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

Developers

Ivo A. Hendriks and Alfred C. O. Vertegaal.

Description of the technology

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.

The technology allows to carry out the lysine-deficient (K0) method for efficient identification of SUMO-ylated lysines by mass spectrometry. 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 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 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.

Practical application

Proteins that are modified by SUMO are predominantly localized to the nucleus, and SUMO regulates virtually all nuclear processes, including cell cycle control and DNA repair. SUMO has been also implicated in diseases such as cancer, Alzheimer’s and Parkinson’s, thereby marking SUMO as a potential therapeutic target. Thus, the technology as a method of identification of SUMO-sites can turn up to be useful in the field of drug discovery for search and development of new drug, targeting SUMO, for therapies of age-associated neurodegenerative diseases.

Laboratories

• Department of Molecular Cell Biology, Leiden University Medical Center, Leiden (the Netherlands)

Links

Publications

• Hendriks, I.A. and Vertegaal A.C.O. «A high-yield double-purification proteomics strategy for the identification of SUMO sites." 11 Nature Protocols (2016): 630–1649.
• Hendriks, I.A. et al. «Uncovering global SUMOylation signaling networks in a site-specific manner." 21 Nat. Struct. Mol. Biol. (2014): 927–936.
• Eifler, K. & Vertegaal, A.C. «Mapping the SUMOylated landscape." FEBS J. (2015).