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A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis

Nature Protocols volume 5, pages 1447–1459 (2010)Cite this article

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Abstract

Ubiquitination can have profound effects on the stability and function of cellular proteins. Mass spectrometry (MS) can be used to map the specific amino acid residues that are conjugated to ubiquitin in a target protein. However, the purification required for proteomic analysis can be challenging. In this paper, we describe a bimolecular affinity purification scheme for the isolation of a specific ubiquitinated protein in which affinity moieties are fused to ubiquitin and to a target protein of interest. After ubiquitin conjugation in vivo, the protein target acquires two affinity tags, allowing the specific purification of its ubiquitin-modified forms. To prevent deubiquitination after lysis or the copurification of interacting cofactors, this procedure is performed after protein denaturation using polyhistidine and biotinylation tags. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified in large amounts of sufficient purity for MS analysis and for mapping of ubiquitin acceptor sites.

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Figure 1: Sequential purification of ubiquitinated proteins.
Figure 2: Fusion of target proteins to the biotinylation tag.
Figure 3: Purification results for selected target proteins.
Figure 4: Mass spectrometry analysis of a ubiquitinated protein.

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Acknowledgements

This work was supported by an R01 DK073639 grant and the University of Texas DOCS Award to E.B., and by an R01 DE19249 grant to K.S.J.-E.J. We thank J. Strahler and M. Hurley at the Michigan Proteome Consortium for their initial assistance in sample analysis.

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Author notes

  1. Ezra Burstein

    Present address: Present address: Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.,

Authors and Affiliations

  1. Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA

    Gabriel N Maine & Ezra Burstein

  2. Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan, USA

    Gabriel N Maine

  3. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Haiying Li & Iram W Zaidi

  4. Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

    Venkatesha Basrur & Kojo S J Elenitoba-Johnson

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  1. Gabriel N Maine
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Contributions

E.B. conceived and supervised the project. The specific tandem affinity purification strategy used here was devised by G.N.M. and E.B. The initial experimental conditions were determined by G.N.M., and subsequent repeats of the purification scheme, including some of the data shown here for COMMD1, were performed by I.W.Z. and H.L. V.B. and K.S.J.-E.J. contributed to the mass spectrometry analysis of COMMD1.

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Correspondence to Ezra Burstein.

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Maine, G., Li, H., Zaidi, I. et al. A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis. Nat Protoc 5, 1447–1459 (2010). https://doi.org/10.1038/nprot.2010.109

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