The overall goal of this procedure is to generate and pre fractionate peptides from formal and fixed and paraffin embedded or FFPE material for proteomic analysis. This is accomplished by isolation of the desired tissue by microdissection, followed by cell lysis. The second step is to generate peptides by sample processing.
Using multi enzyme digestion filter aided sample preparation. The peptides are then quantified by measuring their trytophan fluorescence. The final step is fractionation of the peptides using a strong anion exchanger based separation technique for peptide fractionation.
Ultimately, the prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10, 000 proteins. The main advantage of this technique over existing methods, like in gel or in solution digestion, is that it is applicable to minute amounts of clinical samples such as a laser microdisect cells. The method provides samples containing perfection and pure peptides.
What facilitates proteomic exploration of human tissue and diseases? The entire protocol consists of techniques developed and published in the past. These include high recovery fasp, pipet tip ation, and peptide content determination based on tryptophan fluorescence demonstrating the procedure will be Mrs.
Catina settle a technician from our laboratory To begin prepare tissue slices for microdisect cut sections with a microtome. The thickness of the slices depends on the sample. Usually microdissection works well with slices of seven to 10 micrometers.
After irradiating the membrane slides with UV light for 45 minutes, mount the sections on them before drying at 37 degrees Celsius for two hours. De parize the mounted sections by two successive incubations in consecutive jars of xylene for two and a half minutes and one and a half minutes. Then rehydrate the sections by consecutive.
One minute incubations in absolute ethanol, 70%ethanol. And finally, water. After staining the sections with Meyer's Hematin for 20 seconds.
Rinse with water for one minute and allow to air dry. Collect the cell population of interest using a laser capture microdissection system. When using the palm instrument, collect the samples in the adhesive caps using three microliters of tissue lysis buffer for 10 nanoliters of the microdisect tissue.
Apply the buffer to the micro dissected material in the cap and close the tube. Collect the suspension following a short centrifugation. Then lice the micros dissected tissue at 99 degrees Celsius in a heating block with agitation for one hour.
Clarify the crude extract by centrifugation at 16, 000 Gs and 18 degrees Celsius for 10 minutes. Mix up to 50 microliters of the clarified lysate with 200 microliters of the UA solution in an ultra filtration unit. Then centrifuge at 14, 000 GS until less than 10 microliters of the solution remains in the filter.
Usually this step requires 10 to 15 minutes of centrifugation. Next, pipette 200 microliters of the UA solution to the ultra filtration unit and centrifuge. As before, empty the collection tube before pipetting 50 microliters of the I A A solution into the ultra filtration unit.
Mix at 600 RPM in a thermo mixer at room temperature for one minute and centrifuge. As before, continue ultra filtration and centrifugation steps as outlined in the text protocol accompanying this video. Then transfer the filtration units to new collection tubes.
Pipette 40 microliters of digestion buffer with endo proteinase lic C before mixing at 600 RPM in the thermo mixer for one minute. Incubate the units in a wet chamber at 37 degrees Celsius for 18 hours. After centrifuging the filter units as done previously, pipette 160 microliters of the DB buffer and centrifuge.
The filter units the pooled flow through contains the peptides released by endo protease lic C.Next collect the triptych peptides as described in the text protocol. The measurement of the peptide content in the protein digests can be performed in diluted buffer because the tryptophan residues are well accessible to the solvent. To quantify peptides first pipette 0.2 milliliters of assay buffer DB into the quartz Q vet.
Then place the Q vet into the instruments qve holder and record the emission spectrum for the blank. Prepare a calibration curve using 0.1 microgram steps by adding one microliter aliquots of the tryptophan standard solution to the Q vet and gently mixing with the buffer db. After cleaning the Q vet, pipette the sample in and record the sample spectrum.
Proceed to calculate the protein and peptide concentration as described in the text protocol before fractionation. Prepare the SAX tip column by stacking six layers of EM.Import ion membrane in a common 0.2 milliliter pipette tip. Make two SAX tip columns per sample.
Next, prepare the stage tip by stacking three layers of M four C 18. In a 0.2 milliliter pipette tip, make six stage tips for each sample. To begin.
Fractionation dilute the peptide solutions obtained by digestions with ly C with 0.2 milliliters of BRI and Robinson universal buffer. pH 11 similarly dilute the peptide solutions obtained by digestions with trypsin with 0.2 milliliters of Britain and Robinson Universal buffer. pH five assemble the SAX tip column in the centrifugal tube adapter lid before washing and acqui equilibrating the SAX tip column and the C 18 stage tips as outlined in the text protocol.
Next, assemble the SAX tip column in the C 18 stage tip. Load the sample solutions into the Equilibrated SAX tip columns and centrifuge the columns at 5, 000 Gs for three minutes. Wash the pipette tip columns with 0.1 milliliters of the diluted samples.
Facilitate the flow by centrifugation at 5, 000 Gs.Transfer the SAX tip column to the next C 18 stage tip and continue to elute the peptides as described in the text protocol. After washing the C 18 stage tips with 0.05 milliliters of buffer a elute the fractions with 0.05 milliliters of buffer B into vials, later used for injection of the samples into a liquid chromatography system assembled with a mass spectrometer representative. Results of protein extraction and digestion are shown here.
Lysis of 250 nanoliters of tissue, which corresponds to a micro dissected area of 25 square millimeters from a 10 micrometer slice, results in approximately 45 micrograms of total protein following the two-step consecutive digestion in the fasts format with endo protease slice C and trypsin results in yields of more than 50%of the total protein. Shown here is a representative fluorescence measurement of peptide content, the spectra of the Trytophan standard and the sample show maxima at 350 to 360 nanometers. The intensity of the fluorescence is proportional to the trytophan content.
After watching this video, you should have a good understanding of how to prepare samples from high tissue for proteomic analysis using microdissection technique, sample processing, and peptide fractionation Once mastered, this technique can be done in three to four hours plus a time needed for protein indigestion. While attempting this procedure, it's important to remember to use freshly prepared tissue slices After its development. This technique paved the way for researchers in the field of proteomics to explore the proteomes of tissues and cells and the abnormalities in humans using arval for in fixed and paren embedded material.
