Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).
Total time
~4–6 hours per sample (excluding mass spectrometry analysis and data processing)
Model organism
Mouse (hepatocellular carcinoma models); HepG2 human hepatoma cells
Steps
1
Prepare human hepatoma cells for lysis
Harvest cultured hepatoma cells and lyse them using appropriate buffer conditions to release cellular proteins for subsequent SUMO enrichment.
▶ 01:58
2
Prepare mouse liver tumor tissue for lysis
Dissect and process liver tumor tissue from mouse models, followed by mechanical or enzymatic lysis to generate tissue lysates.
▶ 03:27
3
Bind GST-SUBEs to glutathione-agarose beads
Incubate GST-tagged SUMO-binding entities or GST control protein with glutathione-agarose resin to immobilize the binding domains.
▶ 03:54
4
Perform GST pull-down assay on lysates
Mix prepared cell or tissue lysates with GST-SUBE-coated beads to capture SUMO-conjugated proteins, then wash and elute bound material.
▶ 04:52
5
Identify SUMO targets by mass spectrometry
Perform tandem mass spectrometry on enriched SUMO-modified proteins to identify and quantify target substrates.
▶ 05:59
6
Analyze results via Western blot and proteomics
Validate enriched SUMO targets by Western blotting and integrate mass spectrometry data for comprehensive proteome characterization.
▶ 06:36