ABSTRACT The small ubiquitin-like modifier (SUMO) is a protein modifier that is post-translationally coupled to thousands of lysines in more than a thousand proteins. An understanding of

which lysines are modified by SUMO is critical in unraveling its function as a master regulator of all nuclear processes, as well as its involvement in diseases such as cancer. Here we

describe a protocol for the lysine-deficient (K0) method for efficient identification of SUMOylated lysines by mass spectrometry (MS). To our knowledge, the K0 method is the only currently

available method that can routinely identify >1,000 SUMO sites in mammalian cells under standard growth conditions. The K0 strategy relies on introducing a His10-tagged SUMO wherein all

after digestion with trypsin. This remnant possesses a unique mass signature and readily generates diagnostic ions in the fragment ion scans, which increases SUMO-site identification

confidence. The K0 method can be applied in any mammalian cell line or in any model system that allows for integration of the K0-SUMO construct. From the moment of cell lysis, the K0 method

takes ∼7 d to perform. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution

Subscribe to this journal Receive 12 print issues and online access $259.00 per year only $21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full

article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs *

Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ANTIBODY TOOLKIT REVEALS N-TERMINALLY UBIQUITINATED SUBSTRATES OF UBE2W Article Open access 29 July 2021 PROTEOMIC STRATEGIES

FOR CHARACTERIZING UBIQUITIN-LIKE MODIFICATIONS Article 29 July 2021 PEPPI-MS: GEL-BASED SAMPLE PRE-FRACTIONATION FOR DEEP TOP-DOWN AND MIDDLE-DOWN PROTEOMICS Article 16 January 2025

REFERENCES * Hendriks, I.A. et al. Uncovering global SUMOylation signaling networks in a site-specific manner. _Nat. Struct. Mol. Biol._ 21, 927–936 (2014). Article  CAS  Google Scholar  *

Dasso, M. Emerging roles of the SUMO pathway in mitosis. _Cell Div._ 3, 5 (2008). Article  Google Scholar  * Jackson, S.P. & Durocher, D. Regulation of DNA damage responses by ubiquitin

and SUMO. _Mol. Cell_ 49, 795–807 (2013). Article  CAS  Google Scholar  * Eckermann, K. SUMO and Parkinson's disease. _Neuromolecular. Med._ 15, 737–759 (2013). Article  CAS  Google

Scholar  * Flotho, A. & Melchior, F. Sumoylation: a regulatory protein modification in health and disease. _Annu. Rev. Biochem._ 82, 357–385 (2013). Article  CAS  Google Scholar  * Lee,

L., Sakurai, M., Matsuzaki, S., Arancio, O. & Fraser, P. SUMO and Alzheimer's disease. _Neuromolecular. Med._ 15, 720–736 (2013). Article  CAS  Google Scholar  * Becker, J. et al.

Detecting endogenous SUMO targets in mammalian cells and tissues. _Nat. Struct. Mol. Biol._ 20, 525–531 (2013). Article  CAS  Google Scholar  * Impens, F., Radoshevich, L., Cossart, P. &

Ribet, D. Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli. _Proc. Natl. Acad. Sci. USA_ 111, 12432–12437 (2014). Article  CAS  Google Scholar  *

Bernier-Villamor, V., Sampson, D.A., Matunis, M.J. & Lima, C.D. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and

RanGAP1. _Cell_ 108, 345–356 (2002). Article  CAS  Google Scholar  * Johnson, E.S. Protein modification by SUMO. _Annu. Rev. Biochem._ 73, 355–382 (2004). Article  CAS  Google Scholar  *

Ulrich, H.D. The fast-growing business of SUMO chains. _Mol. Cell_ 32, 301–305 (2008). Article  CAS  Google Scholar  * Guzzo, C.M. et al. RNF4-dependent hybrid SUMO-ubiquitin chains are

signals for RAP80 and thereby mediate the recruitment of BRCA1 to sites of DNA damage. _Sci. Signal._ 5, ra88 (2012). Article  Google Scholar  * Hay, R.T. SUMO-specific proteases: a twist in

the tail. _Trends Cell Biol._ 17, 370–376 (2007). Article  CAS  Google Scholar  * Mann, M. & Jensen, O.N. Proteomic analysis of post-translational modifications. _Nat. Biotechnol._ 21,

255–261 (2003). Article  CAS  Google Scholar  * Eifler, K. & Vertegaal, A.C. Mapping the SUMOylated landscape. _FEBS J._ (2015). * Denison, C. et al. A proteomic strategy for gaining

insights into protein sumoylation in yeast. _Mol. Cell. Proteomics_ 4, 246–254 (2005). Article  CAS  Google Scholar  * Tammsalu, T. et al. Proteome-wide identification of SUMO2 modification

sites. _Sci. Signal._ 7, rs2 (2014). Article  Google Scholar  * Lamoliatte, F. et al. Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling. _Nat. Commun._ 5,

5409 (2014). Article  CAS  Google Scholar  * Hendriks, I.A., D'Souza, R.C., Chang, J.G., Mann, M. & Vertegaal, A.C. System-wide identification of wild-type SUMO-2 conjugation sites.

_Nat. Commun._ 6, 7289 (2015). Article  Google Scholar  * Matic, I. et al. Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic

cluster SUMOylation motif. _Mol. Cell_ 39, 641–652 (2010). Article  CAS  Google Scholar  * Xiao, Z. et al. System-wide analysis of SUMOylation dynamics in response to replication stress

reveals novel SUMO target proteins and acceptor lysines relevant for genome stability. _Mol. Cell. Proteomics._ 14, 1419–1434 (2015). Article  CAS  Google Scholar  * Hendriks, I.A.,

Treffers, L.W., Verlaan-de Vries, M., Olsen, J.V. & Vertegaal, A.C. SUMO-2 orchestrates chromatin modifiers in response to DNA damage. _Cell Rep._ 10, 1778–1791 (2015). Article  CAS 

Google Scholar  * Knuesel, M., Cheung, H.T., Hamady, M., Barthel, K.K. & Liu, X. A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like

modifier 1 (SUMO-1) and a computational program. _Mol. Cell Proteomics_ 4, 1626–1636 (2005). Article  CAS  Google Scholar  * Wohlschlegel, J.A. Identification of SUMO-conjugated proteins and

their SUMO attachment sites using proteomic mass spectrometry. _Methods Mol. Biol._ 497, 33–49 (2009). Article  CAS  Google Scholar  * Bylebyl, G.R., Belichenko, I. & Johnson, E.S. The

SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast. _J. Biol. Chem._ 278, 44113–44120 (2003). Article  CAS  Google Scholar  * Wohlschlegel, J.A., Johnson, E.S., Reed, S.I.

& Yates, J.R. III Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. _J. Proteome. Res._ 5, 761–770 (2006).

Article  CAS  Google Scholar  * Bruderer, R. et al. Purification and identification of endogenous polySUMO conjugates. _EMBO Rep._ 12, 142–148 (2011). Article  CAS  Google Scholar  *

Barysch, S.V., Dittner, C., Flotho, A., Becker, J. & Melchior, F. Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal

antibodies. _Nat. Protoc._ 9, 896–909 (2014). Article  CAS  Google Scholar  * Cubenas-Potts, C. et al. Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes.

_Proteomics_ 15, 763–772 (2015). Article  CAS  Google Scholar  * Tammsalu, T. et al. Proteome-wide identification of SUMO modification sites by mass spectrometry. _Nat. Protoc._ 10,

1374–1388 (2015). Article  CAS  Google Scholar  * Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for

proteomics using StageTips. _Nat. Protoc._ 2, 1896–1906 (2007). Article  CAS  Google Scholar  * Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment.

_J. Proteome. Res._ 10, 1794–1805 (2011). Article  CAS  Google Scholar  * Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass

accuracies and proteome-wide protein quantification. _Nat. Biotechnol._ 26, 1367–1372 (2008). Article  CAS  Google Scholar  * Tiscornia, G., Singer, O. & Verma, I.M. Production and

purification of lentiviral vectors. _Nat. Protoc._ 1, 241–245 (2006). Article  CAS  Google Scholar  * Stacey, G.N. & Masters, J.R. Cryopreservation and banking of mammalian cell lines.

_Nat. Protoc._ 3, 1981–1989 (2008). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful for support from the European Research Council (grant 310913 to

A.C.O.V.) and the Netherlands Organization for Scientific Research (NWO; grant 700.59.006 to A.C.O.V.). We acknowledge R.C.J. D'Souza, B. Yang and M. Mann for assistance with the

initial optimization of the MS methodology. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands Ivo

A Hendriks & Alfred C O Vertegaal Authors * Ivo A Hendriks View author publications You can also search for this author inPubMed Google Scholar * Alfred C O Vertegaal View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS I.A.H. generated cell lines, optimized the entire purification method, created biological samples,

optimized the MS procedure, performed MS data analysis and conducted bioinformatics analysis. A.C.O.V. conceived the method and supervised the project. I.A.H. and A.C.O.V. wrote the

manuscript. CORRESPONDING AUTHOR Correspondence to Alfred C O Vertegaal. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED

SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 QQTGG AND PYROQQTGG VARIABLE MODIFICATIONS Screenshot of the QQTGG and pyroQQTGG (PyroQ) variable modifications as displayed in the Andromeda

software. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figure 1 and Supplementary Note (PDF 258 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS

ARTICLE CITE THIS ARTICLE Hendriks, I., Vertegaal, A. A high-yield double-purification proteomics strategy for the identification of SUMO sites. _Nat Protoc_ 11, 1630–1649 (2016).

https://doi.org/10.1038/nprot.2016.082 Download citation * Published: 11 August 2016 * Issue Date: September 2016 * DOI: https://doi.org/10.1038/nprot.2016.082 SHARE THIS ARTICLE Anyone you

share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the

Springer Nature SharedIt content-sharing initiative