Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The overall goal of this procedure is to characterize the composition of extracellular matrices from tissues by mass spectrometry. This is accomplished by first disrupting the selected tissue until it is completely homogenized. The second step involves decellularizing the tissue to enrich for extracellular matrix proteins while concomitantly depleting any intracellular components.

Next, the ECM enriched protein sample is denatured in the final step, the denatured ECM enriched protein sample is dec glycosylated and digested into peptides. Western blood analysis is used to monitor the quality of the decellularization and ECM protein enrichment. Ultimately, analysis by mass spectrometry of the peptides permits characterization of the composition of the ECM for that particular tissue.

This method can provide insight into the composition of the extracellular matrix of normal tissues, but can also be applied to other systems such as disease tissue like cancer. To begin, prepare all the buffers as indicated in text protocol. Table one, including the addition of protease inhibitors.

Then disrupt 100 milligrams of tissue in 500 microliters of buffer C with a tissue homogenizer until a uniform suspension is obtained. Transfer the homogenate to a tube rotator and incubate for 20 minutes At four degrees Celsius pipe had 10 to 20 microliters of the sample into a tube labeled as total tissue extract or starting material flash. Freeze the sample in liquid nitrogen and store it at minus 80 degrees Celsius.

Next, centrifuge the homogenate at 16, 000 times G for 20 minutes at four degrees Celsius. Collect the supernatant in a clean tube and label it as the cytosolic or C fraction.Flash. Freeze the C fraction and store it at minus 80 degrees Celsius.

Pipette there. Suspend the pellet in 400 microliters of buffer double, and then incubate on a tube rotator for 20 minutes at four degrees Celsius. Next, centrifuge the sample and discard the supernatant to extract the nuclear proteins First.

Resuspend the pellet in 150 microliters of buffer N and incubate on a tube rotator for 30 minutes at four degrees Celsius. Next, centrifuge the sample and collect the supernatant in a clean tube. Repeat the incubation in buffer N after centrifusion.

Combine the supernatants and label them as the nuclear or N fraction.Flash. Freeze the N fraction for storage at minus 80 degrees Celsius. Add 100 microliters of buffer M and resus, suspend the pellet.

Next, incubate the sample on a tube rotator for 30 minutes at four degrees Celsius before centrifuging it. Collect the supernatant in a clean tube and label it as the membrane or M fraction flash. Freeze the M fraction and store it at minus 80 degrees Celsius.

Re suspend the pellet in 200 microliters of buffer CS by vigorously pipetting, and then incubate the sample on a tube rotator for 30 minutes. At room temperature, centrifuge the sample for 30 minutes at room temperature and collect the supernatant in a clean tube and label it as the cytoskeletal or CS fraction. The pellet should now be markedly smaller.

Re suspend the pellet in 150 microliters of buffer C.Transfer the sample to a tube rotator and incubate it for 20 minutes at four degrees Celsius. Centrifuge for 20 minutes at four degrees Celsius and collect the supernatant. Next, combine the supernatant with the one from the CS buffer in the tube already labeled as the cytoskeletal or CS fraction.Flash.

Freeze the CS fraction and store it at minus 80 degrees Celsius. To wash the pellet resus. Suspend it in 500 microliters of PBS and then incubate on a tube rotator for five minutes at four degrees Celsius.

Centrifuge the tube and discard the supernatant. Wash a total of three times. To obtain the ECM enriched pellet store a small fraction of the ECM fraction for Western block QC analysis of the experiment.

Flash freeze the fractions in liquid nitrogen and store at minus 80 degrees Celsius. To begin digestion. First, re suspend the ECM enriched pellet in eight molar urea and add a solution of Dihi three etol in water to make a final concentration of 10 millimolar.

Transfer the tube to an incubator and shake it at 1400 RPM for two hours at 37 degrees Celsius. Next, cool the sample to room temperature and add a solution of reconstituted iota acetamide in water to a final concentration of 25 millimolar. Incubate the mixture in the dark for 30 minutes.

At room temperature, dilute to a two molar urea concentration with 100 millimolar ammonium bicarbonate, and then add P PGA F.Transfer the sample to an incubator and shake it at 1400 RPM for two hours. At 37 degrees Celsius, add endo protease, slice C to the mixture and shake at 1400 RPM for two hours at 37 degrees Celsius. Next, add trypsin and shake at 1400 RPM overnight at 37 degrees Celsius.

The cloudy solution should become clear after the overnight digest at a second deli quad of trypsin and shake for an additional two hours to inactivate the trypsin pipette 50%tri fluoro acetic acid into the mixture in one microliter increments until the pH is less than two. Monitor the pH by spotting one microliter of the peptide solution on pH paper. Centrifuge the solution for five minutes at room temperature.

Finally, collect the supernatant in a clean, low retention tube. Store the ECM peptide solution at minus 20 degrees Celsius. The Western blots from the decellularization of mirroring lung tissue show extraction of intracellular components such ASIN beta one actin GA, dh, and histones in the intermediate fractions.

These components are almost completely absent from the ECM fraction, although some minor histone contamination remains. In contrast, collagen one is highly enriched in the ECM fraction, but is not detected in the other fractions, suggesting that no collagen is lost during the intermediate steps of the extraction. Similar results were obtained with a human memory carcinoma.

Xenograft collagen one was significantly enriched in the ECM fraction with only a small loss to the CS fraction. Some residual actin contamination remained in the ECM fraction After its developments. This technique paved the way for biologists and clinicians to explore the complexity of the extracellular matrix of normal and disease tissues.