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Mass Spectrometric Determination of Protein Ubiquitination

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Book cover Post-Translational Modification of Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1934))

Abstract

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. This “tag” can be used to distinguish a ubiquitinated peptide from the unmodified version, and can be incorporated into automated database searching. Several tags are discussed, the GGK and LRGGK tags, resulting from complete and incomplete tryptic digestion of the protein, and the STLHLVLRLRGG tag from a gluC-digested protein.

A ubiquitinated peptide has two N-termini—one from the original peptide and the other from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain, and any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the “normal” peptide will vary in mass according to the peptide being modified and will therefore not be of general diagnostic use. These diagnostic ions, found through examination of the MS/MS spectra of model ubiquitinated tryptic and gluC peptides, have not previously been reported. These ions can be used to trigger precursor ion scanning in automated MS/MS data acquisition scanning modes.

Viorel Mocanu is deceased.

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Acknowledgments

The study on the fragmentation of ubiquitinated peptides [33] was funded by a gift from an anonymous donor to support research in proteomics and cystic fibrosis, and grants from the Cystic Fibrosis Foundation (CFFTI STUTTS01U0) and from NIH (ES11997, 5U54HD035041-07, and P30 CA 16086-25). Current funding for CHB and CEP is from Genome Canada and Genome British Columbia through the Genomic Innovations Network (GIN), for operations (204PRO) and technology development (214PRO), and the Genomics Technology Platform (264PRO). CHB is also grateful for support from the Leading Edge Endowment Fund (University of Victoria), and from the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, Quebec, Canada). CHB is also grateful for support from the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, Quebec, Canada).

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Author notes

  1. Carol E. Parker

    Present address: Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada

  2. Maria R. E. Warren Hines

    Present address: Hagn Scientific, LLC, Efland, NC, USA

  3. Susanna F. Greer

    Present address: American Cancer Society, Atlanta, GA, USA

  4. Christoph H. Borchers

    Present address: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada

  5. Christoph H. Borchers

    Present address: Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC, Canada

  6. Christoph H. Borchers

    Present address: Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada

Authors and Affiliations

  1. Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Carol E. Parker, Maria R. E. Warren Hines, Viorel Mocanu & Christoph H. Borchers

  2. Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada

    Christoph H. Borchers

  3. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Susanna F. Greer

  4. Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada

    Christoph H. Borchers

  5. Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC, Canada

    Christoph H. Borchers

  6. Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada

    Christoph H. Borchers

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Correspondence to Christoph H. Borchers .

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  1. Recombinant Research and Development, Octapharma Biopharmaceuticals GmbH, Heidelberg, Germany

    Christoph Kannicht

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Dedicated to the memory of our friend and colleague, Viorel Mocanu

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Parker, C.E., Warren Hines, M.R.E., Mocanu, V., Greer, S.F., Borchers, C.H. (2019). Mass Spectrometric Determination of Protein Ubiquitination. In: Kannicht, C. (eds) Post-Translational Modification of Proteins. Methods in Molecular Biology, vol 1934. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9055-9_13

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