System-Wide Identification of Wild-Type SUMO-2 Conjugation Sites

SUMOylation is a reversible post-translational modification regulating all nuclear processes. Identification of SUMOylation sites by mass spectrometry has been hampered by bulky tryptic fragments, which thus far necessitated the use of mutated SUMO. Here, we present a dataset generated through a SUMO-specific protease-based methodology which circumvents this problem, dubbed Protease-Reliant Identification of SUMO Modification (PRISM). PRISM allows for detection of SUMOylated proteins as well as identification of specific sites of SUMOylation while using wild-type SUMO. The method is generic and could be widely applied to study lysine post-translational modifications. PRISM was employed in combination with high-resolution mass spectrometry to identify SUMOylation sites from HeLa cells under standard growth conditions and in response to heat shock. 751 wild-type SUMOylation sites on endogenous proteins were identified, including 200 dynamic SUMO sites in response to heat shock. Thus, we have developed the first method capable of quantitatively studying wild-type mammalian SUMO at the site-specific and system-wide level.