Issue 10, 2010
From the journal:

Molecular BioSystems

Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry†

Hamid Mirzaei,a   Richard S. Rogers,a   Barbara Grimes,a   Jimmy Eng,ab   Alan Aderema  and  Ruedi Aebersold*acd  

* Corresponding authors

a Institute for Systems Biology, Seattle, USA

b Dept. of Genome Sciences, University of Washington, USA

c Institute of Molecular Systems Biology, ETH Zurich (Swiss Federal Institute of Technology Zurich), Wolfgang-Pauli-Str. 16, ETH Hönggerberg, HPT E 75, CH-8093 Zürich, Switzerland
E-mail: aebersold@imsb.biol.ethz.ch
Fax: +41 44 633 10 51
Tel: +41 44 633 31 70

d Faculty of Science, University of Zurich, Zurich, Switzerland

Abstract

Protein ubiquitination is an essential post-translational modification (PTM) involved in the regulation of a variety of cellular functions, including transcription and protein degradation. Proteins can be both mono- or poly-ubiquitinated. Poly-ubiquitin chains vary in the manner by which the ubiquitin proteins are linked and their total length. Different poly-ubiquitin structures are thought to specify different fates for the target protein but the correlation between poly-ubiquitin structures and their specific cellular function(s) is not well understood. We have developed a set of specific and quantitative targeted mass spectrometry assays to determine the frequency of different types of inter-ubiquitin linkages in poly-ubiquitin chains relative to the total ubiquitin concentration. We chemically synthesized heavy isotope labeled reference peptides that represent the products generated by tryptic digestion of the known forms of inter-ubiquitin links for the yeast Saccharomyces cerevisiae and human, in addition to all peptides from tryptic digestion of a single ubiquitin molecule for these two species. We used these peptides to develop optimized Selected Reaction Monitoring (SRM) assays for their unambiguous detection in biological samples. We used these assays to profile the frequency of the different types of inter-ubiquitin linkages in a mixture of in vitro assembled human poly-ubiquitin chains and 15 isolated poly-ubiquitinated proteins from S. cerevisiae. We then applied the method to detect toxin induced changes in the poly-ubiquitination profile in complex and enriched protein samples.

Graphical abstract: Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry

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Article information

DOI
https://doi.org/10.1039/C005242F
Article type
Paper
Submitted
27 Apr 2010
Accepted
25 Jun 2010
First published
06 Aug 2010

Mol. BioSyst., 2010,6, 2004-2014

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Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry

H. Mirzaei, R. S. Rogers, B. Grimes, J. Eng, A. Aderem and R. Aebersold, Mol. BioSyst., 2010, 6, 2004 DOI: 10.1039/C005242F

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