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.