SUMO-binding entities make it possible to isolate endogenous SUMOylated proteins in vivo. This is rather challenging due to the presence of active SUMO-specific protease and the low fractions of SUMOylated proteins in vivo. The isolation of the SUMOylated proteome by using SUMO-binding entities is advantageous as it defers an increase in the overall affinity for SUMO substrates.
This is due to the fact that SUBEs are common in proteins that comprise tandem repeats of SIMs thereby recognizing specifically SUMO molecules on modified proteins. The use of SUMO-binding entities for the isolation and characterization of the SUMOylated proteome in liver cancer is a fast and sensitive method. It provides past information on the rather unknown role of the SUMOylation pathway in liver cancer in a potential therapeutic approach.
SUMO-binding entities can be used to isolate and characterize the SUMOylated proteome in other tissues besides the liver, both from human specimens and animal models, as well as in several cellular models. Although this technique is easy to perform, care should be taken when analyzing several samples at the same type. cooler cube to the several steps involved.
Therefore, we recommend careful leveling off of the tubes before initiating this experiment. The visual demonstration of the use of SUMO-binding entities facilitates the revolution of this protocol especially due to the difficulties of handling the bits and the loss of material during the protocol. Human hepatoma cells have been previously prepared by plating cells in P100 plates at a density of 1.2 to 1.5 million cells per dish and maintain them in standard growth media at 37 degrees Celsius.
When ready to use the cells, aspirate the media from the plates and wash them with five millimeters of sterile PBS. Put the plate on ice and add 500 microliters of lysis buffer supplemented according to the manuscript directions. Then use a cell-scraper to gently scrape the cells off the bottom of the plate into the lysis medium.
Alternatively, harvest the cells by trypsinization and add one milliliter of trypsin-EDTA to the plate making sure that the cells are covered. Put the plate in a 37 degree Celsius incubator for five minutes to allow them to detach from the plate, then add two milliliters of pre-warmed growth medium to stop the trypsinization. Centrifuge the cells at 150 times G for 10 minutes and aspirate the supernatant.
Then wash the cells with PBS and centrifuge for another 10 minutes. Aspirate the supernatant and add 500 microliters of lysis buffer. Centrifuge the lyse cells at 15000 times G and four degrees Celsius for 10 minutes, then transfer the supernatants to a fresh tube and discard the pellet.
When ready to use the harvested mouse livers, homogenize 75 milligrams of fresh or snapped frozen fragments in one milliliter of ice-cold lysis buffer. Run the homogenizer according to the manuscript directions. After tissue homogenization, centrifuge the samples at 15000 times G and four degrees Celsius for 10 minutes.
Transfer the supernatant to a fresh tube and discard the pellet. Prepare glutathione beads by adding one milliliter of de-ionized water to 70 milligrams of beads and reconstituting them overnight at four degress Celsius. After swelling, wash the beads three times with 10 milliliters of de-ionized water or PBS, centrifuging at 300 times G for five minutes after each wash.
After the washes, resuspend the beads in one milliliter of PBS to obtain a 50%slurry for each sample at 100 micrograms of GST-SUBEs or GST control to 100 microliters of the glutathione bead slurry and 500 microliters of PBS. Incubate the mixture at four degrees Celsius for at least two hours while rotating, then recover the beads by centrifugation at 300 times G for five minutes and resuspend them in PBS. Take 10%of the previously prepared cell lysate and dilute it in an equal volume of 3X boiling buffer.
Add 450 microliters of the clarified lysate to 100 microliters of the glutathione bead slurry. Incubate the lysate with the beads at four degrees Celsius for at least two hours while slowly rotating. After the incubation, spin down the beads at 300 times G for five minutes and collect the supernatant for analysis.
Transfer 10%of the total volume to a separate tube and add an equal volume of 3X boiling buffer. Wash the remaining sample three times by adding one milliliter of ice-cold PBS with 05%Tween 20 and spinning it down at 300 times G and four degrees Celsius for one minute. Carefully aspirate the supernatant making sure that no liquid remains.
Then elute the sample with 15 microliters of 3X boiling buffer and 15 microliters of lysis buffer. Run a western blot analysis according to the manuscript directions. If performing Mass-Spectrometry, desalt the peptides using stage-tip C18 microcolumns and resuspend them in 0.1%formic acid prior to analysis.
Load the samples onto an LC-MS system and analyze them in triplicate. Proceed with protein identification and abundance calculation using the associated software. Perform statistical analysis according to the manuscript directions.
This protocol has been used to isolate SUMOylated proteins in mice with glycine N-methyltransferase deficiency which causes spontaneous development of liver cancer and their wild type littermates. Ponceau S staining was performed on the input, flow-through, and BOUND Fractions from the SUBEs pull-down assay. Western blot analysis of LKB1 captured with SUBEs shows that the levels of LKB1 SUMOylation are augmented in liver tumors.
Human hepatoma cells and non-transformed liver epithelial cells were used to investigate the capacity of the SUMO-trap to interact with naturally SUMOylated proteins. First, conventional protein staining was used to visualize the total material captured with SUBEs. Then Mass-Spectrometry showed that 742 proteins were enriched in the hepatoma cell SUBEs sample and 577 were enriched in the liver epithelial non-transformed cell SUBEs sample.
When performing experiments with SUBEs, it is important to maintain cells and tissues on ice and add specific milliliters to the lysis buffer in order to maintain the purity of SUMOylated. After visualization of the SUMOylated proteome with SUBEs, we can find the characterization by using either western blot analysis or Mass-Spectrometry. The application of the SUBEs to isolate and characterize the SUMOylated proteome in liver cancer tissues and hepatoma cells is paving the way to explore the role of the post-translational modifications of SUMO in liver cancer which may provide a potential therapeutic mechanism.
