Abstract
Post-translational modifications (PTMs) have key roles in regulating protein-protein interactions in living cells. However, it remains a challenge to identify these PTM-mediated interactions. Here we develop a new lysine-based photo-reactive amino acid, termed photo-lysine. We demonstrate that photo-lysine, which is readily incorporated into proteins by native mammalian translation machinery, can be used to capture and identify proteins that recognize lysine PTMs, including 'readers' and 'erasers' of histone modifications.
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Acknowledgements
We acknowledge support from the Hong Kong Research Grants Council General Research Fund (GRF 17303114, GRF 17127915), Early Career Scheme (ECS) (HKU 709813P ) and Collaborative Research Fund (CRF C7037-14G). We acknowledge the University of Hong Kong for an e-SRT on Integrative Biology and grants from the Seed Funding Program (201411159101, 201409160027, 201311159007 and 201309176090). We thank X. Li for discussion. We thank E. Verdin (University of California, San Francisco), Q. Hao (The University of Hong Kong), H. Li (Tsinghua University), R.C. Trievel (University of Michigan), K. Guan (Fudan University) and H. Sun (City University of Hong Kong) for providing the plasmids.
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T.Y., X.-M.L. and X.D.L. designed the experiments and wrote the paper. T.Y. synthesized the small-molecule compounds and peptides and performed all of the photo-lysine–based cross-linking experiments in vitro. X.-M.L. performed all of the photo-lysine–based cross-linking experiments in living cells and prepared the MS sample. X.B. performed the ITC experiments. X.B. and Y.M.E.F. conducted the MS-based proteomics experiments. X.-M.L., X.B. and X.D.L. analyzed the MS/MS data.
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Supplementary information
Supplementary Text and Figures
Supplementary Results, Supplementary Figures 1–14 and Supplementary Notes 1–4. (PDF 12956 kb)
Supplementary Table 1
The estimated incorporation rates of photo-Lys at the identified sites. (XLSX 14 kb)
Supplementary Table 2
The estimated incorporation rates of photo-Lys at the identified histone sites. (XLSX 11 kb)
Supplementary Table 3
Summary of the identified histone- and chromatin-binding proteins. (XLSX 17 kb)
Supplementary Data Set 1
Proteins quantified in HeLa S3 cells with/without photo-Lys labeling. (XLSX 222 kb)
Supplementary Data Set 2
Proteins quantified in SILAC experiment to identify histone-/chromatin-binding proteins (XLSX 193 kb)
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Yang, T., Li, XM., Bao, X. et al. Photo-lysine captures proteins that bind lysine post-translational modifications. Nat Chem Biol 12, 70–72 (2016). https://doi.org/10.1038/nchembio.1990
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DOI: https://doi.org/10.1038/nchembio.1990
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