This protocol is important because it is simple to perform and within reach of any laboratory. This protocol allows the isolation of HLA or MHC type I or type II peptides from human or mouse samples. This method can address scientific questions related to all research areas involving the immune system, whether in cancer, virology, or autoimmune diseases.
This protocol was built to be accessible to anyone working in a research laboratory. Just follow the steps. Begin activating the Sepharose cyanogen bromide beads by weighing 80 milligrams per sample and transferring them to a 15-milliliter conical tube.
Add five milliliters of one-millimolar hydrochloric acid. Pipette up and down five times to facilitate resuspension of the dried beads, and then fill the conical tube with an additional 8.5 milliliters of one-millimolar hydrochloric acid. Rotate the beads at 20 RPM for 30 minutes at room temperature using a rotator device.
Centrifuge the beads at 200x G for two minutes at room temperature and remove the supernatant by aspiration. Add 500 microliters of coupling buffer to the beads palette, then transfer the bead suspension to a new, two-milliliter centrifuge tube and set it aside. For coupling the antibodies to these beads, add two milligrams of the selected antibody in a new, two-milliliter microcentrifuge tube and make up the volume to one milliliter with a coupling buffer solution to get a final concentration of two milligrams per milliliter.
Transfer the previously prepared bead solution and add it to the antibody solution. Then, rotate the microcentrifuge tube at 20 RPM for 120 minutes using a rotator device, centrifuge the beads, then remove the supernatant as described previously. Now begin blocking and washing of the beads by first adding one milliliter of 0.2-molar glycine to the microcentrifuge tube containing the antibody-coupled beads, and rotating at 20 RPM for 60 minutes at room temperature using a rotator device.
After centrifuging and removing the supernatant, wash the beads with one milliliter PBS, remove the supernatant by centrifugation, and store the beads in one milliliter of PBS at four degrees Celsius until use. To isolate MHC class I or II peptides, thaw a frozen palette of 100 million cells by warming the bottom of the tube with a palm. Add 500 microliters of PBS to the palette and pipette up and down until the suspension is homogenous.
Transfer the suspension in a new, two-milliliter microcentrifuge tube and add an equal volume of cell lysis buffer. Rotate at 10 RPM for 60 minutes at four degrees Celsius using a rotator device. Centrifuge the cell lysate at 18, 000x G for 20 minutes at four degrees Celsius with full brake, and transfer the supernatant in a new, two-milliliter microcentrifuge tube.
Recover the antibody-coupled beads by centrifugation and removing the supernatant. Then, transfer the cell lysate supernatant to the antibody-coupled beads, and incubate at 10 RPM for 14 to 18 hours overnight at four degrees Celsius using a rotator device. On day two, remove the bottom lid of the polypropylene column, place the column onto the polypropylene column rack, and install an empty container underneath to collect flow-through.
Now rinse the polypropylene column with 10 milliliters of buffer A and let it drain by gravity. If the flow speed of liquid elution is too slow, further cut the bottom tip of the polypropylene column. Measure and collect the beads/lysate mixture and transfer it into the polypropylene column.
Let the liquid mixture elute by gravity. Optionally, collect and freeze 20 microliters of aliquot for Western blotting. Now wash the beads retained in the polypropylene column by adding 10 milliliters of buffer A and letting it elute by gravity.
Remove polypropylene column from the rack and place it on the top of a new, two-milliliter microcentrifuge tube, holding the column and tube together with hand. Add 300 microliters of 1%TFA to the polypropylene column and mix the beads by piping up and down five times. Then, transfer the eluate in a new, two-milliliter microcentrifuge tube.
For desalting and eluting the MHC peptides, add 200 microliters of methanol on top of the C-18 column and centrifuge at at 1, 546x G for three minutes to discard the flow-through. Add 200 microliters of 80%acetonitrile in 0.1%TFA on top of the C-18 column and again centrifuge to discard the flow-through. Then, add 200 microliters of 0.1%TFA, discarding the flow-through by centrifugation.
Finally, load 200 microliters of the MHC peptide complexes on top of the C-18 column, discard the flow through by centrifugation, and repeat until the complete volume is loaded. Next, add 200 microliters of 0.1%TFA, centrifuge to discard the flow-through, transfer the C-18 column into a new, two-milliliter microcentrifuge tube, and elute MHC peptides by adding 150 microliters of 28%acetonitrile in 0.1%TFA. After centrifugation, collect the flow-through in a new, 1.5-milliliter microcentrifuge tube.
Repeat the elution process twice for a total volume of 450 microliters, and freeze at minus 20 degrees Celsius until analyzed by LC-MS-MS. Use these purified peptides for identification of MHC class I and II peptides in a mass spectrometer. The binding efficiency of beads was illustrated by a significant decrease in signal staining intensity of light and heavy chains when beads are covalently-bound to the CNBr beads, compared to the antibody before coupling.
The isolation of MHC peptide complexes following acidic elution was confirmed by the strong detection signal of the MHC peptide complexes using anti-HAL-ABC heavy-chain antibody. The intra-and inter-individual reproducibility of the results using the current protocol was also estimated. The average coefficient of variation for the number of peptides detected across three different biological replicates was small, which varied from 1.9 to 11%However, the identity of peptides varied considerably, and the proportion of peptides reproducibly-detected across three biological replicates ranged from 39%to 63%The heat maps generated showed predicted MHC binding strength of the identified peptides.
For the mouse MHC class I and class II peptides, the predicated strong or weak binders were 89 and 87%respectively. For human HLA class I and class II peptides, the predicated strong or weak binders were 97 and 70%respectively. The peptide binding motifs of the mouse and human for LHC class I and class II peptides were also generated.
Prior to the optimization of this protocol, the isolation of murine MHC class I and II peptides was quite more difficult. This optimized protocol will facilitate the isolation of murine MHC class I and II peptides from in vivo mouse models, for instance, routinely used for the validation of many diverse, disease-related studies.
