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Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder

Nature Chemical Biology volume 12, pages 959–966 (2016)Cite this article

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Abstract

We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified ∼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.

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Figure 1: The Src superbinder as an efficient reagent for affinity purification of phosphotyrosyl peptides.
Figure 2: The pTyr superbinders compare favorably to antibodies in pTyr enrichment.
Figure 3: Ultra-deep Tyr phosphoproteomics by superbinder AP-MS/MS.
Figure 4: Functional features of the Tyr phosphoproteome.
Figure 5: Superbinder-based quantitative phosphoproteomics identified kinases that are potential cancer drivers.

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Acknowledgements

This work was supported, in part, by funds from the China State Key Basic Research Program Grants (2012CB910101 and 2016YFA0501402 to M.Y. and 2013CB911202 to H.Z.), the National Natural Science Foundation of China (21235006, 21321064 and 21535008 to H.Z., and 81361128015 to M.Y. and S.S.-C.L.), the Canadian Cancer Society (to S.S.-C.L.), the Canadian Institute of Health Research (to S.S.-C.L. and M.Y.) and the Ontario Research Fund (to S.S.-C.L.). L.L. is a recipient of “Distinguished Expert of Overseas Tai Shan Scholar”. M.Y. is a recipient of the National Science Fund of China for Distinguished Young Scholars (21525524). S.S.-C.L. holds a Canadian Research Chair in Functional Genomics and Cellular Proteomics.

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Author notes

  1. Yangyang Bian, Lei Li and Mingming Dong: These authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China

    Yangyang Bian, Mingming Dong, Kai Cheng, Yan Wang, Mingliang Ye & Hanfa Zou

  2. University of Chinese Academy of Sciences, Beijing, China

    Yangyang Bian & Mingming Dong

  3. Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.,

    Lei Li, Xuguang Liu, Tomonori Kaneko, Huadong Liu, Courtney Voss, Xuan Cao, David Litchfield & Shawn S-C Li

  4. School of Basic Medical Sciences, Qingdao University, Qingdao, China

    Lei Li

  5. Department of Oncology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.,

    David Litchfield & Shawn S-C Li

  6. The Children's Health Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.,

    Shawn S-C Li

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Contributions

S.S.-C.L., H.Z., M.Y. and M.D. conceived and designed the project. Y.B. and M.D. carried out the pTyr enrichment experiments and MS analysis under the supervision of H.Z., M.Y. and S.S.-C.L. L.L., M.Y., Y.B., M.D., K.C., T.K. and S.S.-C.L. analyzed the data. X.L., Y.W. and X.C. performed the cellular and biochemical experiments with input from S.S.-C.L. and D.L. C.V. and H.L. prepared the superbinder reagents. S.S.-C.L., L.L. and M.Y. wrote the manuscript with input from H.Z., M.D., Y.B. and D.L.

Corresponding authors

Correspondence to Mingliang Ye, Shawn S-C Li or Hanfa Zou.

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Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–19. (PDF 9135 kb)

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Supplementary Datasets 1–8. (XLSX 41064 kb)

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Bian, Y., Li, L., Dong, M. et al. Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder. Nat Chem Biol 12, 959–966 (2016). https://doi.org/10.1038/nchembio.2178

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