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A conditional proteomics approach to identify proteins involved in zinc homeostasis

Nature Methods volume 13, pages 931–937 (2016)Cite this article

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Abstract

Zinc signaling and dynamics play significant roles in many physiological responses and diseases. To understand the physiological roles of zinc in detail, comprehensive identification of proteins under high concentration of mobile zinc ion is crucial. We developed a 'conditional proteomics' approach to identify proteins involved in zinc homeostasis based on a chemical proteomic strategy that utilizes designer zinc-responsive labeling reagents to tag such proteins and quantitative mass spectrometry for their identification. We used this method to elucidate zinc dyshomeostasis induced by nitric-oxide-triggered oxidative stress in glioma cells, and we unveiled dynamic changes of the zinc-related proteomes. Moreover, we characterized unknown zinc-rich vesicles generated by oxidative stress as endoplasmic-reticulum- and Golgi-related vesicles.

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Figure 1: Conditional proteomics by Zn2+-enhanced chemical labeling.
Figure 2: Activation of AIZin reagents by Zn2+ coordination.
Figure 3: Conditional protein labeling in live C6 cells treated with SNOC.
Figure 4: Proteomic analysis of labeled proteins in NO-stimulated C6 cells.

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Acknowledgements

We thank S. Kitagawa and D. Umeyama (Kyoto University, Japan) for X-ray structure determination. This work was supported by the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) of Molecular Technology for I.H. and a Research Fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists for T.M. (25-4988) and Y.N. (16J10316).

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Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan

    Takayuki Miki, Masashi Awa, Yuki Nishikawa, Shigeki Kiyonaka & Itaru Hamachi

  2. Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Masaki Wakabayashi & Yasushi Ishihama

  3. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan

    Itaru Hamachi

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  1. Takayuki Miki
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Contributions

T.M. and I.H. designed the project. T.M., M.A., and Y.N. performed synthesis and chemical labeling. T.M., M.W., and Y.I. performed LC–MS/MS analysis. T.M. and S.K. constructed the expression plasmids. The manuscript was written by T.M. and I.H., according to discussion with all authors.

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Correspondence to Itaru Hamachi.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–19 and Supplementary Note (PDF 14425 kb)

Supplementary Table 1

The list of labeled peptides by AIZin-1 in HeLa cell lysates (XLSX 37 kb)

Supplementary Table 2

The protein list and statistical analyses for proteomic data of labeled proteins in NO-stimulated C6 cells. (XLSX 366 kb)

Supplementary Table 3

The peptide sequences identified in the proteomic analysis of labeled proteins in NO-stimulated C6 cells. (XLSX 507 kb)

Supplementary Table 4

The protein list for proteomic analysis of labeled proteins in NO-stimulated C6 cells in the presence or absence of TPEN. (XLSX 84 kb)

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Miki, T., Awa, M., Nishikawa, Y. et al. A conditional proteomics approach to identify proteins involved in zinc homeostasis. Nat Methods 13, 931–937 (2016). https://doi.org/10.1038/nmeth.3998

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