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Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

  • Focus: MS in the Characterization of Microorganisms: Research Article
  • Published:
Journal of The American Society for Mass Spectrometry

Abstract

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

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Acknowledgments

The authors thank Drs. Matthew Monroe, Brooke Deatherage-Kaiser, and Alexandra Rutledge for comments, input, and suggestions. This work was supported by the National Institute of Allergy and Infectious Diseases (NIH/DHHS through interagency agreement Y1-AI-4894-01; project website www.SysBEP.org) and the National Institute for General Medical Sciences (GM094623). Proteomics capabilities were developed under support from the U.S. Department of Energy (DOE) Office of Biological and Environmental Research (BER), NIH grant 5P41RR018522-10 and National Institute of General Medical Sciences grant 8 P41 GM103493-10. Significant portions of this work were performed using EMSL, a DOE/BER national scientific user facility located at Pacific Northwest National Laboratory. The Pacific Northwest National Laboratory is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

Data Availability

The LC-MS/MS results are available at www.SysBEP.org that includes links to the raw proteomics data.

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  1. Daniel Lopez-Ferrer

    Present address: Caprion Proteomics US, LLC, Menlo Park, CA, 94025, USA

Authors and Affiliations

  1. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA

    Ernesto S. Nakayasu, Charles Ansong, Joseph N. Brown, Feng Yang, Daniel Lopez-Ferrer, Wei-Jun Qian, Richard D. Smith & Joshua N. Adkins

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  1. Ernesto S. Nakayasu
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Nakayasu, E.S., Ansong, C., Brown, J.N. et al. Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes. J. Am. Soc. Mass Spectrom. 24, 1214–1223 (2013). https://doi.org/10.1007/s13361-013-0619-8

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