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

Hamid Mirzaei, Richard S. Rogers, Barbara Grimes, Jimmy Eng, Alan Aderem, Ruedi Aebersold

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30 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2004-2014
Number of pages11
JournalMolecular BioSystems
Volume6
Issue number10
DOIs
StatePublished - Oct 1 2010

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ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

Cite this

Mirzaei, H., Rogers, R. S., Grimes, B., Eng, J., Aderem, A., & Aebersold, R. (2010). Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry. Molecular BioSystems, 6(10), 2004-2014. https://doi.org/10.1039/c005242f