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Assessment of Ubiquitin Chain Topology by Targeted Mass Spectrometry

  • Joseph Longworth
  • Gunnar DittmarEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1977)

Abstract

Protein homeostasis is essential for the survival of cells. It is closely related to the functioning of the ubiquitin-proteasome system, which utilizes the small protein ubiquitin as a posttranslational modifier (PTM). Clinically, the modification is of great importance as its disruption is the cause of many diseases. Unlike other PTMs, ubiquitin can encode several cellular signals by being attached as a single molecule or as a chain of several ubiquitins in various conformations. Thus, ubiquitin signaling is dependent not only on the site of attachment but also on the chain type, the so-called ubiquitin chain topology.

The most reliable quantification method for the chain topology uses a bottom-up targeted mass spectrometry-based proteomics technique. While similar to other targeted proteomics techniques, the measurement of ubiquitination chain topology is complicated. First, the ubiquitin chains in the sample have to be biochemically stabilized. Second, the selection of peptides for the analysis is restricted to a given set harboring the PTMs and does not allow for optimization for amenability to mass spectrometry-based quantification. Instead, the topology-characteristic peptides are fixed. We here present such a methodology, including notes for a successful application.

Key words

Ubiquitin Chain topology Mass spectrometry Proteomics Parallel reaction monitoring (PRM) Quantitative proteomics Posttranslational modification (PTM) 

Notes

Acknowledgments

The authors would like to thank Dr. Antoine Lesur for his helpful advice on various technical issues examined in this chapter and reviewing the manuscript.

References

  1. 1.
    Goldstein G, Scheid M, Hammerling U et al (1975) Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells. Proc Natl Acad Sci U S A 72:11–15CrossRefGoogle Scholar
  2. 2.
    Pickart CM, Eddins MJ (2004) Ubiquitin: structures, functions, mechanisms. Biochim Biophys Acta 1695:55–72CrossRefGoogle Scholar
  3. 3.
    Diehl JA, Fuchs SY, Haines DS (2010) Ubiquitin and cancer. Genes Cancer 1:679–680CrossRefGoogle Scholar
  4. 4.
    Senft D, Qi J, Ronai ZA (2018) Ubiquitin ligases in oncogenic transformation and cancer therapy. Nat Rev Cancer 18:69–88CrossRefGoogle Scholar
  5. 5.
    Zheng Q, Huang T, Zhang L et al (2016) Dysregulation of ubiquitin-proteasome system in neurodegenerative diseases. Front Aging Neurosci 8:303CrossRefGoogle Scholar
  6. 6.
    Atkin G, Paulson H (2014) Ubiquitin pathways in neurodegenerative disease. Front Mol Neurosci 7:63CrossRefGoogle Scholar
  7. 7.
    Zinngrebe J, Montinaro A, Peltzer N, Walczak H (2014) Ubiquitin in the immune system. EMBO Rep 15:28–45CrossRefGoogle Scholar
  8. 8.
    Hu H (2016) Ubiquitin signaling in immune responses. Cell Res 26:27Google Scholar
  9. 9.
    Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479CrossRefGoogle Scholar
  10. 10.
    Beaudette P, Popp O, Dittmar G (2016) Proteomic techniques to probe the ubiquitin landscape. Proteomics 16:273–287CrossRefGoogle Scholar
  11. 11.
    Akutsu M, Dikic I, Bremm A (2016) Ubiquitin chain diversity at a glance. J Cell Sci 129:875–880CrossRefGoogle Scholar
  12. 12.
    Kwon YT, Ciechanover A (2017) The ubiquitin code in the ubiquitin-proteasome system and autophagy. Trends Biochem Sci 42:873–886CrossRefGoogle Scholar
  13. 13.
    Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198–207CrossRefGoogle Scholar
  14. 14.
    Chait BT (2006) Mass spectrometry: bottom-up or top-down? Science 314:65–66CrossRefGoogle Scholar
  15. 15.
    Goldknopf IL, Busch H (1977) Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24. Proc Natl Acad Sci U S A 74:864–868CrossRefGoogle Scholar
  16. 16.
    Lesur A, Domon B (2015) Advances in high-resolution accurate mass spectrometry application to targeted proteomics. Proteomics 15:880–890CrossRefGoogle Scholar
  17. 17.
    MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966–968CrossRefGoogle Scholar
  18. 18.
    Nielsen ML, Vermeulen M, Bonaldi T et al (2008) Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 5:459–460CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Proteomics of Cellular SignallingLuxembourg Institute of HealthStrassenLuxembourg

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