This protocol allows us to investigate antigens present on the cell surface of various cell types to find antigens specific to that type of cell or tissue. The technique we present here allows for enrichment of cell membrane proteins out of a whole cell lysate, ultimately for analysis by mass spectrometry-based proteomics. So one specific application of this protocol is to apply it to tumor cells to look for antigens present on the surface of those cancer cells and not normal cells that could serve as novel targets for cancer immunotherapies.
One thing to note about this protocol is that it can be important to optimize your sample input for your experiment of interest. So it may take several iterations or titrations to find the optimal conditions. So demonstrating the procedure will be Akul Naik, a junior specialist from our laboratory.
To begin, remove the frozen biotin-labeled cell pellet from minus 80 degrees Celsius and thaw it on ice. Add 500 microliters of 2X Lysis Buffer to the pellet, mix thoroughly, and vortex vigorously for 30 seconds. Sonicate the cell lysate on ice, and centrifuge the lysate at 17, 200 G for 10 minutes at 4 degrees Celsius to pellet any remaining debris.
While the lysate is centrifuging, add 100 microliters of NeutrAvidin Agarose resin beads to a filtration column attached to the vacuum manifold. Apply a gentle vacuum and wash the beads by flowing 3 milliliters of Wash Buffer 1 over them. Remove the filtration column from the vacuum manifold, cap the bottom, and add the clarified cell lysate to the column with the beads.
Cap the top of the column and incubate the lysate with the beads on an end-to-end rotor for 120 minutes at 4 degrees Celsius. After completing the lysate incubation, place the filtration column back onto the vacuum manifold, and apply a gentle vacuum. Wash the beads by flowing five milliliters of Wash Buffer 1 over them.
Repeat the washing step with five milliliters of Wash Buffer 2 and Wash Buffer 3. Once the final Wash Buffer has flowed entirely through the beads, remove the column from the manifold. Then add 100 microliters of lyse solution to the beads and transfer them to a 1.7-milliliter low protein binding micro centrifuge tube with a pipette.
Briefly spin the tube to settle the beads and remove 50 microliters of the solution. Next, place the tube on a heat block shaker at 65 degrees Celsius for 10 minutes at 1000 RPM shaking. Resuspend the dried trypsin in the digest tube from the kit by adding 210 microliters of the resuspend solution and pipetting it up and down several times to ensure all the dried trypsin is resuspended.
At the end of bead incubation, remove it from the heat block shaker and add 50 microliters of the resuspended trypsin solution to the beads. Incubate the tube at 37 degrees Celsius, shaking at 500 RPM for at least 90 minutes to digest the protein. Once the digestion is complete, transfer the solution containing the beads to a spin column and insert the column into a 1.7-milliliter low protein binding micro centrifuge tube.
Spin the tube to separate the digested peptide solution from the beads. Next, add 100 microliters of the stop solution to stop the digestion reaction and acidify the peptide solution. Using a tube adapter, place a desalting column in a 1.7-milliliter low protein binding micro centrifuge tube.
Add the entire acidified peptide solution to the column. Spin the column at 3, 800 G for 3 minutes, so that the solution flows through the column. Discard the flow-through as the peptides are now bound to the column.
Add 200 microliters of Wash 1 solution to the column, spin at 3, 800 G for 3 minutes at room temperature, and then discard the flow-through. Repeat the washing step with 200 microliters of Wash 2 solution. Transfer the column to a new labeled 1.7-milliliter low protein binding micro centrifuge tube.
Add 100 microliters of elute solution to the column and spin at 3, 800 G for 3 minutes at room temperature. The peptides are now eluted off the column into the tube. Place the peptide solution in a vacuum centrifuge and allow it to dry overnight.
Place the dried down peptides at minus 80 degrees Celsius until ready to quantify and analyze on the mass spectrometer. Proteome analysis was performed to characterize the enriched cell surface proteins using liquid chromatography tandem mass spectrometry. A final peptide concentration of approximately 630 nanograms per microliter was obtained based on the colorimetric peptide quantification assay.
Mass spectrometry data analyzed using MaxQuant and subsequent dataset analysis of the cell surface proteins yielded 601 surface proteins in the sample, approximately 27%of all the proteins identified. A plot representing the distribution of the identified protein in Andromeda score is shown here, and the scatter plot illustrates the sample-to-sample correlation. The box plots depicted the run-to-run variation.
And the sample-wise distribution plot represented the data quality of replicates. The most important thing to keep in mind is where the peptides are at each step. Initially, they're in solution, then, they're bound to the beads until the digestion, then, they're bound to desalting column until they're finally eluted.
Following this procedure, there are various ways to validate a handful of the proteins identified, such as targeted flow cytometry and targeted proteomics. This will give more detailed information about the expression levels of these proteins across the sample.
