Protocol for SUMOylome analysis using a SUMO-T86K mutant combined with Lys-C digestion

Small ubiquitin-like modifier (SUMO) modification, also known as SUMOylation, is a critical post-translational modification. Here, we present a protocol using the SUMO-T86K (Thr86→Lys) mutant and Lys-C digestion, followed by enrichment of modified peptides and mass spectrometry, to identify SUMOylated proteins and modification sites. This strategy overcomes a major limitation of the commonly used SUMO-T86R (Thr86→Arg) approach with trypsin digestion, in which the resulting diglycine (GG) remnant is indistinguishable from ubiquitin modifications. For complete details on the use and execution of this protocol, please refer to Wu et al.¹.