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Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

Nature Methods volume 8, pages 691–696 (2011)Cite this article

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Abstract

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.

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Figure 1: Ubiquitin pools and assay overview.
Figure 2: Assay validation.
Figure 3: Effect of acute proteasome inhibition on ubiquitin pools.
Figure 4: Ub-PSAQ analysis of ubiquitin pools in cytosolic and histone-enriched fractions from HEK293 cells.

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Acknowledgements

We thank E. Bennett, M. Reese and M. Bowen for discussions and critical reading of the manuscript. This work was funded in part by a grant (NS 04842) from the US National Institute of Neurological Disorders and Stroke.

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Authors and Affiliations

  1. Department of Biology, Stanford University, Stanford, California, USA

    Stephen E Kaiser, Brigit E Riley, R Sean Trevino & Ron R Kopito

  2. Caprion Proteomics U.S., LLC, Menlo Park, California, USA

    Thomas A Shaler, Christopher H Becker & Howard Schulman

Authors
  1. Stephen E Kaiser
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  7. Ron R Kopito
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Contributions

S.E.K. and R.R.K. devised the Ub-PSAQ strategy with contributions from B.E.R. and T.A.S.; S.E.K. prepared all protein affinity reagents and standards, performed the experiments and analyzed the data with input from B.E.R. and T.A.S.; B.E.R. prepared all cellular samples; T.A.S. performed all mass spectrometry analyses; and R.S.T. contributed to mass spectrometry data analysis. S.E.K. and R.R.K. wrote the manuscript, and B.E.R. contributed to figure preparation. B.E.R., T.A.S. and R.S.T. contributed to editing. C.H.B. and H.S. contributed to conceptual and experimental design. All authors discussed the results and manuscript.

Corresponding author

Correspondence to Ron R Kopito.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 2003 kb)

Supplementary Data 1

Overview of Ub-PSAQ analysis using the samples described in Figure 3b as an example. (XLS 225 kb)

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Kaiser, S., Riley, B., Shaler, T. et al. Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools. Nat Methods 8, 691–696 (2011). https://doi.org/10.1038/nmeth.1649

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