A high-yield double-purification proteomics strategy for the identification of SUMO sites


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 lysines have been substituted to arginines. Lysine deficiency renders the SUMO immune to digestion by the endoproteinase Lys-C, which in turn allows for stringent and high-yield tandem purification through the His10 tag. In addition, the His10-tagged SUMO also contains a C-terminal Q87R mutation, which accommodates generation of SUMO-site peptides with a QQTGG mass remnant 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.

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Figure 1: The K0-SUMO method for the identification of SUMOylation sites.
Figure 2: The QQTGG remnant allows for dual-mode detection of SUMOylation sites with its ability to cyclize into pyroglutamate-QTGG (pyroQQTGG).
Figure 3: Schematic overview of the K0 method.
Figure 4: Typical chromatography profiles when analyzing tryptic digests of K0-SUMO samples.


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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.

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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.

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Correspondence to Alfred C O Vertegaal.

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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)

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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

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