Assessment of Ubiquitin Chain Topology by Targeted Mass Spectrometry

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


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) 



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.


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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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