Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry

Abstract

Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-REx HEK293T cells and validated several newly identified linear polyubiquitin targets. We demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for NFκB signaling.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The design and in vitro validation of INT-Ub.
Figure 2: In vivo validation of INT-Ub.
Figure 3: The use of INT-Ub.7KR for the identification of novel linear polyUb-modified substrates.
Figure 4: Mass-spectrometry-based identification of novel linear polyUb-modified substrates.
Figure 5: Validation of novel linear polyUb-modified substrates.
Figure 6: TRAF6 is a novel LUBAC substrate in NFκB signaling.

Similar content being viewed by others

References

  1. Varshavsky, A. Regulated protein degradation. Trends Biochem. Sci. 30, 283–286 (2005).

    CAS  PubMed  Google Scholar 

  2. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    CAS  PubMed  Google Scholar 

  3. Kulathu, Y. & Komander, D. Atypical ubiquitylation — the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523 (2012).

    CAS  PubMed  Google Scholar 

  4. Kirisako, T. et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 25, 4877–4887 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Matsumoto, M.L. et al. Engineering and structural characterization of a linear polyubiquitin-specific antibody. J. Mol. Biol. 418, 134–144 (2012).

    CAS  PubMed  Google Scholar 

  6. Phu, L. et al. Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals. Mol. Cell. Proteomics 10, M110.003756 (2011).

    PubMed  Google Scholar 

  7. Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation. Nat. Cell Biol. 11, 123–132 (2009).

    CAS  PubMed  Google Scholar 

  8. Asaoka, T. et al. Linear ubiquitination by LUBEL has a role in Drosophila heat stress response. EMBO Rep. 17, 1624–1640 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Damgaard, R.B. et al. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol. Cell 46, 746–758 (2012).

    CAS  PubMed  Google Scholar 

  10. Shimizu, Y., Taraborrelli, L. & Walczak, H. Linear ubiquitination in immunity. Immunol. Rev. 266, 190–207 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Tokunaga, F. Linear ubiquitination-mediated NF-κB regulation and its related disorders. J. Biochem. 154, 313–323 (2013).

    CAS  PubMed  Google Scholar 

  12. Okamura, K. et al. Survival of mature T cells depends on signaling through HOIP. Sci. Rep. 6, 36135 (2016).

    PubMed  PubMed Central  Google Scholar 

  13. Teh, C.E. et al. Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis. Nat. Commun. 7, 13353 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Sasaki, Y. et al. Defective immune responses in mice lacking LUBAC-mediated linear ubiquitination in B cells. EMBO J. 32, 2463–2476 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Smit, J.J. et al. The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J. 31, 3833–3844 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Stieglitz, B. et al. Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP. Nature 503, 422–426 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011).

    CAS  PubMed  Google Scholar 

  19. Tokunaga, F. et al. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011).

    CAS  PubMed  Google Scholar 

  20. Keusekotten, K. et al. OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell 153, 1312–1326 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Rivkin, E. et al. The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis. Nature 498, 318–324 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Fiil, B.K. et al. OTULIN restricts Met1-linked ubiquitination to control innate immune signaling. Mol. Cell 50, 818–830 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Rodgers, M.A. et al. The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation. J. Exp. Med. 211, 1333–1347 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Satpathy, S. et al. Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation. Mol. Syst. Biol. 11, 810 (2015).

    PubMed  PubMed Central  Google Scholar 

  25. Emmerich, C.H. et al. Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains. Proc. Natl. Acad. Sci. USA 110, 15247–15252 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Smahi, A. et al. Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti. Nature 405, 466–472 (2000).

    CAS  PubMed  Google Scholar 

  27. Döffinger, R. et al. X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling. Nat. Genet. 27, 277–285 (2001).

    PubMed  Google Scholar 

  28. Filipe-Santos, O. et al. X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production. J. Exp. Med. 203, 1745–1759 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Xu, G., Paige, J.S. & Jaffrey, S.R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat. Biotechnol. 28, 868–873 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Wagner, S.A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell. Proteomics 10, M111.013284 (2011).

    PubMed  PubMed Central  Google Scholar 

  31. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Danielsen, J.M.R. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol. Cell. Proteomics 10, M110.003590 (2011).

    PubMed  Google Scholar 

  33. Meierhofer, D., Wang, X., Huang, L. & Kaiser, P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J. Proteome Res. 7, 4566–4576 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926 (2003).

    CAS  PubMed  Google Scholar 

  35. Rahighi, S. et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation. Cell 136, 1098–1109 (2009).

    CAS  PubMed  Google Scholar 

  36. Baker, R.T. et al. Using deubiquitylating enzymes as research tools. Methods Enzymol. 398, 540–554 (2005).

    CAS  PubMed  Google Scholar 

  37. Povlsen, L.K. et al. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nat. Cell Biol. 14, 1089–1098 (2012).

    CAS  PubMed  Google Scholar 

  38. Haas, T.L. et al. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol. Cell 36, 831–844 (2009).

    CAS  PubMed  Google Scholar 

  39. Takiuchi, T. et al. Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN. Genes Cells 19, 254–272 (2014).

    CAS  PubMed  Google Scholar 

  40. Lewis, M.J. et al. UBE2L3 polymorphism amplifies NF-κB activation and promotes plasma cell development, linking linear ubiquitination to multiple autoimmune diseases. Am. J. Hum. Genet. 96, 221–234 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Müller-Rischart, A.K. et al. The E3 ligase Parkin maintains mitochondrial integrity by increasing linear ubiquitination of NEMO. Mol. Cell 49, 908–921 (2013).

    PubMed  Google Scholar 

  42. Khan, M., Syed, G.H., Kim, S.J. & Siddiqui, A. Hepatitis B virus-induced Parkin-dependent recruitment of linear ubiquitin assembly complex (LUBAC) to mitochondria and attenuation of innate immunity. PLoS Pathog. 12, e1005693 (2016).

    PubMed  PubMed Central  Google Scholar 

  43. Lamothe, B. et al. Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation. J. Biol. Chem. 282, 4102–4112 (2007).

    CAS  PubMed  Google Scholar 

  44. Yang, W.L. et al. The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science 325, 1134–1138 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang, M. et al. HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSα. Mol. Cell 55, 31–46 (2014).

    PubMed  PubMed Central  Google Scholar 

  46. Deng, L. et al. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).

    CAS  PubMed  Google Scholar 

  47. Aguileta, M.A. et al. The E3 ubiquitin ligase Parkin is recruited to the 26 S proteasome via the proteasomal ubiquitin receptor Rpn13. J. Biol. Chem. 290, 7492–7505 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Bremm, A. & Komander, D. Synthesis and analysis of K11-linked ubiquitin chains. Methods Mol. Biol. 832, 219–228 (2012).

    CAS  PubMed  Google Scholar 

  49. Lin, D.Y., Diao, J., Zhou, D. & Chen, J. Biochemical and structural studies of a HECT-like ubiquitin ligase from Escherichia coli O157:H7. J. Biol. Chem. 286, 441–449 (2011).

    CAS  PubMed  Google Scholar 

  50. Umebayashi, K., Stenmark, H. & Yoshimori, T. Ubc4/5 and c-Cbl continue to ubiquitinate EGF receptor after internalization to facilitate polyubiquitination and degradation. Mol. Biol. Cell 19, 3454–3462 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Stieglitz, B., Morris-Davies, A.C., Koliopoulos, M.G., Christodoulou, E. & Rittinger, K. LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep. 13, 840–846 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Catanzariti, A.M., Soboleva, T.A., Jans, D.A., Board, P.G. & Baker, R.T. An efficient system for high-level expression and easy purification of authentic recombinant proteins. Prot. Sci. 13, 1331–1339 (2004).

    CAS  Google Scholar 

  53. Licchesi, J.D. et al. An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains. Nat. Struct. Mol. Biol. 19, 62–71 (2011).

    PubMed  PubMed Central  Google Scholar 

  54. Martin, S.R. & Schilstra, M.J. Circular dichroism and its application to the study of biomolecules. Methods Cell Biol. 84, 263–293 (2008).

    CAS  PubMed  Google Scholar 

  55. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    CAS  PubMed  Google Scholar 

  56. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  PubMed  Google Scholar 

  57. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  PubMed  Google Scholar 

  58. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    CAS  PubMed  Google Scholar 

  59. Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

    CAS  PubMed  Google Scholar 

  60. Vizcaíno, J.A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Asada (Nippon Medical School), R.T. Baker (Clinical Genomics), A. Bremm (Goethe University School of Medicine), C. Behrends (Goethe University School of Medicine), J. Chen (Rockefeller University), K.-H. Chun (Yonsei University College of Medicine), I. Dikic (Goethe University School of Medicine), H. Jiang (National Institute of Biological Sciences), J.U. Jung (University of Southern California), M. Kobayashi (Kanazawa University), C. White (Rosalind Franklin University of Medicine and Science) and A. Wittinghofer (Max Planck Institute of Molecular Physiology) for providing reagents and M. Akutsu, A. Carpy, J. Lopez-Mosqueda, M. Olma and S. Wahl for initial help with the project. We thank S. Schaubeck for excellent technical assistance. We are especially grateful to J.-I. Inoue (University of Tokyo) for providing us with TRAF6−/− MEFs and J.W. Bowman for providing us with the detailed LUBAC purification protocol. We thank A. Bremm, J. Lopez-Mosqueda and B. Srinivasan for discussions, comments and reading of the manuscript. We would also like to thank Reviewer 1 for their comments, which helped us to substantially improve the manuscript. K.K. was supported by the UPStream grant (EU, FP7, ITN project 290257).

Author information

Authors and Affiliations

Authors

Contributions

K.H. and K.K. developed the concept and designed the experiments. K.H. developed the INT-Ub tool, prepared the constructs and performed initial validation experiments. K.K. optimized the method, carried out all the cell biology and biochemical experiments, prepared all the inducible cell lines and samples for MS experiments and analyzed the results, including MS data. B.M., C.T. and M.F.-W. generated and analyzed MS data. B.S., I.P. and S.K. performed and analyzed CD and NMR experiments. K.H. and K.K. wrote the manuscript with contribution from all authors.

Corresponding author

Correspondence to Koraljka Husnjak.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The design and in vitro validation of INT-Ub.

(A) Ub chain building potential of various Ub mutants (top). Ub molecule with an insertion point of STREP-tag II (black) and the following color-coded residues: K (red), M (orange), G76 (blue) and I44 (yellow). This Figure was generated with PyMOL software by modifying a 1UBQ.pdb file (bottom). (B) Coomassie staining of recombinant Ub variants used for CD measurements in (C) Far-UV CD spectra of wild-type Ub (blue), Ub.7KR (red), INT-Ub (green) and INT-Ub.7KR (violet). (D) and (E) A direct comparison of in vitro ubiquitination reaction for untagged Ub, INT-Ub and INT-Ub.ΔGG. (F) Pull-down assay between INT-Ub and UBDs that can recognise single Ub moieties. (G) A direct comparison of diUb and INT-diUb ability to bind linear polyUb-specific UBD UBAN. (H) A direct comparison of USP2-cc DUB assay with diUb and INT-diUb. (I) A direct comparison of OTULIN DUB assay with diUb and INT-diUb. Blot and gel images for Supplementary Figure 1b,f-i were cropped to improve the conciseness of the presentation (full blots and gel images are available in Supplementary Figure 12).

Supplementary Figure 2 In vivo validation of INT-Ub.

(A) A comparison of INT-Ub (left panel), INT-Ub.7KR (middle panel), INT-Ub.ΔGG (right panel) and endogenous Ub levels in T-REx HEK293T cells upon doxycycline induction. To visualise levels of unconjugated Ub proteins, lysates were treated with USP2-cc. (B) A comparison of the effect of untagged Ub and Ub.ΔGG on NFκB transcriptional activity (control for Figure 2f). NFκB transcriptional activity was measured by a luciferase assay. Results are shown as means and s.e.m. (n=3). n.s=no statistically significant difference (p>0.05), determined by the two tailed Student’s t-test. (C) The effect of OTULIN C129A overexpression on the abundance of HMW linear polyubiquitinated proteins, was estimated by the linear Ub-specific Lub9 antibody upon denaturing INT-Ub.7KR Strep-Tactin PD. (D) The effect of TNFa stimulation (15 min) on the appearance of HMW ubiquitinated forms of NEMO upon denaturing INT-Ub.7KR Strep-Tactin PD. Blot images for Supplementary Figure 2a,c,d were cropped to improve the conciseness of the presentation (full blots are available in Supplementary Figure 12).

Supplementary Figure 3 Mass-spectrometry-based identification of novel linear polyUb-modified substrates.

(A) A schematic representation of the preliminary SILAC-based MS proteomic approach for the identification of linear polyUb-modified substrates upon 15 min of TNFα stimulation. Note that the SILAC labels were reversed in the subsequent triplicate MS analysis. (B) Scatter plots shown as a function of protein intensity and SILAC ratios (left: M/L, right: H/L) for preliminary MS data in (A) and Supplementary Table 1. SILAC ratios of log2>1 are marked in red. Several putative LUBAC substrates selected for the final validation are indicated in the plots. The number of identified protein pairs is marked in left upper part of each plot. (C) Experimental reproducibility of three independent experimental replicates of MS proteomic experiments from Figure 4a. The Pearson Correlation Coefficients are depicted in the left upper part of each plot. (D) Expression levels of putative linear polyUb-modified proteins in two different sets of T-REx HEK293T cell lines used in MS screens. Blot images for Supplementary Figure 3d were cropped to improve the conciseness of the presentation (full blots are available in Supplementary Figure 12).

Supplementary Figure 4 Validation of novel linear polyUb-modified substrates.

(A) Effect of active LUBAC overexpression on the appearance of HMW species above expected protein size for transiently transfected HA-tagged selected MS hits. Inactive LUBAC (HOIP C885A/HOIL-1L) was used as negative control. (B) Interaction studies between selected MS candidates and LUBAC components HOIP and HOIL-1. HOIP and/or HOIL-1L were transiently overexpressed in HEK293T cells and used for MBP or GST pull-down assays with recombinant putative substrates. (C) In vitro ubiquitination assays with recombinant LUBAC components and recombinant HIS-tagged putative substrates. (D) In vitro ubiquitination sample containing recombinant LUBAC complex and TRAF6 was treated with recombinant OTULIN to confirm TRAF6 modification by linear polyUb chains. (E) MS/MS spectra showing the presence of linear Ub signature peptide GGMQIFVK in IP sample, in which transiently overexpressed HA-TRAF6 and HOIP/HOIL-1L were immunoprecipitated with HA agarose under denaturing conditions. (F) Analysis of the presence of HMW species of endogenous SEPT2 (panel 1), HDAC6 (panel 2), VDAC1 (panel 3) and TRAF6 (panel 4) in the presence of ectopic LUBAC complex in HEK293T cells by denaturing linear Ub IP. Blot images for Supplementary Figure 4 (except e) were cropped to improve the conciseness of the presentation (full blots are available in Supplementary Figures 13, 14).

Supplementary Figure 5 TRAF6 is a novel LUBAC substrate in NFκB signalling.

(A) The effect of HOIP silencing on LUBAC-dependent linear polyubiquitination of TRAF6 upon IL-1β-stimulation. (B) MS/MS spectra showing identified ubiquitinated residues of TRAF6. HA-TRAF6 and LUBAC components were transiently overexpressed in HEK293T cells and used for GST pull-down with NEMO UBANx3 and further processed for MS analysis. (C) In vitro ubiquitination assays with recombinant LUBAC components and either HIS-tagged TRAF6 C70A or TRAF6 C70A K339/K497/K518R mutant. (D) Levels of recombinant HIS-tagged TRAF6 C70A and C70A K339/K497/ K518R mutants. The difference in protein size observed on gradient gels (Supplementary Figure 5c) is not visible on non-gradient 10 % SDS-PAGE. (E) IκBα phosphorylation and degradation kinetics in reconstituted TRAF6-/- MEFs. Cells were stimulated with IL-1β (10 ng/ml) for the indicated time periods. Quantification of the protein levels was performed by ImageJ software. Mean of three independent experimental replicas was calculated and is shown below figure panels (compared to time point 0 for each cell line). (F) Expression levels of various HA-TRAF6 variants stably reconstituted in TRAF6-/- MEFs. Blot images for Supplementary Figure 5 (except b) were cropped to improve the conciseness of the presentation (full blots are available in Supplementary Figure 15).

Source data

Supplementary Figure 6 Full blot images for Figures 1 and 2.

Full blot names indicate their position in the original Figures 1 and 2. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 7 Full blot images for Figure 3.

Full blot names indicate their position in the original Figure 3. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 8 Full blot images for Figure 5a.

Full blot names indicate their position in the original Figure 5a. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 9 Full blot images for Figure 5b.

Full blot names indicate their position in the original Figure 5b. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 10 Full blot images for Figure 5c.

Full blot names indicate their position in the original Figure 5c. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 11 Full blot images for Figure 6.

Full blot names indicate their position in the original Figure 6. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 12 Full blot images for Supplementary Figures 1, 2, 3.

Full blot names indicate their position in the original Supplementary Figures 1, 2, 3. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 13 Full blot images for Supplementary Figures 4a–d.

Full blot names indicate their position in the original Supplementary Figures 4a-d. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 14 Full blot images for Supplementary Figure 4f.

Full blot names indicate their position in the original Supplementary Figure 4f. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary Figure 15 Full blot images for Supplementary Figure 5.

Full blot names indicate their position in the original Supplementary Figure 5. Unless specifically indicated next to the blot, all the blots had the same protein marker (with 2 bands marked with +), as indicated by the schematic protein marker representation. Red frames indicate cropped parts used in the original figures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 3–5 (PDF 2356 kb)

Supplementary Table 1

Results of the mass spectrometry experiments. (XLSX 1454 kb)

Supplementary Table 2

Results of the triplicate SILAC-based mass spectrometryexperiment with INT-Ub variants (10 min of TNFα stimulation). (XLSX 4985 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kliza, K., Taumer, C., Pinzuti, I. et al. Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry. Nat Methods 14, 504–512 (2017). https://doi.org/10.1038/nmeth.4228

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.4228

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research