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Detecting endogenous SUMO targets in mammalian cells and tissues

Abstract

SUMOylation is an essential modification that regulates hundreds of proteins in eukaryotic cells. Owing to its dynamic nature and low steady-state levels, endogenous SUMOylation is challenging to detect. Here, we present a method that allows efficient enrichment and identification of endogenous targets of SUMO1 and the nearly identical SUMO2 and 3 (SUMO 2/3) from vertebrate cells and complex organ tissue. Using monoclonal antibodies for which we mapped the epitope, we enriched SUMOylated proteins by immunoprecipitation and peptide elution. We used this approach in combination with MS to identify SUMOylated proteins, which resulted in the first direct comparison of the endogenous SUMO1- and SUMO2/3-modified proteome in mammalian cells, to our knowledge. This protocol provides an affordable and feasible tool to investigate endogenous SUMOylation in primary cells, tissues and organs, and it will facilitate understanding of SUMO's role in physiology and disease.

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Figure 1: Two well-known monoclonal antibodies work efficiently in denaturing SUMO1 and SUMO2/3 immunoprecipitation.
Figure 2: Identification of epitope-spanning peptides for monoclonal SUMO1 21C7 and SUMO2 8A2 antibodies.
Figure 3: Enrichment of endogenously SUMOylated proteins by immunoprecipitation and peptide elution from HeLa cells.
Figure 4: A new monoclonal anti-SUMO1 antibody works efficiently in immunoprecipitation and peptide elution.
Figure 5: Analysis of the endogenous SUMO1- and SUMO2/3-modified proteome of HeLa cells.
Figure 6: Endogenous SUMO targets can be enriched efficiently from mouse liver.

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Acknowledgements

We gratefully acknowledge M. Matunis (Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA) for making hybridomas for monoclonal anti-SUMO antibodies available to the community and H. de Thé (Université Paris Diderot, Sorbonne Paris Cité, Hôpital St. Louis 1, Paris, France) for generously providing anti-PML antibodies, R. Ramaker and N. Stankovic for help with antibody characterization and all lab members for sharing reagents and advice. This work was supported by the Deutsche Forschungsgemeinschaft (SPP1365, ME 2279/3 to F.M.) and the EU Network of Excellence Rubicon (to F.M.) and by fellowships of the Excellence Cluster CellNetworks (to S.V.B.) and the Deutsche Krebshilfe (to S.H.).

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J.B. and S.V.B. designed and carried out most experiments and wrote the manuscript. C.D. carried out experiments and wrote the manuscript. S.K., H.-H.H. and H.U. carried out MS and data analyses. M.B.D. and S.H. helped with mouse liver experiments. F.M. guided the project, designed experiments and wrote the manuscript.

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Correspondence to Frauke Melchior.

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Becker, J., Barysch, S., Karaca, S. et al. Detecting endogenous SUMO targets in mammalian cells and tissues. Nat Struct Mol Biol 20, 525–531 (2013). https://doi.org/10.1038/nsmb.2526

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