction Protein ubiquitination is one of the most frequent posttranslational modifications in eukaryotes; it is estimated that more than 30% of all human proteins undergo ubiquitination (Wilhelm et al., 2014). Ubiquitination is performed by a hierarchal system of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), with substrate specificity determined by the E3 (Hershko & Ciechanover, 1998). More than 600 E3 ubiquitin ligases are encoded in the human genome, and these enzymes coordinate the ubiquitination of thousands of substrates (Deshaies & Joazeiro, 2009). Development of an antibody to the ubiquitin remnant motif Lys-ε-Gly-Gly (diGly), which is exposed upon tryptic digestion of ubiquitinated proteins, has enabled global proteomic analysis of ubiquitinated proteins (Kim et al., 2011; Xu, Paige, & Jaffrey, 2010). Characterization of the network of E3–substrate relationships has been challenging and is not yet fully achieved, but is essential for understanding the affected biological pathways in cells and be useful for drug development. Various methods have been utilized to identify substrates for a given E3, including cell-based genome-wide screens, two-hybrid screens, and coimmunoprecipitation (co-IP) paired with mass spectrometry (MS) (O’Connor & Huibregtse, 2017). Ultimately, the candidate substrates identified in these screens must be validated by verifying their ubiquitination by the E3 of interest. One of the most reliable methods for detecting ubiquitination is an in vitro reconstitution system using recombinant E1, E2, E3, ubiquitin, ATP, and the purified candidate protein (Petroski & Deshaies, 2005). Drawbacks of the reconstitution system include the difficulty of preparing active recombinant enzymes, the possibility that posttranslational modification of substrates may be necessary for their ubiquitination, and the potential for different substrate specificities in vitro and in vivo. More conveniently, ubiquitination is often detected by IP followed by western blotting (WB) in overexpression systems consisting of ubiquitin, E3, and target substrate in the presence of a proteasome inhibitor. The detected ubiquitination, however, sometimes occurs independently of the expressed E3, as described in Section 2.1. Furthermore, some protocols for detecting ubiquitination of immunoprecipitated substrates by using an antiubiquitin antibody may be unreliable due to ubiquitination of a protein that has coimmunoprecipitated with the substrate. Tandem ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains have been developed for isolation of polyubiquitinated
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Detection of ubiquitination activity using TR-TUBE
Substrate DUBs
UB E
E3 TR-TUBE
Ub Ub Ub Ub
TR -T
Ub
Ub Ub Ub
Substrate
Substrate Pro
tea
so
me
Substrate
Fig. 1 Concept of the TR-TUBE method. Polyubiquitin chains on substrates are masked by exogenously expressed TR-TUBE, and thereby protected from both DUBs and the proteasome. Ubiquitinated proteins can be enriched by immunoaffinity purification of TR-TUBE from cells exogenously expressing an E3, ubiquitin, and TR-TUBE.
proteins from cell lysates (Hjerpe et al., 2009). The ubiquitination state in cells is not stable because polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and most ubiquitinated proteins are degraded by the proteasome. TUBEs can protect polyubiquitinated proteins in cell lysates from proteasomal degradation and DUBs as efficiently as their respective specific inhibitors (Hjerpe et al., 2009; Ordureau et al., 2014). To detect the ubiquitination state of any given protein easily, we developed a method in which TUBEs are exogenously expressed and stabilize ubiquitinated substrates in cells by binding to and effectively masking their polyubiquitin chains (Fig. 1) (Yoshida et al., 2015). To apply this technique for identification of E3 substrates by MS, we constructed a trypsin-resistant tandem UBA domain of human UBQLN1 (TR-TUBE) that can interact with all eight types of polyubiquitin chain linkages: M1, K6, K11, K27, K29, K33, K48, and K63 (Yoshida et al., 2015). In this chapter, we describe methods for identifying substrates of a given E3 using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with MS, as well as for detection of E3 ligase activity.
2. Detection of substrate ubiquitination by a given E3 using TR-TUBE 2.1 Comparison of TR-TUBE and ubiquitin overexpression methods The SCFSKP2 complex is one of best-characterized ubiquitin ligases, and ubiquitinates the cyclin-dependent kinase inhibitor p27/CDKN1B in a phosphorylation-dependent manner (Carrano, Eytan, Hershko, &
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Pagano, 1999). We compared the efficiency of detecting p27 ubiquitination by SCFSKP2 using the TR-TUBE method to the conventional system overexpressing tagged ubiquitin and SKP2 (Fig. 2) (Yoshida et al., 2015). With the conventional system, ubiquitination of endogenous p27 by overexpressed SKP2 was not detected in immunoprecipitated ubiquitin, even in cells treated with the proteasome inhibitor MG132 (Fig. 2, lanes 1–4). Although ubiquitination of overexpressed p27 was detectable in ubiquitin immunoprecipitates from cells treated with MG132, overexpression of SKP2 failed to increase the amount p27 ubiquitination (lanes 5–8). By contrast, expressing TR-TUBE enabled detection of p27 ubiquitination by endogenous SCFSKP2 (lane 9), and exogenously expressing SKP2 increased ubiquitination of endogenous p27 even without proteasome inhibitor (lanes 9 and 11). Thus, ubiquitination of candidate substrates can easily be validated using cells coexpressing a specific E3 in combination with TR-TUBE. With the TR-TUBE method, antibodies that can detect endogenous proteins are necessary for detecting ubiquitination of candidate substrates. Because we often failed to detect E3-dependent ubiquitination of overexpressed substrates using the TR-TUBE method, we establish a stable IP: αFLAG FLAG-tagged Ub TR-TUBE HA-tagged emp p27 emp p27 – + – + – + – + HA-Skp2 – + – + – + – + – + –+ – + –+ MG132 kDa 250 150 100 75 50 37
(Ub)n -p27
p27
25 20 1 2 3 4 5 6 7 8 9 10111213141516
αp27 Fig. 2 Detection efficiency of p27 ubiquitinated by SCFSKP2 using the TR-TUBE method and a conventional overexpression method. 293T cells expressing FLAG-ubiquitin or FLAG-TR-TUBE with or without HA-SKP2 and/or HA-p27 were treated with MG132 or left untreated. Cells were harvested at 48 h posttransfection, and immunoprecipitates prepared using anti-FLAG antibody were analyzed by western blotting. Reprint from Yoshida, Y., Saeki, Y., Murakami, A., Kawawaki, J., Tsuchiya, H., Yoshihara, H., et al. (2015). A comprehensive method for detecting ubiquitinated substrates using TR-TUBE. Proceedings of the National Academy of Sciences of the United States of America, 112(15), 4630–4635.
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cell line that expresses low levels of tagged substrate if an antibody is not available. Although it is possible to use various cell lines to express the candidate substrate, we use 293T or HEK293 cells because they have a high transfection efficiency. 2.1.1 Equipment • Standard cell culture equipment • High speed refrigerated microcentrifuge • Sonicator • Rotator • Standard SDS-PAGE equipment • Gel blotting equipment • ImageQuant LAS 4000 2.1.2 Buffers and reagents • Phosphate-buffered saline (PBS) • TBS–N: 10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 • Transfection reagent: 1 mg/mL polyethylenimine (PEI; Polyscience Inc. CAT#23966) dissolved in 70°C water, adjusted to pH 7.5 with 1 N HCl, and filter sterilized with 0.2 μm Millex (Millipore). Store at 4°C • Opti-MEM j Reduced serum media (Thermo Fisher Scientific) • cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche): 1 tablet dissolved in 2 mL distilled water (25 stock solution) • Anti-DDDDK monoclonal antibody-conjugated agarose (MBL International) • Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate (Perkin Elmer) 2.1.3 Procedure 1. Culture 293T cells in a 10-cm cell culture dish with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum for 24 h. Start the culture with 1.3 106 cells. 2. Mix 3.5 μg FLAG-tagged TR-TUBE and 3.5 μg HA-tagged E3 plasmids in 1 mL Opti-MEM, and then add 21 μL PEI. Mix well, and incubate at room temperature for 15 min. Prepare another transfection mixture of TR-TUBE and HA-empty vector as a control. 3. Gently add each transfection mixture to a separate dish of cells. Culture for an additional 48 h.
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4. Harvest cells by centrifugation at 2000 g for 3 min, wash twice with PBS, and lyse with 500 μL TBS–N containing 1 cOmplete EDTAfree Protease Inhibitor Cocktail. Sonicate briefly on ice. 5. Centrifuge the homogenate at 20,000 g for 20 min at 4°C. 6. Transfer the supernatant to a new tube, and add 15 μL of anti-DDDDK monoclonal antibody-conjugated agarose that has been washed three times with TBS–N. 7. Rotate the tube for 1.5 h at 4°C. 8. Centrifuge the immunoprecipitate at 5000 g for 1 min at 4°C, and discard the supernatant. 9. Wash the agarose beads with 1 mL ice-cold TBS–N five times, centrifuging after each wash. 10. Remove the buffer thoroughly, add 30 μL SDS-PAGE sample buffer, and incubate sample for 5 min at 95°C. 11. Subject 1/3 of the eluate to SDS-PAGE in a 4%–20% acrylamide gel and Western blot using an antibody against the substrate candidate and HRP-labeled secondary antibody. 12. Visualize the candidate substrate protein using the ECL system and a LAS 4000 imager. The ubiquitinated protein is detected as a highmolecular weight smear. 2.1.4 Notes 1. It is necessary to confirm that 293T cells express the candidate substrate protein. We examine expression by Western immunoblotting using lysates prepared from untreated cells and cells treated with 10 μM MG132 (Peptide Institute, Inc.) for 4 h. 2. If the protein is not detected in 293T cells or an antibody is not available, cells stably expressing a tagged version of the candidate protein should be established. We use the pBABE-puro retrovirus vector system (Addgene plasmid 1764) for low-level expression. 3. To detect candidate ubiquitination by the coexpressed E3, a control prepared from cells that express only TR-TUBE is required. If the E3 of interest is a complex-type ligase such as the SCF complex, a dominant negative mutant that lacks ubiquitin ligase activity but maintains substrate binding may be used as a negative control. 4. If the antibody to the candidate protein was generated in mice, it is better to elute immunoprecipitates using the FLAG peptide (Sigma-Aldrich), not SDS-PAGE sample buffer, to reduce background derived from IgG. 5. TR-TUBE can stabilize all eight types of uniformly linked polyubiquitin chains as well as multiply monoubiquitinated substrates.
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3. Identification of substrates for a given E3 ligase using TR-TUBE The anti-diGly antibody is a useful tool for efficiently identifying peptides derived from ubiquitinated proteins, and it has been used for direct immunoprecipitation of trypsinized lysates prepared from cells treated with a proteasome inhibitor. As described earlier, an increase of substrate ubiquitination by overexpressed E3 is seldom detected even when proteasomal activity is inhibited. Because the proteins ubiquitinated by an overexpressed E3 accumulate in cells expressing TR-TUBE, peptides derived from the substrates of the overexpressed E3 may be isolated by diGly peptide immunoprecipitation following affinity isolation of ubiquitinated proteins using TR-TUBE.
3.1 Equipment • • • • •
SpeedVac EASY-nLC 1000 Liquid Chromatography System (Thermo Fisher Scientific, LC120) C18 analytical column (ReproSil-Pur 3 μm, 75 μm inner diameter, and 12 cm length, Nikkyo Technos) Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) Proteome Discoverer software version 1.3 (Thermo Fisher Scientific)
3.2 Buffers and reagents • • • • •
•
PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signaling #5562) Cross-linking reagent: dimethyl pimelimidate (DMP) Wash buffer: 0.2 M triethanolamine pH 8.2 Quenching buffer: 0.2 M ethanolamine pH 8.0 Stock solutions: 1 M Ammonium bicarbonate (AMBIC) 50 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 0.2 M Methyl methanethiosulfonate (MMTS) in isopropanol 10 IAP buffer (500 mM MOPS pH 7.2, 100 mM Na2HPO4, 500 mM NaCl); included in PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit Trypsin Gold, MS grade (Promega): 1 mg/mL trypsin in 50 mM acetic acid
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3.3 Procedure This example uses the SCF complex-type E3 and a series of samples consisting of three cell lines expressing FLAG-TR-TUBE/HA-empty (control), FLAG-TR-TUBE/HA-F box protein ΔF (dominant negative form, used as negative control), and FLAG-TR-TUBE/HA-F box protein. F-box proteins are the substrate-recognition subunits of SCF ligases and must have the F-box motif to associate with the rest of the SCF complex. At least three independent assays are necessary. One sample is prepared from a 10-cm cell culture dish as described in Section 2.1.2, and the procedure of immunoaffinity purification is the same as in Section 2.1.3, steps 1–8. Here we describe the procedure after immunoprecipitation of samples. Fig. 3 shows an overview of the procedure and the time required for the identification of substrates of a given E3. 1. To reduce the injurious effects that abundant antibody-derived protein contaminants have on the detection of diGly peptides, we use the chemically cross-linked anti-diGly antibody beads (see Note 1 later). Transfer 25 μL of diGly antibody beads in PTMScan Ubiquitin Remnant Motif Kit to a new tube, add 500 μL wash buffer, mix gently, and centrifuge the tube at 2000 g for 30 s at 4°C. Remove the buffer, and wash the beads again. After removing the buffer a second time, add 500 μL 25 mM DMP in wash buffer and rotate for 45 min at room temperature. Centrifuge, then wash the beads twice. After removing the buffer from the second wash, add 1 mL quenching buffer, and rotate the tube for 2 h at 4°C. Remove the supernatant, and wash three times with IAP buffer. Add 50 μL IAP buffer and keep the beads on ice until use. 2. Follow the procedure detailed in Section 2.1.3, steps 1–8. 3. Wash the anti-DDDDK (anti-FLAG) monoclonal antibody-conjugated agarose beads, which will contain bound FLAG-tagged TR-TUBE and TR-TUBE-associated proteins from the immunoprecipitation, with 1 mL ice-cold TBS–N five times and twice with 200 μL 50mM AMBIC by centrifuging at 5000 g for 1 min at 4°C. 4. Centrifuge each immunoprecipitate, remove the supernatant thoroughly, add 50 μL 50 mM AMBIC and 5 μL 50 mM TCEP to the beads, and then incubate the suspension for 30 min at 50°C with gently agitated to be reduced cysteine residues in proteins. 5. Add 2.5 μL of 0.2 M MMTS, and alkylate the proteins on beads for 10 min at room temperature.
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FLAG-TR-TUBE/empty (cont)
Transfection Culture
[
FLAG-TR-TUBE/E3 FLAG-TR-TUBE/dominant-negative E3 (neg. cont)
]
~2 day Cell lysis (~1 h) Immunoaffinity enrichment of ubiquitinated protein by TR-TUBE (~2.5 h)
E
Ub Ub Ub Ub
Substrate
~1 day
TR
Substrate
TR
-T
Ub Ub Ub Ub
-T
Alkylation on beads (~1 h)
UB
UB
E
Cross-linking of antidiGly antibody (~4.5 h)
+ Trypsin (~15 h)
Peptide filtration (~1 h) Immunoaffinity enrichment of diGly-containing peptide (~3.5 h) Peptide purification by C18 tip (~1.5 h)
GG KGG GG KK-
~1 day ~2 day LC-MS/MS analysis Fig. 3 Overview of the procedure for identifying substrates of a given E3 ligase using two enrichment methods, TR-TUBE and di-Gly antibody. 293T cells in a 10-cm cell culture dish are transfected with FLAG-TR-TUBE in combination with an empty vector or E3 expression vector (if available, a dominant negative mutant may also be used as a negative control). At 48 h posttransfection, cells are lysed and immunoprecipitated using an anti-FLAG antibody. The bead-bound proteins are alkylated and digested with trypsin. Ubiquitinated peptides are enriched using cross-linked anti-diGly antibody, and analyzed by LC-MS/MS.
6. Add 1 μg of trypsin to the alkylated proteins and incubate overnight at 37°C. 7. Transfer the beads suspension to an Ultrafree-MC GV 0.22 μm filter (Millipore) with a new 1.5-mL tube, and centrifuge at 5000 g for 1 min at 4°C. Add 70 μL solution (10 μL of 10 IAP buffer, 50 μL of distilled water, and 10 μL of 25 cOmplete EDTA-free Protease
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Inhibitor Cocktail) to the original tube, transfer additional solution with remaining beads to the Ultrafree filter, and centrifuge. Repeat. The total flow-through is 200 μL peptide solution. Add 18 μL of cross-linked anti-diGly antibody bead slurry (6 μL beads) prepared in step 1 to each peptide solution, and rotate gently for 2 h at 4°C. Centrifuge at 2000 g for 30 s at 4°C and remove the supernatant. Add 250 μL 1 IAP buffer to the beads, mix gently by tapping, centrifuge, and remove supernatant. Wash the beads twice with IAP buffer and three times with distilled water. Add 20 μL of 0.15% TFA to the beads, mix gently, let stand at room temperature for 10 min, and centrifuge. Transfer the supernatant to a new tube. Repeat peptide elution twice, and combine the three eluents. Apply the pooled eluents to a GL-Tip SDB (GL Sciences) for peptide purification according to the manufacturer’s instructions. Elute the peptide with 30 μL of 80% acetonitrile/0.1% TFA, and evaporate acetonitrile by placing samples in a SpeedVac for 10 min at 30°C (samples should not be fully dry but have less than 5 μL liquid remaining). Add 40 μL of 0.1% TFA to the remaining peptide solution, and repeat purification with a new GL-Tip SDB. Evaporate acetonitrile by placing samples in a SpeedVac, then adjust volume to 12 μL by adding 0.1% TFA. Apply 10 μL of the resulting samples to a nanoflow UHPLC coupled to a Q Exactive mass spectrometer. Process the raw MS output data using Proteome Discoverer software version 1.3. Assemble the results of the control(s) and E3-expressing sample into one multiconsensus report using Proteome Discoverer software to identify specific substrates of the E3. Compare the ion scores and the total numbers of identified sequences (PSMs: peptide spectrum matches) of ubiquitinated peptides (peptides containing Lys-ε-Gly-Gly). Select the candidates whose PSM numbers and protein scores reproducibly increased in the sample of E3-expressing cells in at least three independent analyses. Confirm the candidate substrates’ ubiquitination by the E3 in an IP-WB using the TR-TUBE method as described in Section 2.
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3.4 Notes 1. Chemical cross-linking of anti-diGly antibody beads in the kit is necessary to reduce antibody contamination in the peptide elution with 0.15% TFA (Tammsalu et al., 2015; Udeshi et al., 2013). Although the crosslinking efficiency of BS3 (bis(sulfosuccinimidyl)suberate) is higher than DMP, it seems to reduce binding ability to the diGly peptide. Some antibody can be removed by two rounds of purification with a GL-Tip SDB. 2. Since TR-TUBE enriches proteins with polyubiquitin chains, an excess amount of starting materials increases the number of peptides derived from ubiquitin, which compromises the detection of less abundant peptides. 3. Following immunoprecipitation of the ubiquitinated protein by TR-TUBE, the proteins eluted with FLAG peptide can be used for alkylation and protein digestion. In that case, we elute proteins three times with 25 μL of 200 μg/mL FLAG peptide (Sigma) in 50 mM AMBIC. 4. Our MS conditions are described later. We load the peptide directly onto a C18 analytical column. Peptides are separated using a 150 min two-step gradient (0%–40% in 120 min, 40%–100% in 20 min, and 100% for 10 min in Solvent B (0.1% formic acid in 100% acetonitrile)) at a constant flow rate of 300 nL/min. For ionization, we use a 1.8 kV liquid junction voltage and 250°C capillary temperature. The Q Exactive is operated in the data-dependent MS/MS mode, using Xcalibur software, with survey scans acquired at a resolution of 70,000 at m/z 200. The 10 most abundant isotope patterns with charge 2–4 are selected from the survey scans with an isolation window of 2.0 m/z, and fragmented by higher-energy collisional dissociation with normalized collision energies of 28. The maximum ion injection times for the survey and MS/MS scans are 60 ms, and the ion target values are set to 3e6 for the survey scan and 1e5 for the MS/MS scan. Ions selected for MS/MS are dynamically excluded for 5 s. 5. The raw MS data are processed using Proteome Discoverer software with the settings shown later. The MS/MS spectra are searched against a SwissProt database (version 2012_10 of UniProtKB/Swiss-Prot protein database) using the MASCOT search engine. The precursor and fragment mass tolerances are set to 10 ppm and 20 milli mass unit, respectively. Methionine oxidation, protein amino-terminal acetylation,
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pyroglutamate formation, serine/threonine/tyrosine phosphorylation, and diGly modification of lysine side chains are set as variable modifications, and cysteine methylthio modification is set as a static modification for database searching. Peptide identification is filtered at a 1% false discovery rate.
4. Conclusion This chapter describes the TR-TUBE method for detecting the activity of a given ubiquitin ligase and identifying its substrates. Ubiquitination of proteins is transient, but expression of exogenous TR-TUBE preserves ubiquitination states in cells. In cells expressing TR-TUBE, an overexpressed E3 ubiquitinates its endogenous substrates using ubiquitination-related factors present in cells and the ubiquitinated substrates are maintained, allowing detection of the specific E3 activity. The TR-TUBE method is a simple alternative to an in vitro reconstitution system. A combination of two enrichment strategies, TR-TUBE and use of the anti-diGly antibody, provides a useful method for identifying substrates of an E3 from a small amount of cell lysate.
References Carrano, A. C., Eytan, E., Hershko, A., & Pagano, M. (1999). SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nature Cell Biology, 1(4), 193–199. Deshaies, R. J., & Joazeiro, C. A. (2009). RING domain E3 ubiquitin ligases. Annual Review of Biochemistry, 78, 399–434. Hershko, A., & Ciechanover, A. (1998). The ubiquitin system. Annual Review of Biochemistry, 67, 425–479. Hjerpe, R., Aillet, F., Lopitz-Otsoa, F., Lang, V., England, P., & Rodriguez, M. S. (2009). Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitinbinding entities. EMBO Reports, 10(11), 1250–1258. Kim, W., Bennett, E. J., Huttlin, E. L., Guo, A., Li, J., Possemato, A., et al. (2011). Systematic and quantitative assessment of the ubiquitin-modified proteome. Molecular Cell, 44(2), 325–340. O’Connor, H. F., & Huibregtse, J. M. (2017). Enzyme-substrate relationships in the ubiquitin system: Approaches for identifying substrates of ubiquitin ligases. Cellular and Molecular Life Sciences, 74(18), 3363–3375. Ordureau, A., Sarraf, S. A., Duda, D. M., Heo, J. M., Jedrychowski, M. P., Sviderskiy, V. O., et al. (2014). Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Molecular Cell, 56(3), 360–375. Petroski, M. D., & Deshaies, R. J. (2005). In vitro reconstitution of SCF substrate ubiquitination with purified proteins. Methods in Enzymology, 398, 143–158. Tammsalu, T., Matic, I., Jaffray, E. G., Ibrahim, A. F., Tatham, M. H., & Hay, R. T. (2015). Proteome-wide identification of SUMO modification sites by mass spectrometry. Nature Protocols, 10(9), 1374–1388.
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Udeshi, N. D., Svinkina, T., Mertins, P., Kuhn, E., Mani, D. R., Qiao, J. W., et al. (2013). Refined preparation and use of anti-diglycine remnant (K-epsilon-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments. Molecular & Cellular Proteomics, 12(3), 825–831. Wilhelm, M., Schlegl, J., Hahne, H., Gholami, A. M., Lieberenz, M., Savitski, M. M., et al. (2014). Mass-spectrometry-based draft of the human proteome. Nature, 509(7502), 582–587. Xu, G., Paige, J. S., & Jaffrey, S. R. (2010). Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nature Biotechnology, 28(8), 868–873. Yoshida, Y., Saeki, Y., Murakami, A., Kawawaki, J., Tsuchiya, H., Yoshihara, H., et al. (2015). A comprehensive method for detecting ubiquitinated substrates using TR-TUBE. Proceedings of the National Academy of Sciences of the United States of America, 112(15), 4630–4635.
Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE Yukiko Yoshida, Yasushi Saeki, Hikaru Tsuchiya, Keiji Tanaka* Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan *Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Detection of substrate ubiquitination by a given E3 using TR-TUBE 2.1 Comparison of TR-TUBE and ubiquitin overexpression methods 3. Identification of substrates for a given E3 ligase using TR-TUBE 3.1 Equipment 3.2 Buffers and reagents 3.3 Procedure 3.4 Notes 4. Conclusion References
2 3 3 7 7 7 8 11 12 12
Abstract Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).
Methods in Enzymology ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2018.12.032
#
2019 Elsevier Inc. All rights reserved.
1
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1. Introduction Protein ubiquitination is one of the most frequent posttranslational modifications in eukaryotes; it is estimated that more than 30% of all human proteins undergo ubiquitination (Wilhelm et al., 2014). Ubiquitination is performed by a hierarchal system of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), with substrate specificity determined by the E3 (Hershko & Ciechanover, 1998). More than 600 E3 ubiquitin ligases are encoded in the human genome, and these enzymes coordinate the ubiquitination of thousands of substrates (Deshaies & Joazeiro, 2009). Development of an antibody to the ubiquitin remnant motif Lys-ε-Gly-Gly (diGly), which is exposed upon tryptic digestion of ubiquitinated proteins, has enabled global proteomic analysis of ubiquitinated proteins (Kim et al., 2011; Xu, Paige, & Jaffrey, 2010). Characterization of the network of E3–substrate relationships has been challenging and is not yet fully achieved, but is essential for understanding the affected biological pathways in cells and be useful for drug development. Various methods have been utilized to identify substrates for a given E3, including cell-based genome-wide screens, two-hybrid screens, and coimmunoprecipitation (co-IP) paired with mass spectrometry (MS) (O’Connor & Huibregtse, 2017). Ultimately, the candidate substrates identified in these screens must be validated by verifying their ubiquitination by the E3 of interest. One of the most reliable methods for detecting ubiquitination is an in vitro reconstitution system using recombinant E1, E2, E3, ubiquitin, ATP, and the purified candidate protein (Petroski & Deshaies, 2005). Drawbacks of the reconstitution system include the difficulty of preparing active recombinant enzymes, the possibility that posttranslational modification of substrates may be necessary for their ubiquitination, and the potential for different substrate specificities in vitro and in vivo. More conveniently, ubiquitination is often detected by IP followed by western blotting (WB) in overexpression systems consisting of ubiquitin, E3, and target substrate in the presence of a proteasome inhibitor. The detected ubiquitination, however, sometimes occurs independently of the expressed E3, as described in Section 2.1. Furthermore, some protocols for detecting ubiquitination of immunoprecipitated substrates by using an antiubiquitin antibody may be unreliable due to ubiquitination of a protein that has coimmunoprecipitated with the substrate. Tandem ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains have been developed for isolation of polyubiquitinated
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Detection of ubiquitination activity using TR-TUBE
Substrate DUBs
UB E
E3 TR-TUBE
Ub Ub Ub Ub
TR -T
Ub
Ub Ub Ub
Substrate
Substrate Pro
tea
so
me
Substrate
Fig. 1 Concept of the TR-TUBE method. Polyubiquitin chains on substrates are masked by exogenously expressed TR-TUBE, and thereby protected from both DUBs and the proteasome. Ubiquitinated proteins can be enriched by immunoaffinity purification of TR-TUBE from cells exogenously expressing an E3, ubiquitin, and TR-TUBE.
proteins from cell lysates (Hjerpe et al., 2009). The ubiquitination state in cells is not stable because polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and most ubiquitinated proteins are degraded by the proteasome. TUBEs can protect polyubiquitinated proteins in cell lysates from proteasomal degradation and DUBs as efficiently as their respective specific inhibitors (Hjerpe et al., 2009; Ordureau et al., 2014). To detect the ubiquitination state of any given protein easily, we developed a method in which TUBEs are exogenously expressed and stabilize ubiquitinated substrates in cells by binding to and effectively masking their polyubiquitin chains (Fig. 1) (Yoshida et al., 2015). To apply this technique for identification of E3 substrates by MS, we constructed a trypsin-resistant tandem UBA domain of human UBQLN1 (TR-TUBE) that can interact with all eight types of polyubiquitin chain linkages: M1, K6, K11, K27, K29, K33, K48, and K63 (Yoshida et al., 2015). In this chapter, we describe methods for identifying substrates of a given E3 using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with MS, as well as for detection of E3 ligase activity.
2. Detection of substrate ubiquitination by a given E3 using TR-TUBE 2.1 Comparison of TR-TUBE and ubiquitin overexpression methods The SCFSKP2 complex is one of best-characterized ubiquitin ligases, and ubiquitinates the cyclin-dependent kinase inhibitor p27/CDKN1B in a phosphorylation-dependent manner (Carrano, Eytan, Hershko, &
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Pagano, 1999). We compared the efficiency of detecting p27 ubiquitination by SCFSKP2 using the TR-TUBE method to the conventional system overexpressing tagged ubiquitin and SKP2 (Fig. 2) (Yoshida et al., 2015). With the conventional system, ubiquitination of endogenous p27 by overexpressed SKP2 was not detected in immunoprecipitated ubiquitin, even in cells treated with the proteasome inhibitor MG132 (Fig. 2, lanes 1–4). Although ubiquitination of overexpressed p27 was detectable in ubiquitin immunoprecipitates from cells treated with MG132, overexpression of SKP2 failed to increase the amount p27 ubiquitination (lanes 5–8). By contrast, expressing TR-TUBE enabled detection of p27 ubiquitination by endogenous SCFSKP2 (lane 9), and exogenously expressing SKP2 increased ubiquitination of endogenous p27 even without proteasome inhibitor (lanes 9 and 11). Thus, ubiquitination of candidate substrates can easily be validated using cells coexpressing a specific E3 in combination with TR-TUBE. With the TR-TUBE method, antibodies that can detect endogenous proteins are necessary for detecting ubiquitination of candidate substrates. Because we often failed to detect E3-dependent ubiquitination of overexpressed substrates using the TR-TUBE method, we establish a stable IP: αFLAG FLAG-tagged Ub TR-TUBE HA-tagged emp p27 emp p27 – + – + – + – + HA-Skp2 – + – + – + – + – + –+ – + –+ MG132 kDa 250 150 100 75 50 37
(Ub)n -p27
p27
25 20 1 2 3 4 5 6 7 8 9 10111213141516
αp27 Fig. 2 Detection efficiency of p27 ubiquitinated by SCFSKP2 using the TR-TUBE method and a conventional overexpression method. 293T cells expressing FLAG-ubiquitin or FLAG-TR-TUBE with or without HA-SKP2 and/or HA-p27 were treated with MG132 or left untreated. Cells were harvested at 48 h posttransfection, and immunoprecipitates prepared using anti-FLAG antibody were analyzed by western blotting. Reprint from Yoshida, Y., Saeki, Y., Murakami, A., Kawawaki, J., Tsuchiya, H., Yoshihara, H., et al. (2015). A comprehensive method for detecting ubiquitinated substrates using TR-TUBE. Proceedings of the National Academy of Sciences of the United States of America, 112(15), 4630–4635.
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cell line that expresses low levels of tagged substrate if an antibody is not available. Although it is possible to use various cell lines to express the candidate substrate, we use 293T or HEK293 cells because they have a high transfection efficiency. 2.1.1 Equipment • Standard cell culture equipment • High speed refrigerated microcentrifuge • Sonicator • Rotator • Standard SDS-PAGE equipment • Gel blotting equipment • ImageQuant LAS 4000 2.1.2 Buffers and reagents • Phosphate-buffered saline (PBS) • TBS–N: 10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 • Transfection reagent: 1 mg/mL polyethylenimine (PEI; Polyscience Inc. CAT#23966) dissolved in 70°C water, adjusted to pH 7.5 with 1 N HCl, and filter sterilized with 0.2 μm Millex (Millipore). Store at 4°C • Opti-MEM j Reduced serum media (Thermo Fisher Scientific) • cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche): 1 tablet dissolved in 2 mL distilled water (25 stock solution) • Anti-DDDDK monoclonal antibody-conjugated agarose (MBL International) • Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate (Perkin Elmer) 2.1.3 Procedure 1. Culture 293T cells in a 10-cm cell culture dish with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum for 24 h. Start the culture with 1.3 106 cells. 2. Mix 3.5 μg FLAG-tagged TR-TUBE and 3.5 μg HA-tagged E3 plasmids in 1 mL Opti-MEM, and then add 21 μL PEI. Mix well, and incubate at room temperature for 15 min. Prepare another transfection mixture of TR-TUBE and HA-empty vector as a control. 3. Gently add each transfection mixture to a separate dish of cells. Culture for an additional 48 h.
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4. Harvest cells by centrifugation at 2000 g for 3 min, wash twice with PBS, and lyse with 500 μL TBS–N containing 1 cOmplete EDTAfree Protease Inhibitor Cocktail. Sonicate briefly on ice. 5. Centrifuge the homogenate at 20,000 g for 20 min at 4°C. 6. Transfer the supernatant to a new tube, and add 15 μL of anti-DDDDK monoclonal antibody-conjugated agarose that has been washed three times with TBS–N. 7. Rotate the tube for 1.5 h at 4°C. 8. Centrifuge the immunoprecipitate at 5000 g for 1 min at 4°C, and discard the supernatant. 9. Wash the agarose beads with 1 mL ice-cold TBS–N five times, centrifuging after each wash. 10. Remove the buffer thoroughly, add 30 μL SDS-PAGE sample buffer, and incubate sample for 5 min at 95°C. 11. Subject 1/3 of the eluate to SDS-PAGE in a 4%–20% acrylamide gel and Western blot using an antibody against the substrate candidate and HRP-labeled secondary antibody. 12. Visualize the candidate substrate protein using the ECL system and a LAS 4000 imager. The ubiquitinated protein is detected as a highmolecular weight smear. 2.1.4 Notes 1. It is necessary to confirm that 293T cells express the candidate substrate protein. We examine expression by Western immunoblotting using lysates prepared from untreated cells and cells treated with 10 μM MG132 (Peptide Institute, Inc.) for 4 h. 2. If the protein is not detected in 293T cells or an antibody is not available, cells stably expressing a tagged version of the candidate protein should be established. We use the pBABE-puro retrovirus vector system (Addgene plasmid 1764) for low-level expression. 3. To detect candidate ubiquitination by the coexpressed E3, a control prepared from cells that express only TR-TUBE is required. If the E3 of interest is a complex-type ligase such as the SCF complex, a dominant negative mutant that lacks ubiquitin ligase activity but maintains substrate binding may be used as a negative control. 4. If the antibody to the candidate protein was generated in mice, it is better to elute immunoprecipitates using the FLAG peptide (Sigma-Aldrich), not SDS-PAGE sample buffer, to reduce background derived from IgG. 5. TR-TUBE can stabilize all eight types of uniformly linked polyubiquitin chains as well as multiply monoubiquitinated substrates.
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3. Identification of substrates for a given E3 ligase using TR-TUBE The anti-diGly antibody is a useful tool for efficiently identifying peptides derived from ubiquitinated proteins, and it has been used for direct immunoprecipitation of trypsinized lysates prepared from cells treated with a proteasome inhibitor. As described earlier, an increase of substrate ubiquitination by overexpressed E3 is seldom detected even when proteasomal activity is inhibited. Because the proteins ubiquitinated by an overexpressed E3 accumulate in cells expressing TR-TUBE, peptides derived from the substrates of the overexpressed E3 may be isolated by diGly peptide immunoprecipitation following affinity isolation of ubiquitinated proteins using TR-TUBE.
3.1 Equipment • • • • •
SpeedVac EASY-nLC 1000 Liquid Chromatography System (Thermo Fisher Scientific, LC120) C18 analytical column (ReproSil-Pur 3 μm, 75 μm inner diameter, and 12 cm length, Nikkyo Technos) Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) Proteome Discoverer software version 1.3 (Thermo Fisher Scientific)
3.2 Buffers and reagents • • • • •
•
PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signaling #5562) Cross-linking reagent: dimethyl pimelimidate (DMP) Wash buffer: 0.2 M triethanolamine pH 8.2 Quenching buffer: 0.2 M ethanolamine pH 8.0 Stock solutions: 1 M Ammonium bicarbonate (AMBIC) 50 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 0.2 M Methyl methanethiosulfonate (MMTS) in isopropanol 10 IAP buffer (500 mM MOPS pH 7.2, 100 mM Na2HPO4, 500 mM NaCl); included in PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit Trypsin Gold, MS grade (Promega): 1 mg/mL trypsin in 50 mM acetic acid
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3.3 Procedure This example uses the SCF complex-type E3 and a series of samples consisting of three cell lines expressing FLAG-TR-TUBE/HA-empty (control), FLAG-TR-TUBE/HA-F box protein ΔF (dominant negative form, used as negative control), and FLAG-TR-TUBE/HA-F box protein. F-box proteins are the substrate-recognition subunits of SCF ligases and must have the F-box motif to associate with the rest of the SCF complex. At least three independent assays are necessary. One sample is prepared from a 10-cm cell culture dish as described in Section 2.1.2, and the procedure of immunoaffinity purification is the same as in Section 2.1.3, steps 1–8. Here we describe the procedure after immunoprecipitation of samples. Fig. 3 shows an overview of the procedure and the time required for the identification of substrates of a given E3. 1. To reduce the injurious effects that abundant antibody-derived protein contaminants have on the detection of diGly peptides, we use the chemically cross-linked anti-diGly antibody beads (see Note 1 later). Transfer 25 μL of diGly antibody beads in PTMScan Ubiquitin Remnant Motif Kit to a new tube, add 500 μL wash buffer, mix gently, and centrifuge the tube at 2000 g for 30 s at 4°C. Remove the buffer, and wash the beads again. After removing the buffer a second time, add 500 μL 25 mM DMP in wash buffer and rotate for 45 min at room temperature. Centrifuge, then wash the beads twice. After removing the buffer from the second wash, add 1 mL quenching buffer, and rotate the tube for 2 h at 4°C. Remove the supernatant, and wash three times with IAP buffer. Add 50 μL IAP buffer and keep the beads on ice until use. 2. Follow the procedure detailed in Section 2.1.3, steps 1–8. 3. Wash the anti-DDDDK (anti-FLAG) monoclonal antibody-conjugated agarose beads, which will contain bound FLAG-tagged TR-TUBE and TR-TUBE-associated proteins from the immunoprecipitation, with 1 mL ice-cold TBS–N five times and twice with 200 μL 50mM AMBIC by centrifuging at 5000 g for 1 min at 4°C. 4. Centrifuge each immunoprecipitate, remove the supernatant thoroughly, add 50 μL 50 mM AMBIC and 5 μL 50 mM TCEP to the beads, and then incubate the suspension for 30 min at 50°C with gently agitated to be reduced cysteine residues in proteins. 5. Add 2.5 μL of 0.2 M MMTS, and alkylate the proteins on beads for 10 min at room temperature.
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Detection of ubiquitination activity using TR-TUBE
FLAG-TR-TUBE/empty (cont)
Transfection Culture
[
FLAG-TR-TUBE/E3 FLAG-TR-TUBE/dominant-negative E3 (neg. cont)
]
~2 day Cell lysis (~1 h) Immunoaffinity enrichment of ubiquitinated protein by TR-TUBE (~2.5 h)
E
Ub Ub Ub Ub
Substrate
~1 day
TR
Substrate
TR
-T
Ub Ub Ub Ub
-T
Alkylation on beads (~1 h)
UB
UB
E
Cross-linking of antidiGly antibody (~4.5 h)
+ Trypsin (~15 h)
Peptide filtration (~1 h) Immunoaffinity enrichment of diGly-containing peptide (~3.5 h) Peptide purification by C18 tip (~1.5 h)
GG KGG GG KK-
~1 day ~2 day LC-MS/MS analysis Fig. 3 Overview of the procedure for identifying substrates of a given E3 ligase using two enrichment methods, TR-TUBE and di-Gly antibody. 293T cells in a 10-cm cell culture dish are transfected with FLAG-TR-TUBE in combination with an empty vector or E3 expression vector (if available, a dominant negative mutant may also be used as a negative control). At 48 h posttransfection, cells are lysed and immunoprecipitated using an anti-FLAG antibody. The bead-bound proteins are alkylated and digested with trypsin. Ubiquitinated peptides are enriched using cross-linked anti-diGly antibody, and analyzed by LC-MS/MS.
6. Add 1 μg of trypsin to the alkylated proteins and incubate overnight at 37°C. 7. Transfer the beads suspension to an Ultrafree-MC GV 0.22 μm filter (Millipore) with a new 1.5-mL tube, and centrifuge at 5000 g for 1 min at 4°C. Add 70 μL solution (10 μL of 10 IAP buffer, 50 μL of distilled water, and 10 μL of 25 cOmplete EDTA-free Protease
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Inhibitor Cocktail) to the original tube, transfer additional solution with remaining beads to the Ultrafree filter, and centrifuge. Repeat. The total flow-through is 200 μL peptide solution. Add 18 μL of cross-linked anti-diGly antibody bead slurry (6 μL beads) prepared in step 1 to each peptide solution, and rotate gently for 2 h at 4°C. Centrifuge at 2000 g for 30 s at 4°C and remove the supernatant. Add 250 μL 1 IAP buffer to the beads, mix gently by tapping, centrifuge, and remove supernatant. Wash the beads twice with IAP buffer and three times with distilled water. Add 20 μL of 0.15% TFA to the beads, mix gently, let stand at room temperature for 10 min, and centrifuge. Transfer the supernatant to a new tube. Repeat peptide elution twice, and combine the three eluents. Apply the pooled eluents to a GL-Tip SDB (GL Sciences) for peptide purification according to the manufacturer’s instructions. Elute the peptide with 30 μL of 80% acetonitrile/0.1% TFA, and evaporate acetonitrile by placing samples in a SpeedVac for 10 min at 30°C (samples should not be fully dry but have less than 5 μL liquid remaining). Add 40 μL of 0.1% TFA to the remaining peptide solution, and repeat purification with a new GL-Tip SDB. Evaporate acetonitrile by placing samples in a SpeedVac, then adjust volume to 12 μL by adding 0.1% TFA. Apply 10 μL of the resulting samples to a nanoflow UHPLC coupled to a Q Exactive mass spectrometer. Process the raw MS output data using Proteome Discoverer software version 1.3. Assemble the results of the control(s) and E3-expressing sample into one multiconsensus report using Proteome Discoverer software to identify specific substrates of the E3. Compare the ion scores and the total numbers of identified sequences (PSMs: peptide spectrum matches) of ubiquitinated peptides (peptides containing Lys-ε-Gly-Gly). Select the candidates whose PSM numbers and protein scores reproducibly increased in the sample of E3-expressing cells in at least three independent analyses. Confirm the candidate substrates’ ubiquitination by the E3 in an IP-WB using the TR-TUBE method as described in Section 2.
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3.4 Notes 1. Chemical cross-linking of anti-diGly antibody beads in the kit is necessary to reduce antibody contamination in the peptide elution with 0.15% TFA (Tammsalu et al., 2015; Udeshi et al., 2013). Although the crosslinking efficiency of BS3 (bis(sulfosuccinimidyl)suberate) is higher than DMP, it seems to reduce binding ability to the diGly peptide. Some antibody can be removed by two rounds of purification with a GL-Tip SDB. 2. Since TR-TUBE enriches proteins with polyubiquitin chains, an excess amount of starting materials increases the number of peptides derived from ubiquitin, which compromises the detection of less abundant peptides. 3. Following immunoprecipitation of the ubiquitinated protein by TR-TUBE, the proteins eluted with FLAG peptide can be used for alkylation and protein digestion. In that case, we elute proteins three times with 25 μL of 200 μg/mL FLAG peptide (Sigma) in 50 mM AMBIC. 4. Our MS conditions are described later. We load the peptide directly onto a C18 analytical column. Peptides are separated using a 150 min two-step gradient (0%–40% in 120 min, 40%–100% in 20 min, and 100% for 10 min in Solvent B (0.1% formic acid in 100% acetonitrile)) at a constant flow rate of 300 nL/min. For ionization, we use a 1.8 kV liquid junction voltage and 250°C capillary temperature. The Q Exactive is operated in the data-dependent MS/MS mode, using Xcalibur software, with survey scans acquired at a resolution of 70,000 at m/z 200. The 10 most abundant isotope patterns with charge 2–4 are selected from the survey scans with an isolation window of 2.0 m/z, and fragmented by higher-energy collisional dissociation with normalized collision energies of 28. The maximum ion injection times for the survey and MS/MS scans are 60 ms, and the ion target values are set to 3e6 for the survey scan and 1e5 for the MS/MS scan. Ions selected for MS/MS are dynamically excluded for 5 s. 5. The raw MS data are processed using Proteome Discoverer software with the settings shown later. The MS/MS spectra are searched against a SwissProt database (version 2012_10 of UniProtKB/Swiss-Prot protein database) using the MASCOT search engine. The precursor and fragment mass tolerances are set to 10 ppm and 20 milli mass unit, respectively. Methionine oxidation, protein amino-terminal acetylation,
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pyroglutamate formation, serine/threonine/tyrosine phosphorylation, and diGly modification of lysine side chains are set as variable modifications, and cysteine methylthio modification is set as a static modification for database searching. Peptide identification is filtered at a 1% false discovery rate.
4. Conclusion This chapter describes the TR-TUBE method for detecting the activity of a given ubiquitin ligase and identifying its substrates. Ubiquitination of proteins is transient, but expression of exogenous TR-TUBE preserves ubiquitination states in cells. In cells expressing TR-TUBE, an overexpressed E3 ubiquitinates its endogenous substrates using ubiquitination-related factors present in cells and the ubiquitinated substrates are maintained, allowing detection of the specific E3 activity. The TR-TUBE method is a simple alternative to an in vitro reconstitution system. A combination of two enrichment strategies, TR-TUBE and use of the anti-diGly antibody, provides a useful method for identifying substrates of an E3 from a small amount of cell lysate.
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