The overall goal of the following experiment is to use a combined proteomic approach to identify potential prognostic markers and or therapeutic targets in human diseases. This is achieved by isolating primary leukemic cells from patient's peripheral blood, carrying good or bad prognosis, and expressing both CD 19 and CD five surface markers to use for proteomic analysis as a second step. Leukemic cell lysates are resolved by two dimensional electrophoresis or two de, and the comparison of proteomic maps allows the identification of spots differentially expressed among good versus bad prognosis patients, which can subsequently be identified by mass spectrometry or ms.
Next different assays can be used in order to validate and dissect the role of the identified proteins in the natural history of the disease. The results show that different phosphorylation status of HS one detected by two de gels, correlates with the clinical outcome of the patients. The implication of this technique extend towards the therapy of chronic lymphocytic leukemia because it can help to identify protein involved in the progression of the disease that can be exploit as prognostic marker and or therapeutic targets demonstrating this procedure will be together with me.
Pamela ran to technician in the lab and a postdoc from an external lab Prior to starting this procedure, collect peripheral blood in vacutainer blood collection tubes with sodium heparin. Transfer blood into a 15 milliliter tube and add human B cell enrichment cocktail at 50 microliters per milliliter of whole blood. Then mix well and incubate for 20 minutes at room temperature.
Following incubation, dilute the sample with an equal volume of PBS plus 2%FBS mix gently and carefully overlay blood over the fly call making sure not to break the interface. Centrifuge the sample at 400 times G at room temperature for 20 minutes with no break. When finished, carefully aspirate the interface to recover the cells and place them into a new tube.
Wash the cells once with PBS after centrifugation and supernatant removal.Resus. Suspend the pellet in the remaining supernatant by manually flicking the tube back and forth following dilution. With complete RPMI medium, count the cells using the Trian blue exclusion method.
Next, add the following antibodies to 100 microliters of the purified cell suspension in a fax tube. Incubate the sample for 20 minutes at four degrees Celsius in the dark. After washing the sample with four milliliters of PBS, check the purity by flow cytometry.
Check the percent of the chronic lymphocytic leukemia or CLL cells, which Coex express CD 19 and CD five on their surface. Ensure that the preparations are virtually devoid of NK t lymphocytes and monocytes by checking the percent of CD three, CD 14, and CD 1656 in the plot, which should be near zero at this point. Solubilize CLL cell pellets with a final volume of 380 microliters of two dimensional electrophoresis buffer.
Apply one milligram of the protein samples to 18 IPG strips to perform iso electro focusing. After the run, equilibrate the strips in two milliliters of equilibration buffer, supplemented with 2%of fresh DTT for 15 minutes at room temperature. After discarding the solution, add two milliliters of equilibration buffer, supplemented with 2.5%I oto acetamide.
Then incubate the sample for 15 minutes at room temperature on a rocking platform. Following this, dry the strips on paper without touching the gel to remove the excess solution. Once dry, load each strip on top of a nine to 16%gradient SDS page gel.
Add a small volume of SDS electrophoresis buffer on top of the gel to facilitate loading of the strip. Next, seal the gel by adding approximately five milliliters of 0.8%agar solution to prevent floating of the strip. Once the electrophoresis unit has been assembled, fill it with SDS Electrophoresis Buffer.
Then perform the run at 60 milliamps per gel for four hours, four degrees Celsius, or by using a cooling system. After removing the gel from the electrophoretic unit, place it in the proper staining box. Prepare about 200 milliliters of each solution.
Carefully add each solution to the staining box containing the gel. Perform all incubations on a rocking platform. After the staining, remove the stop solution and leave the gel in a solution of 1%acetic acid until acquisition.
For the migration assay, resus suspend the previously purified cells. In complete RPMI prepare the lower transwell chamber by adding 600 microliters of complete RPMI with or without the specific stimuli. To measure the induced and the spontaneous migration respectively, place the upper chamber on top and let the system equilibrate for 30 minutes at 37 degrees Celsius in the incubator.
When finished, seed 10 to the six cells per 200 microliters in the upper chamber and incubate the plate for four hours at 37 degrees Celsius in the incubator. Following incubation, remove the upper chamber and collect the media in the lower chamber. Then wash the lower chamber once with one milliliter of PBS.
After collecting the PBS in a tube, centrifuge the sample for five minutes at 300 times G following supernatant removal. Resuspend the sample in 500 microliters of PBS by flow cytometry. Count the number of migrated cells after one minute of acquisition.
Check the number of cells in a bi dimensional dot plot considering the side scatters of the cells and the number of events visualized in one minute, which is the number to use for calculation. Isolated primary leukemic cell samples from CLL patients were grouped into two main subsets based on each patient's clinical features and analyzed by two DE, and MS masses within a certain mass tolerance. Were assigned to peptide sequences in the database and then assembled into a protein which is considered identified if it passes a certain probability score.
By this analysis, proteins mainly involved in cytoskeletal activity and metabolic processes were identified among these proteins. Hematopoietic lineage, cell specific protein or hs. One whose differential phosphorylation strongly associated with the clinical course of the disease was differentially expressed.
In particular, patients carrying a single spot experience a bad clinical outcome while patients with two spots have good prognosis. CLL cells carrying HS one as one spot, were found to have an impaired cytoskeletal activity in terms of migration, adhesion and actin polymerization compared to hypo phosphorylated HS.One, two spot samples. Thus explaining a different clinical behavior.
After watching this video, you should have a good understanding of how to use proteomic to identify biological candidates involved in the progression of the disease that can be used as prognostic marker and or therapeutic targets.
