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 • Steps for establishing a doxycycline-inducible OSC line • Procedures for anti-FLAG-based enrichment of SUMO-conjugated proteins with NEM • Instructions for capturing SUMO-modified lysines via K-ε-GG remnant enrichment • Guidance on identifying SUMOylated proteins and modification sites by Orbitrap LC-MS/MS Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.