Inclusion of enrichment strategies for wild-type SUMOylated proteins or SUMO-isopeptides pre-MS analyses at the protein-level enrichment involves the use of (i) affinity- or epitope-tagged SUMOylated proteins resulting in subsequent isolation and purification of SUMOylated proteins . These enrichment strategies represent a marked improvement in directing analytical-based proteomic analyses toward wild-type SUMOylation; (ii) anti-SUMO antibodies, which have the capability to recognize, isolate, and purify SUMOylated proteins [8, 79]; and (iii) SUMO affinity traps containing the presence of sumo interacting motif (SIM) sequences to capture SUMOylated proteins .
A recent enrichment strategy has been developed toward improving analytical- based proteomic analysis toward wild-type SUMOylation. This strategy is termed Protease-Reliant Identification of SUMO modification (PRISM) and has been developed and applied to enable the LC-MS/MS analysis of wild-type SUMO(2) ylation . Briefly, the PRISM workflow involves the use of (a) affinity-tagged SUMO(2) proteins expressed at controlled levels to purify SUMOylated proteins, (b) covalent attachment of the primary amino groups of lysine residues on SUMOylated proteins on to beads via acetylation, resulting in the acetylation and subsequent blocking of free lysine residues not involved in SUMO(2)ylation, thus enabling the concentration of SUMO(2)ylated proteins and subsequent removal of free SUMO(2) proteins that are not involved in the SUMOylation of target proteins, and (c) the utilization of the specificity of SENP2s for SUMO(2)/(3) proteins to specifically remove the SUMO(2) iso-chain from the SUMO(2)ylated proteins resulting in the reintroduction of previously SUMO(2)ylated lysine residues as free lysine residues, subsequently resulting in the generation of peptides rather than isopeptides post-tryptic digestion. Trypsinization of these proteins resulted in the free lysine residues of the peptides enabling the identification of 751 wild-type SUMO(2)ylation sites on 400 proteins on a global scale in HeLa cells . The removal of the SUMO(2) iso-chains from the SUMO(2)ylated proteins using SENP2s presents an attractive analytical advantage during LC-MS/MS under low-energy type CID conditions by eliminating the dominant complex fragmentation pattern associated with the full-length tryptic iso-chain  and the need for analysis using specialist bioinformatic software previously used for SUMOylation [34, 35]. However, acetylation of multiple lysine residues can result in the generation of much larger peptides of higher charge state post-tryptic digestion due to multiple tryptic miscleavages, potentially resulting in complex low-energy CID spectra, which may affect the performance of bioinformatic interpretation of the spectra. As alluded to in the COFRADIC approach  to the analysis of ubiquitination, acetylation is not 100% efficient in blocking all free lysine residues; therefore, a proportion (determined to be 3% at the peptide level in data generated from the PRISM strategy ) of the SUMOylation sites identified could potentially be due to false-positive identifications. However, this enrichment strategy is a vast improvement in directing analytical-based proteomic analyses away from mutagenic approaches  and more toward wild-type SUMOylation on a global scale.
Recently, an enrichment strategy was developed at the SUMO-isopeptide level and involved:
- 1) The use of a wild-type alpha-lytic protease (WaLP)-specific enzyme, which has been shown to have semi-specific C-terminal cleavage toward threonine residues at a frequency of 30% and generate peptide lengths amenable to good analytical performance . It has been suggested that this enzyme would be used in the generation of a GG iso-chain specifically on SUMO- isopeptides.
- 2) The use of the K-e-GG antibody to enrich for the wild-type alpha-lytic- derived (K)-GG iso-chain in the same way it does for the tryptically derived (K)-GG iso-chain . A 30% cleavage frequency is similar to the combined frequency of the atypically tryptic SUMO(2)/(3)-isopeptides detected in the CRA(K)-enabled approach.
The concept of this strategy sounds promising and future presentation and publication of data will be of high interest to the community.