Monitoring protease activity with the Protein-Probe

assay in a homogeneous, label-free format

1. Introduction
Proteases are enzymes that modify proteins by hydrolyzing (cutting) a peptide bond between two
amino acids. Proteases control multiple processes in the human body, such as digestion, cell cycle,
wound healing, and the activation of the immune system. They also play a role in multiple diseases,
such as cancers, HIV, cardiovascular diseases, and type 2 diabetes. Therefore, protease inhibitors,
which prevent protease activity, have potential as drug molecules. For example, some HIV and
hepatitis C medications are based on protease inhibitors, and studies to find more drug candidates
are ongoing. To effectively study proteases and their inhibitors, assays that can monitor protease
activity in a sensitive, rapid, and easy-to-perform manner are required.

Traditionally, protease activity is studied by separating the digested substrate protein fragments with
liquid chromatography and subsequently quantifying the peptide fragments with mass spectrometry.
However, these methods are not suitable for high-throughput screening (HTS), which is required to
efficiently analyze, for example, a large molecular library of drug compounds. Several of the developed
HTS-compatible methods for protease studies are based on absorbance and fluorescence detection.
The substrates in these assays can be either whole proteins or peptides designed with an appropriate
digestion site. Usually, the substrates must be labeled with reporter molecules.

Förster resonance energy transfer (FRET) is a very popular method for protease studies that requires
fluorescent labels on each side of the digestion site. The labels must be in close proximity to each
other to produce FRET signal, so digestion is observed as a signal loss (acceptor) or increase (donor)
when the labels are separated. The FRET substrate can be a whole protein, although peptides are
often used, as they are easy to design and produce. However, using peptides instead of the natural
substrate proteins of proteases may affect the digestion efficiency. FRET is very sensitive and also
quantitative, but the requirement for two label conjugations makes the assays expensive and
laborious to prepare. These problems persist also with other label-based methods. On the other hand,
absorbance-based assays often use native, unmodified proteins, such as casein, but with sacrificed
sensitivity compared to FRET.

We have developed an assay for studying protease activity in a high-throughput compatible format,
with the possibility to use a wide variety of native, whole proteins as substrates. The method, called
the Protein-Probe, is based on an external, non-specific Eu-probe. Using an external probe removes
the need to label the substrate proteins. The Eu-probe binds to the intact, but not digested, protein
substrate. The binding protects the Eu-probe from the quenching effect of the modulation solution,
leading to an elevated time-resolved luminescence (TRL)-signal. If a protease digests the substrate,
the Eu-probe remains free in the solution and the TRL-signal is quenched (Fig. 1). The Protein-Probe
method is suitable for monitoring protease activity and inhibition in an easy-to-use concept, and
performing the assay on microtiter plates enables a high throughput.

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Time-resolved luminescence signal (counts) A 340 nm B 340 nm
340 nm 620 nm
Eu

620 nm 340 nm

Substrate/
C digested substrate
Protease/
Eu Protease inhibitor
Eu-probe high TRL/
Eu-probe quenched
Protease concentration increase

Figure 1. The principle of the Protein-Probe method for monitoring protease activity and inhibition.
A. The Eu-probe interacts with the intact substrate protein, which leads to a high TRL-signal in the
modulation solution. B. Digestion of the substrate by an active protease prevents the Eu-probe
binding. The emission of the free Eu-probe is quenched, and a low TRL-signal is observed. C.
Inhibitors block the protease activity, and the Eu-probe can bind to the intact substrate, producing
high TRL-signal.

2. Aims
The aim of this work is to monitor the digestion activity and inhibition of pepsin, using the Protein-
Probe QTRL-method in a high-throughput format.

֍ The assay is validated with dose-response measurements of the inhibitor effect on the
protease activity.

֍ The assay performance is assessed by calculating the Z’-factor.

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 3. Laboratory work
3.1 Materials

3.1.1 General
• Assay buffer: 10 % Na2HPO4/Citric acid (pH 3) (McIlvaine buffer), 0.001 % triton
• Detection buffer: 10 % Na2HPO4/Citric acid (pH 4), 0.001 % triton
• Plates: 384-well black low volume plate (Corning, NY, USA)
• Instrumentation: Tecan Spark 20M plate reader (Tecan, Männedorf, Switzerland), using
excitation wavelength of 340 nm and emission wavelength of 620 nm, 800 µs delay time, 400
µs decay time, and 30 flashes with gain 130. PTC-100 Programmable Thermal Controller (MJ
Research, Inc., Watertown, MA, USA).

3.1.2 Digestion reaction (concentrations in 8 µl reaction volume)

• Protease enzyme: 200 nM pepsin (Sigma-Aldrich, St. Louis, MO, USA)
• Protein substrate: 100 nM trastuzumab (Roche, Basel, Switzerland)
• Protease inhibitor: 5–5000 nM pepstatin A (Sigma-Aldrich)

3.1.3 Detection solution (concentrations in 15 µl)

• Modulator molecule: 3.5 µM Q22 (QRET technologies, Turku, Finland)
• Europium labeled peptide: 1 nM Eu3+-peptide (QRET technologies)

3.2 Assay conditions

• Digestion reaction volume: 8 µl
• Detection solution: 15 µl
• Total volume: 23 µL
• Heating: 30 min incubation at 37 C

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3.3 Protocol

3.3.1 Assay protocols

Inhibitor (2 µL)

Protease (2µL) Detection

solution
(Modulator + Eu3+-
peptide)

Denatured Protein
substrate (4µL)

Figure 2. Schematic of the protein digestion assay.

Trastuzumab control measurement – room temperature vs. 85 °C

1. Pipet 100 nM of native trastuzumab to wells 1-6 in 8 µl.

2. Pipet 100 nM heated trastuzumab to wells 7-12 in 8 µl.
3. Pipet Detection solution to wells 1-12 in 15 µl.
4. Measure the TRL-signals using Tecan Spark.

Dose-response assay (inhibitor titration)

Press the tip to the bottom of the well to ensure that the liquid is released from the tip.

1. Pipet 2 µl buffer/inhibitor (pepstatin A; 5–5000 nM) to the wells according to the plate
map, using a single channel pipette.
2. Add 2 µl protease enzyme (pepsin; 200 nM) to each well.
After adding all the substrate, gently tap the plate onto the table to even out the liquid
in the wells.
3. Place the plate for 1-2 min on the slow shaking.
4. Add 4 µl of the substrate protein (already heated in PCR tube at 85 °C for 5 min)
(trastuzumab, 100 nM).
5. Incubate the plate for 30 min at 37 °C.
6. Add 15 µl of the detection solution.
• Don’t pipette in the air, touch the wall of the well when pipetting.

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 7. Measure the TRL-signals using Tecan Spark. Measure again after 10 min.
Z’-factor assay

1. Pipet 2 µl of buffer or inhibitor (pepstatin A; 5 µM) to the wells according to the plate
map.
2. Add 2 µl protease enzyme (pepsin; 200 nM) to each well.
3. Place the plate for 1-2 min on the slow shaking.
4. Add 4 µl of protein substrate (trastuzumab; 100 nM) (already heated in PCR tube at
85 °C for 5 min).
5. Incubate the plate for 30 min at 37 °C.
6. Add 15 µl of the detection solution.
7. Measure the TRL-signals using Tecan Spark. Measure again after 10 min.

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Figure 3. Protease activity assay protocol.

3.3.2 Dose-response assay (inhibitor titration)

The dose-response assay measures what is the effective inhibitor (pepstatin A) concentration for
blocking pepsin function. When the results are plotted using a linear Y axis and a logarithmic X axis,
the resulting curve should be sigmoidal.

The midpoint of the curve represents the IC50 value: the inhibitor concentration at which 50 % of the
protease has been inhibited. The lower the IC50 value is, the more potent the inhibitor. The IC50 value
is calculated from the curve, by plotting average signals and standard deviation (mark it as error bar)
in data analysis software, such as Origin.

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 10000

Relative TRL signal (counts)

8000

6000

4000

2000

1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06

Inhibitor concentration

3.3.3 Protein-Probe method robustness – Z’-factor

To study the robustness of the Protein-Probe method, the Z’-factor will be calculated using standard
deviation of 24 replicates of positive (with 5 µM inhibitor) and 24 replicates of negative controls
(without inhibitor), using the following equation:

"#$%&'(&)( (+,-&%-.' (01) .3 4.$-%-,+ 5.'%).67"#01 .3 '+8&%-,+ 5.'%).6

𝑍! = 1 −
(&,+)&8+ .3 4.$-%-,+ 5.'%).69&,+)&8+ .3 '+8&%-,+ 5.'%).6)

The Z’-factor is used to evaluate, compare, and validate HTS assays. When the Z’-factor is
above 0.5, the assay is robust enough for HTS use.

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4. Report
Write a report from the experiments you have performed (one report per person). The report
includes:

1) brief introduction
2) introduce the principle of the detection
3) aims of the experiment
4) brief methodology section
5) the results you obtained including data analysis and graphs
6) discussion section
7) write a luminescence principle section to the end of the report

Finally, send the report to harri.harma@utu.fi.

4.1 Data analysis

• Calculate the average and coefficient of variation (CV%) of your signals from the replicates.
CV% is (σ/µ)*100. CV% = 15–20% is acceptable for these experiments. Consider taking out the
outliers to improve the CV%.
• Calculate the signal-to-background ratio (S/B). S/B is calculated as µmax/µmin for the Z’-factor
assay, and as µ(of each inhibitor concentration)/µmin for the dose-response measurements.
• Show all data values and average values for Z’-factor and dose-response in a table. Mark the
outliers. Provide S/B ratio for dose-response values and CV%.

In the formulas, µ = average and σ = standard deviation.

4.2 Graphs
• For the dose-response measurement graphs, you can normalize the data and use relative
signals, or use the TRL-signals. No inhibitor = 0% enzyme inhibition and 5 µM inhibitor = 100%
enzyme inhibition. Provide the IC50 value using Origin or Prism programs – do not use excel.
Show error bars.
• For the Z’-factor graph, show the TRL-signal counts and mark the outliers which have been
taken out from the calculations.

4.3 Principles to be included in the report

• Explain the detection principle.
• Explain the luminescence detection principle in the text. Provide a stepwise description of the
excitation and emission processes of time-resolved luminescence including the quenching
mechanism (the QRET principle) using the Jablonski diagram.

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Report deadline: 26 Oct 2023
Example of a simple Jablonski diagram

PIPETTING MAPS:
Trastuzumab control measurement – native vs. denatured:

1-6 7-12
100 nM trastuzumab 100 nM trastuzumab
Row 1 (native) (denatured)

1-6 7-12
Row 1 Detection

ð MEASUREMENT
1-6 7-12
Row 1 Detection

Inhibitor titration:

Pepstatin A (inhibitor) (2 µl):

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 1-6 7-12 13-18 19-24
Row 1 Buffer 5 nM PepA 20 nM PepA 78 nM PepA
Row 2 313 nM PepA 1250 nM PepA 5000 nM PepA Buffer
Row 3 Buffer

Pepsin (protease) (2 µl):

1-6 7-12 13-18 19-24
Row 1 200 nM pepsin 200 nM pepsin 200 nM pepsin 200 nM pepsin
Row 2 200 nM pepsin 200 nM pepsin 200 nM pepsin Buffer
Row 3 Buffer

Trastuzumab (substrate) (4 µl):

1-6 7-12 13-18 19-24
Row 1 100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab
100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab
Row 2
Row 3 Buffer

Detection (15 µl):

1-6 7-12 13-18 19-24
Row 1 Detection solution Detection solution Detection solution Detection solution
Row 2 Detection solution Detection solution Detection solution Detection solution
Row 3 Detection solution Detection solution

Z’-factor assay:
Pepstatin A (inhibitor) (2 µl):
1-6 7-12 13-18 19-24
Row 1 Buffer Buffer Buffer Buffer
Row 2 5 µM pepstatin A 5 µM pepstatin A 5 µM pepstatin A 5 µM pepstatin A

Pepsin (protease) (2 µl):

1-6 7-12 13-18 19-24
Row 1 200 nM pepsin 200 nM pepsin 200 nM pepsin 200 nM pepsin
Row 2 200 nM pepsin 200 nM pepsin 200 nM pepsin 200 nM pepsin

Trastuzumab (substrate) (4 µl):

1-6 7-12 13-18 19-24
Row 1 100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab
Row 2 100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab 100 nM trastuzumab

+ Detection solution to all wells

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