Skip to main content
SpringerLink
Log in
Book cover

Analysis of Post-Translational Modifications and Proteolysis in Neuroscience pp 111–125Cite as

Identification and Quantification of K63-Ubiquitinated Proteins in Neuronal Cells by High-Resolution Mass Spectrometry

  • Protocol
  • First Online:

  • 975 Accesses

Part of the book series: Neuromethods ((NM,volume 114))

Abstract

Protein ubiquitination is a widespread modification serving many roles in neuronal development and function. Moreover, the accumulation of ubiquitinated proteins is a prominent feature of neurodegeneration and oxidative stress related diseases. The emerging diversity of ubiquitin signals beyond protein degradation—based on distinct types of polyubiquitin chains—necessitates tools that specifically and quantitatively investigate its different functions. Polyubiquitin chains linked by lysine 63 (K63) relate to neurodegenerative diseases, but most of their targets and functions have not yet been elucidated. K63-linked ubiquitin has been implicated in DNA repair signaling, endocytosis, and inclusion body clearance. In addition, we recently identified an important role of K63 ubiquitin in regulating translation in response to oxidative stress in yeast. The change in K63 ubiquitination in response to hydrogen peroxide is conserved in mouse hippocampal HT22 cells, highlighting the importance of this modification in higher eukaryotes. In this chapter, we discuss cutting-edge methodologies available to investigate protein ubiquitination in a proteome-wide and quantitative manner, and we present a method to simultaneously isolate and identify the specific targets of K63 ubiquitin. This method relies on the use of a selective K63 ubiquitin isolation tool with subsequent analysis of protein content by high-resolution mass spectrometry. The proposed workflow can be combined with additional methods for ubiquitin analysis and applied to several research models. This approach can also provide the scientific basis for the development of new tools to isolate and identify targets of other ubiquitin linkages.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Komander D (2009) The emerging complexity of protein ubiquitination. Biochem Soc Trans 37:937–953. doi:10.1042/BST0370937

    Article  CAS  PubMed  Google Scholar 

  2. Tai HC, Schuman EM (2008) Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction. Nat Rev Neurosci 9:826–838. doi:10.1038/nrn2499

    Article  CAS  PubMed  Google Scholar 

  3. Dennissen FJ, Kholod N, van Leeuwen FW (2012) The ubiquitin proteasome system in neurodegenerative diseases: culprit, accomplice or victim? Prog Neurobiol 96:190–207. doi:10.1016/j.pneurobio.2012.01.003

    Article  CAS  PubMed  Google Scholar 

  4. Ciechanover A, Brundin P (2003) The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 40:427–446

    Article  CAS  PubMed  Google Scholar 

  5. Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292:1552–1555. doi:10.1126/science.292.5521.1552

    Article  CAS  PubMed  Google Scholar 

  6. Li W et al (2008) Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle’s dynamics and signaling. PLoS One 3:e1487. doi:10.1371/journal.pone.0001487

    Article  PubMed  PubMed Central  Google Scholar 

  7. van Wijk SJ, Timmers HT (2010) The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. FASEB J 24:981–993. doi:10.1096/fj.09-136259

    Article  PubMed  Google Scholar 

  8. Olzmann JA et al (2007) Parkin-mediated K63-linked polyubiquitination targets misfolded DJ-1 to aggresomes via binding to HDAC6. J Cell Biol 178:1025–1038. doi:10.1083/jcb.200611128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tan Z et al (2007) Mutant ubiquitin found in Alzheimer’s disease causes neuritic beading of mitochondria in association with neuronal degeneration. Cell Death Differ 14:1721–1732. doi:10.1038/sj.cdd.4402180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kishino T, Lalande M, Wagstaff J (1997) UBE3A/E6-AP mutations cause Angelman syndrome. Nat Genet 15:70–73. doi:10.1038/ng0197-70

    Article  CAS  PubMed  Google Scholar 

  11. Nascimento RM, Otto PA, de Brouwer AP, Vianna-Morgante AM (2006) UBE2A, which encodes a ubiquitin-conjugating enzyme, is mutated in a novel X-linked mental retardation syndrome. Am J Hum Genet 79:549–555. doi:10.1086/507047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yi JJ, Ehlers MD (2007) Emerging roles for ubiquitin and protein degradation in neuronal function. Pharmacol Rev 59:14–39. doi:10.1124/pr.59.1.4, 59/1/14 [pii]

    Article  CAS  PubMed  Google Scholar 

  13. Hallengren J, Chen PC, Wilson SM (2013) Neuronal ubiquitin homeostasis. Cell Biochem Biophys 67:67–73. doi:10.1007/s12013-013-9634-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479. doi:10.1146/annurev.biochem.67.1.425

    Article  CAS  PubMed  Google Scholar 

  15. Kulathu Y, Komander D (2012) Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat Rev Mol Cell Biol 13:508–523. doi:10.1038/nrm3394

    Article  CAS  PubMed  Google Scholar 

  16. Matsumoto ML et al (2010) K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Mol Cell 39:477–484. doi:10.1016/j.molcel.2010.07.001

    Article  CAS  PubMed  Google Scholar 

  17. Brown NG et al (2014) Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly. Mol Cell 56:246–260. doi:10.1016/j.molcel.2014.09.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hamilton AM, Zito K (2013) Breaking it down: the ubiquitin proteasome system in neuronal morphogenesis. Neural Plast 2013:196848. doi:10.1155/2013/196848

    PubMed  PubMed Central  Google Scholar 

  19. Spence J, Sadis S, Haas AL, Finley D (1995) A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol Cell Biol 15:1265–1273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Peng J et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21:921–926. doi:10.1038/nbt849

    Article  CAS  PubMed  Google Scholar 

  21. Silva GM, Finley D, Vogel C (2015) K63 polyubiquitination is a new modulator of the oxidative stress response. Nat Struct Mol Biol 22:116–123. doi:10.1038/Nsmb.2955

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  23. Paine S et al (2009) Immunoreactivity to Lys63-linked polyubiquitin is a feature of neurodegeneration. Neurosci Lett 460:205–208. doi:10.1016/j.neulet.2009.05.074

    Article  CAS  PubMed  Google Scholar 

  24. Liu C et al (2007) Assembly of lysine 63-linked ubiquitin conjugates by phosphorylated alpha-synuclein implies Lewy body biogenesis. J Biol Chem 282:14558–14566. doi:10.1074/jbc.M700422200

    Article  CAS  PubMed  Google Scholar 

  25. Tan JM et al (2008) Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Hum Mol Genet 17:431–439. doi:10.1093/hmg/ddm320

    Article  CAS  PubMed  Google Scholar 

  26. Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28:868–873. doi:10.1038/nbt.1654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kim W et al (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44:325–340. doi:10.1016/j.molcel.2011.08.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Udeshi ND 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. Mol Cell Proteomics 12:825–831. doi:10.1074/mcp.O112.027094

    Article  CAS  PubMed  Google Scholar 

  29. Ziv I et al (2011) A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis. Mol Cell Proteomics 10:M111 009753. doi:10.1074/mcp.M111.009753

    Article  PubMed  PubMed Central  Google Scholar 

  30. Kirkpatrick DS et al (2006) Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat Cell Biol 8:700–710. doi:10.1038/ncb1436

    Article  CAS  PubMed  Google Scholar 

  31. Xu P et al (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137:133–145. doi:10.1016/j.cell.2009.01.041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Dammer EB et al (2011) Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease. J Biol Chem 286:10457–10465. doi:10.1074/jbc.M110.149633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hjerpe R et al (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10:1250–1258. doi:10.1038/embor.2009.192, embor2009192 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Udeshi ND, Mertins P, Svinkina T, Carr SA (2013) Large-scale identification of ubiquitination sites by mass spectrometry. Nat Protoc 8:1950–1960. doi:10.1038/nprot.2013.120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cox J et al (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4:698–705. doi:10.1038/nprot.2009.36

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the US National Science Foundation EAGER grant MCB-1355462 (CV), by the US National Institutes of Health K99 award ES025835 (GMS), and the Zegar Family Foundation Fund for Genomics Research at New York University (CV).

We thank R. Ratan (Burke Medical Research Institute) for providing the HT22 cells.

Author information

Authors and Affiliations

  1. Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY, 10003, USA

    Gustavo Monteiro Silva, Wei Wei, Sandhya Manohar & Christine Vogel

Authors
  1. Gustavo Monteiro Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sandhya Manohar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christine Vogel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Gustavo Monteiro Silva or Christine Vogel .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Wisconsin-Stout, Menomonie, Wisconsin, USA

    Jennifer Elizabeth Grant

  2. Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers University—New Jersey Medical School Cancer Center Newark, Newark, New Jersey, USA

    Hong Li

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Silva, G.M., Wei, W., Manohar, S., Vogel, C. (2015). Identification and Quantification of K63-Ubiquitinated Proteins in Neuronal Cells by High-Resolution Mass Spectrometry. In: Grant, J., Li, H. (eds) Analysis of Post-Translational Modifications and Proteolysis in Neuroscience. Neuromethods, vol 114. Humana Press, New York, NY. https://doi.org/10.1007/7657_2015_95

Download citation

  • DOI: https://doi.org/10.1007/7657_2015_95

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3470-6

  • Online ISBN: 978-1-4939-3472-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation