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Proteome-wide identification of SUMO modification sites by mass spectrometry

Nature Protocols volume 10, pages 1374–1388 (2015)Cite this article

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Abstract

The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the ɛ-amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific consequences of SUMO modification requires knowledge of the location of conjugation sites, and we have developed a straightforward protocol for the proteome-wide identification of SUMO modification sites using mass spectrometry (MS). The approach described herein requires the expression of a mutant form of SUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) is replaced with a lysine (SUMOKGG). Digestion of SUMOKGG protein conjugates with endoproteinase Lys-C yields a diGly motif attached to target lysines. Peptides containing this adduct are enriched using a diGly-Lys (K-ɛ-GG)-specific antibody and identified by MS. This diGly signature is characteristic of SUMOKGG conjugation alone, as no other ubiquitin-like protein (Ubl) yields this adduct upon Lys-C digestion. We have demonstrated the utility of the approach in SUMOylation studies, but, in principle, it may be adapted for the site-specific identification of proteins modified by any Ubl. Starting from cell lysis, this protocol can be completed in ∼5 d.

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Figure 1: 6His-SUMO2T90K as a biochemical tool for the large-scale identification of SUMOylation sites.
Figure 2: Overview of the PROCEDURE for the enrichment and large-scale identification of SUMO2-modified peptides.
Figure 3: Proteome-wide identification of SUMOylation sites enabled by the efficient enrichment and improved acquisition of modified peptides by MS.
Figure 4: Increased precursor ion count and improved fragment ion sequence coverage enabled by the acquisition of MS/MS spectra with ultrasensitive settings.

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Acknowledgements

T.T. is funded through the EU Seventh Framework Programme (FP7A-PEOPLE-2011-ITN). I.M. was supported by a Sir Henry Wellcome Fellowship (Wellcome Trust 088957/Z/09/Z) and the DFG (Deutsche Forschungsgemeinschaft; Cologne Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases). E.G.J. and M.H.T. are funded through a Cancer Research UK programme grant (C434/A13067). R.T.H. holds a Wellcome Trust Senior Investigator Award (098391/Z/12/Z).

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Author notes

  1. Ivan Matic

    Present address: Present address: Max Planck Institute for Biology of Ageing, Cologne, Germany.,

  2. Triin Tammsalu and Ivan Matic: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK

    Triin Tammsalu, Ivan Matic, Ellis G Jaffray, Michael H Tatham & Ronald T Hay

  2. MRC Protein Phosphorylation and Ubiquitination Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK

    Adel F M Ibrahim

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  1. Triin Tammsalu
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Contributions

T.T. optimized sample processing and conducted sample preparation, MS analysis, data analysis and bioinformatic analysis. R.T.H. and I.M. conceived the enrichment and sample processing strategy. I.M. developed the workflow for the MS analysis and edited the manuscript. E.G.J. purified recombinant proteins and conducted preliminary in vitro assays. A.F.M.I. created the stable cell lines. M.H.T. consulted at multiple stages. T.T., R.T.H. and M.H.T. co-wrote the manuscript.

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Correspondence to Ronald T Hay.

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The authors declare no competing financial interests.

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Tammsalu, T., Matic, I., Jaffray, E. et al. Proteome-wide identification of SUMO modification sites by mass spectrometry. Nat Protoc 10, 1374–1388 (2015). https://doi.org/10.1038/nprot.2015.095

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