Detection of Protease Activity by Fluorescent Peptide Zymography

Summary

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

Abstract

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Introduction

Gel zymography is a biological technique used to measure proteolytic activity within biological samples, such as body fluids or cell culture media^1,2,3. The samples are separated by their molecular weights with electrophoresis through a polyacrylamide gel embedded with a degradable substrate. Common degradable substrates include gelatin, casein, collagen and elastin, which have been used to measure the activity of matrix metalloproteinases (MMPs) -1, -2, -3, -7, -8, -9, and -11, in addition to a variety of cathepsins^{1,2,4,5,6,7,8}. After electrophoresis, the enzymes are renatured and allowed to degrade the protein within the gel. In traditional gel zymography, the gel is stained with a protein dye, such as Coomassie Blue, and protease activity is detected as a loss of signal, i.e., white bands (degradation of protein) on a dark blue background.

Here, we describe a protocol for an alternative method of gel zymography, in which the degradable substrate is a short, fluorogenic peptide covalently incorporated into the polyacrylamide gel (Figure 1). The substitution of synthetic peptides as the degradable substrates enables detection of a wider range of proteases as compared to traditional gel zymography with native proteins⁹. Covalent linkage of the fluorogenic peptide prevents peptide diffusion and migration during gel electrophoresis observed with previous methods^9,10. Furthermore, the use of a fluorogenic substrate enables direct detection of protease activity without additional staining and de-staining steps. The overall goal of this method is the detection of protease activity in biological samples via the covalent incorporation of fluorogenic peptides in zymogram gels.

Protocol

1. Preparation of the Resolving Gel Layer

1. Prepare a 10% polyacrylamide resolving gel solution as per Table 1. Add the Tetramethylethylenediamine (TEMED) and Ammonium Persulfate (APS) immediately prior to pouring the gel as their addition initiates the polymerization reaction.
2. Fill an empty 1.5 mm mini-gel cassette half way (5 mL) with the 10% resolving gel solution.
3. Add a thin layer of isopropanol (~500 µL) to the top of the polyacrylamide gel to produce a level gel and prevent bubbles. Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. When the polyacrylamide in the tube has completely solidified, the reaction is complete (~40 min).

2. Preparation of the Azido-PEG3-maleimide Linker Molecule

1. While the first resolving gel layer is polymerizing, retrieve the azido-PEG3-maleimide kit from the -20 °C storage and allow the components to reach room temperature. There are two components in each kit. Vial 1 contains a maleimide-NHS ester, an off-white to grey solid. Vial 2 contains azido-PEG3-amine, a slightly yellow oil.
   NOTE: The authors suggest using the 25 mg azido-PEG3-maleimide kit as it can only be stored for short periods of time (1-2 hours) at -20 °C after being prepared before it begins to degrade. 25 mg is sufficient to produce 10 peptide gels. Use gels within 3 weeks of preparation.
2. Dissolve the components of Vial 2 in the manufacturer recommended volume of dimethyl sulfoxide (DMSO) and vortex for 30 s to ensure the liquids have been mixed well.
3. Transfer the contents of Vial 1 into a clean, dry 100 mL round-bottom flask containing a stir bar.
   NOTE: Rinse the flask with acetone and dry completely prior to usage to prevent moisture from interfering with the reaction.
4. Immediately insert a rubber septum stopper with a diaphragm that can be punctured with a syringe into the mouth of the flask. Work quickly to prevent moisture from entering the flask.
5. Insert two 18 gauge syringe needles into the diaphragm and connect one to an inert gas tank (e.g. argon gas). Allow the inert gas to fill the flask for 3 min. Mix the components of Vials 1 and 2 under inert gas to prevent undesirable reaction products.
   CAUTION: The second syringe needle is to provide a vent, thereby allowing the atmospheric air contained within the flask to flow out of the flask as it fills with inert gas. Do not forget to include a vent needle!
6. Shut off the inert gas and detach it from the needle. Using a syringe, inject the full contents of Vial 2 into the flask.
7. Remove both needles and syringe and allow the components to mix for 30 min at room temperature while stirring.
8. Remove the rubber septum stopper and transfer the contents to a clean 5 mL centrifuge tube. The azido-PEG3-maleimide solution must be used within 1 hour at room temperature.

3. Preparation of the Peptide Resolving Gel Layer

1. Once the first resolving gel layer has polymerized, pour off the isopropanol layer. Rinse the top of the gel by pipetting 1 mL of deionized water on the top of the gel and then pour off the water.
2. Retrieve the thiol-functionalized fluorescent peptide from -80 °C storage and allow it to thaw at room temperature.
   NOTE: The thiol-functionalized peptide can be prepared as described previously^11,12. Commercially available peptides can also be used but require the addition of a terminal cysteine residue to enable the maleimide-thiol click reaction. Dissolve the peptide to a stock concentration of 10 mM and store it at -80 °C in small (30 uL) aliquots to limit repeated freeze-thaw cycles.
3. Prepare a 10% resolving gel solution containing the azido-PEG3-maleimide linker molecule and the fluorescent peptide as per Table 1. Add the TEMED and APS immediately prior to pouring the gel as their addition initiates the polymerization reaction.
4. Fill half of the remaining portion of the gel cassette (3 mL) with the peptide resolving gel solution.
   NOTE: A multi-layer resolving gel approach reduces the amount of peptide and linker necessary for each gel. The size of the peptide resolving gel layer can be adjusted to accommodate a larger range of molecular weights as needed.
5. Pipette a thin layer of isopropanol (~500 µL) to the top of the polyacrylamide gel to produce a level gel and prevent bubbles. Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. When the polyacrylamide in the tube has completely solidified, the reaction is complete (~40 min).
   NOTE: The fluorescent peptide is light sensitive. Keep the gels covered with aluminum foil to prevent photobleaching during gel preparation, electrophoresis, washing and development.
6. Pour off the isopropanol layer and rinse the top of the peptide resolving gel with deionized water as in step 3.1.
7. If using the gels immediately, proceed to step 4, otherwise, immerse the prepared gels in 100 mL of 1x phosphate buffered saline (PBS) at 4 °C in a plastic box to prevent the gels from drying out. Wrap the box in aluminum foil to prevent photobleaching. Gels can be stored in PBS for up to 3 weeks prior to usage.

4. Preparation of the Stacking Gel

1. Prepare a 5% stacking gel solution as per Table 1. Add the TEMED and APS immediately prior to pouring the gel as their addition initiates the polymerization reaction.
2. Fill the remaining empty portion of the gel cassette (~2 mL) with the stacking gel solution.
3. Quickly insert a 1.5 mm gel comb into the stacking gel layer, making sure no bubbles remain trapped under the wells. Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. When the polyacrylamide in the tube has completely solidified, the reaction is complete (~10 min).
4. Gently remove the comb and the tape from the back of the gel cassette.

5. Preparation of Biological Samples for Electrophoresis

1. Prepare conditioned cell media, cell lysates, tissue homogenates, and MMP standards as described elsewhere under non-reducing conditions². Do not heat samples.
2. For example, prepare conditioned cell media as follows:
   1. Plate 40,000 cells/cm² cells in a 6-well plate in 10% fetal bovine serum (FBS) culture media. Incubate cells in a humidified chamber (5% CO₂ at 37 °C) for 24 hours and allow them to reach 70-80% confluence.
      NOTE: If the cells have not reached the desired confluency after 24 hours, allow them to grow in 10% FBS culture media for an additional 24 hours.
   2. Wash the cells twice with PBS and add 2 mL of serum-free culture media. Incubate the cells in a humidified chamber (5% CO₂ at 37 °C) for an additional 24 hours.
   3. Using a serological pipette, collect the conditioned media from each well. Centrifuge the media at 1200 rpm for 3 minutes to remove any cell debris. Take the supernatant and concentrate using a 15 mL, 10 kDa molecular weight cutoff centrifugal filter unit. Centrifuge the filter units at 4,000 x g for 15 min in a swinging bucket rotor or 5,000 x g for 15 min in a fixed angle rotor.
      NOTE: This step is optional but can enhance the intensity of the proteolytic bands in the peptide zymography gels.
   4. Transfer the concentrated filtrate to a fresh 1.5 mL centrifuge tube. Aliquot and store samples at -80 °C for up to three freeze-thaw cycles.
3. Quantify protein content using a standard protein quantification assay (e.g. BCA, Bradford Assay, etc.).

6. Electrophoresis of Biological Samples in Peptide Zymography Gels

1. Dissolve samples in conventional zymography sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 4% SDS, 0.01% bromophenol blue). For cell and tissue samples, ~30 µg of total protein per well is recommended, and 50-100 ng of protein for MMP standards.
2. Add 400 mL of 1x Tris-Glycine SDS Running Buffer to the gel apparatus. Load up to 35 µL of sample per well. Run the samples at 120 V at 4 °C for 1.5 hours or until the molecular weight standards indicate that the proteases of interest are within the peptide resolving gel layer (which has a visible orange color).
   NOTE: Most MMPs and their variants fall within the range of 35-100 kDa. When the molecular weight standards indicate that those weights are within the peptide resolving gel layer, electrophoresis can be stopped. The same principle can be applied to other classes of proteases with known molecular weights. If there is an interest in detecting multiple proteases over a larger range of molecular weights, reduce the size of the resolving gel layer and increase the size of the peptide resolving gel layer.
3. Following electrophoresis, remove the gels from the plastic cassette and wash gels three times for 10 min each at room temperature under gentle agitation in renaturing buffer containing 2.5% Triton X-100, 1 µM ZnCl₂, and 5 mM CaCl₂ in 50 mM Tris-HCl, pH 7.5.
4. Transfer gels to a developing buffer solution containing 1% Triton X-100, 1 µM ZnCl₂ and 5 mM CaCl₂ in 50 mM Tris-HCl, pH 7.5 for 15 min. Replace with fresh developing buffer solution and incubate gels at 37 °C under gentle agitation for 24 hours, making sure the gels are fully submersed in the solution.

7. Imaging of Peptide Zymography Gels

1. After 24 hours, image gels using a fluorescent gel scanner/imager using the appropriate excitation and emission filters. For example, the peptide gels shown in the representative results are conjugated with Fluorescein and were imaged using an excitation filter of 488 nm and an emission filter of 521 nm. Using the appropriate filters for your fluorophore will maximize the detection of proteolytic activity.
   NOTE: Images can also be taken with a gel imager equipped with a UV transilluminator, often used for the imaging of DNA gels stained with ethidium bromide. Image the gels using the UV transilluminator (365 nm) setting and an emission filter of 590 nm.
2. Conduct densitometric evaluation of band intensities using ImageJ as described elsewhere¹³.

Results

Using the method described here, two fluorescent protease-degradable peptides were incorporated into polyacrylamide gels: GGPQG↓IWGQK(PEG)₂C (abbreviated as QGIW throughout the text and figures) and GPLA↓C^pMeOBzlWARK(PEG)₂C (abbreviated as LACW throughout the text and figures). ↓ indicates the site of cleavage. QGIW is a collagen-I derived sequence designed to detect cellular collagenases¹⁴. LACW is a sequence that has been optimize...

Discussion

Current zymographic techniques rely on the incorporation of native substrates into polyacrylamide gels for the detection of proteolysis. While these techniques have garnered widespread use, they are still limited in the number of proteases they can detect. Here, a protocol was described in which fluorescent, protease-degradable peptides are incorporated into the polyacrylamide resolving gel. Covalent coupling using an azido-PEG3-maleimide linker molecule enables the separation and detection of a wider variety of protease...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mm Empty Gel Cassettes	ThermoFisher Scientific	NC2015
1.5 mm, 10 well Empty Gel Cassette Combs	ThermoFisher Scientific	NC3510
1x Phosphate Buffered Saline	Fisher Scientific	10-010-049
20% SDS Solution	Ambion	AM9820
3x Zymography Sample Buffer	Bio-Rad	1610764
40% (w/v) Acrylamide/Bis (19:1)	Ambion	AM9022
6 Well Tissue Culture Plates	ThermoFisher Scientific	087721B
Amicon Ultra-2 Centrifugal Filter Unit (10 kDa MWCO)	Sigma-Aldrich	UFC201024
Ammounium Persulfate	Sigma-Aldrich	A3678
Azido-PEG3-Maleimide Kit	Click Chemistry Tools	AZ107
Calcium Chloride	ThermoFisher Scientific	BP510100
Dimethyl Sulfoxide	Fisher Scientific	BP231
Isopropanol	Fisher Scientific	A416P
Micro BCA Protein Assay Kit	ThermoFisher Scientific	23235
N N N' N'-Tetramethylethylenediamine (TEMED)	Sigma-Aldrich	T9281
PowerPac Basic Power Supply	Bio-Rad	1645050
Precision Plus Protein Dual Color Standard	Bio-Rad	161-0374
PrecisionGlide Hypodermic Needles	Fisher Scientific	14-826
Round Bottom Flask (100 mL)	Fisher Scientific	50-873-144
Septum Rubber Stopper	Fisher Scientific	50-872-546
Sterile Slip Tip Syringe (1 mL)	Fisher Scientific	14-823-434
Triton X-100	Sigma-Aldrich	X100
Trizma hydrochlroide	Sigma-Aldrich	T5941
Typhoon 9410 Molecular Imager	GE Amersham	8149-30-9410
Zinc Chloride	Sigma-Aldrich	208086