The overall goal of this experiment, is to determine protease specificity in multiple crude extracts. This method can help answer key questions to determine protease specificity. The main advantage of this technique, is that it can identify protease specificity of multiple samples in parlay.

Cool lung tissue samples obtained from male ICR mice in ice. Homogenize the samples at 20, 000 RMP for 30 seconds in a blender. Repeat this step until the samples are completely homogenous.

Do not homogenize for more than one minute continuously in order to avoid an increase in temperature. Transfer 1, 000 microliters of the homogenate to a 1.5 milliliter tube and centrifuge for five minutes at 10, 000 times G at four degrees Celsius. Transfer 500 microliters of the supernatant to a new 1.5 milliliter plastic tube.

Use 10 microliters of the sample to determine the protein concentration. Using methods, such as the bicinchoninic acid assay or the Bradford assay. To prevent freezing, add 80%glycerol to the samples for a final concentration of 50%glycerol.

To clarify the difference in protease specificity depending on the pH, use digestion buffers adjusted to pH three, five, seven, and nine. Into 890 microliter of a digestion buffer, add either 10 microliters of 0.1 microgram per microliter or TPCK trypsin, 0.1 microgram per microliter of staphylococcus aureus V8 protease, or 10 micrograms per microliter of tissue extracts. Prepare negative controls by inactivating protease in tissue extracts by incubation and boiling water for 10 minutes.

Next, add 10 microliters of MNO biotin label substrates to the samples and incubate for three hours at 37 degrees Celsius to digest. After incubation, stop the digestion of substrates with boiling water for 10 minutes. Centrifuge the digested substrates for five minutes at 10, 000 times G at four degrees Celsius.

Transfer the supernatant into a new 1.5 milliliter tube. Add 50 milliliter of 50%streptavidin sepharose beads in one molar tris buffer, containing 0.1%polyoxyethylene octyl phenyl ether. Use pH test paper to ensure that the solution is around pH nine.

It is important to adjust the proper pH to nine or more. Amino biotin cannot bite a beading unless it is pH nine or higher. If the pH of the solution is low, adjust it to approximately pH nine by adding more buffer.

Additionally one molar tris buffer can be added to bring up the pH if needed. Incubate the suspension overnight at four degrees Celsius with continuous gentle mixing on a rotator wheel to bind the MNO biotin labeled substrates to the streptavidin. The beads should remain suspended throughout the incubation.

The following day, centrifuge the samples for one minute at 10, 000 times G at four degrees Celsius. After removing the supernatant, wash the beads by adding 1, 000 microliters of a wash buffer. Then, incubate for 10 minutes at four degrees Celsius with continuous gentle mixing on a rotator.

Centrifuge the beads for one minute at 10, 000 times G at four degrees Celsius. Remove the supernatant and repeat this wash four times. Next, add 100 microliters of 0.2%trifluoroacetic acid to the beads.

Use pH test paper to ensure that the solution is below pH two. If the pH of the solution is high, adjust the pH to below two by adding 1%trifluoroacetic acid. Incubate the suspension for 30 minutes at room temperature with continuous gentle mixing on a rotator wheel.

After centrifuging the beads as before, transfer the supernatant to a new 1.5 milliliter tube. Pipet 20 microliters of acetonitrile onto a C18 resin to activate it. Remove the used acetonitrile and repeat this activation step three times.

Then, equilibrate the C18 resin with 20 microliters of 0.1%trifluoroacetic acid. Remove the eluant and repeat this equilibration step three times. to load the samples, bind them to the resin using a pipet.

Wash the resin with 20 microliters of 0.1%trifluoroacetic acid. Repeat this step five times. Elute the samples with three microliters of 60%acetonitrile in 0.1%trifluoroacetic acid.

Pipet the eluant on a target plate for MALDI-TOF mass spectromitry. Immediately add CHCA solution, saturated in 50%acetonitrile in 0.1%trifluoroacetic acid. Crystallize the mixture by air drying.

Acquire mass spectra of samples, according to the instrument manufacturer's protocol, paying close attention to the mass spectra from 700 to 900 daltons for the cleaved form of the biotin labels substrates. Mass spectra of synthetic substrates are shown here. Peaks corresponding to the precursor molecular weight were detected between 850 and 1, 050.

The cleaved fragments are detected in the range of 700 to 850. Shown here are the spectra for MNO biotin labeled substrates treated with TPCK trypsin. The 839.81 dalton and 796.76 dalton peaks correspond to the fragments cleaved from the C terminus of lysine and arginine respectively.

Shown here are the mass spectra of synthetic substrates digested with V8 protease. The 769.78 dalton and 755.62 dalton peaks correspond to the fragments cleaved from the C terminus of glutamic acid and aspartic acid respectively. The spectra for MNO biotin labels substrates treated with a lung tissue extract at various pH conditions are shown here.

In the acidic condition, at pH five, the molecular weight of a cleaved fragment was 770.13 daltons and 826.66 daltons, indicating cleavage at the C terminus of glutamic acid and tryptophan. In the neutral and basic condition, at pH seven and nine, the molecular weight of a cleaved fragment was increased at the peak of 767 daltons and 840 daltons, indicating cleaved at the C terminus of arginine and lysine. Conversely, the peaks of the glutamic acid and tryptophan were decreased.

In addition, this method cannot detect cleaved fragments with inactivated extracts. Once mastered, this technique can be done in two days if it is performed properly, except for the synthesis of substrate. Additionally, once the substrate is synthesized, it can be measured in several samples.Why?

Attempting this procedure, it's important to remember to operate at the buffer pH, about nine. Generally, the reaction of the avidin amino biotin complex. Following this procedure, it is possible to quantify enzyme activity by performing a CMS.

This method will allow researchers in the proteon field to pave the way for exploring protease expression in several conditions.