MMP9: Matrix Metalloproteinase 9 Assay

4 V. Materials TM TMNM AUATRRCATALOG # MANUFACTURER Assay Components Caliper Life Sciences Discovery System ITEM NAME Components LabChip 3000 Drug Microfluidic System ITEM niiosMP9IhbtrIClice 444278 Calbiochem MMP-9 Inhibitor I Inhibitors MOBrikadJcsnBJ081-1 Burdick and Jackson B 4184 E7889 760050 Caliper Life Sciences Sigma 1-1309 18 M 391333 Sigma DMSO 391338 EDTA, disodium salt, 0.5 M solution J T Baker Coating Reagent 3 Calbiochem Calbiochem Brij-35 Solution Calcium Chloride, Anhydrous HEPES, Sodium Salt ULTROL HEPES, Free Acid ULTROL M9CtltcDmi BOO nentoa SE-244 BIOMOL International Substrate: FITC-AHA-CPLGLKAR-NH 760137-0372R Specific Activity: 92.31U/μg Lot: T4744 Caliper Life Sciences (human, recombinant) MMP9 Catalytic Domain 761043-0266R Caliper Life Sciences Caliper Life Sciences 12-Sipper, with Coating-3 Reagent Off-chip Mobility Shift Chip, Caliper Life Sciences 4-Sipper, with Coating-3 Reagent Off-chip Mobility Shift Chip, Caliper Chip Module FS Caliper Chip Module TC B3TBoo EI 325-0001 PI 136-0001 EI 300-0001 PI 115-0005 BioMol BioMol BioMol BioMol CTT-Gelatinase SB-3CT GM6001 NNGH Ω Water 2 Tufts University Analytical Core Facility Email: cust.support Fax: 1-508-435-3439 www.caliperLS.com 1-508-435-9500 Hopkinton, MA 01748-1668 68 Elm Street Corporate Headquarters Caliper Life Sciences Custom @

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30 μL Stop Solution 60 minute incubation at ambient temperature 15 μL Substrate 60 minute pre-incubation with compound at ambient temperature 1 μL Compound, 15 mL Enzyme 31 μL Reaction FITC-AHA 1.5 μM Substrate ( 1 μL Compound in 100% DMSO 5 nM MMP9 enzyme tagged product net charge of 0.15 at pH 7.5. Upon cleavage the fluorescent- This peptide substrate has a molecular weight of 1215 and Substrate II. Methods as arthritis and metastasis. and tissue remodeling, as well in disease processes, such processes, such as embryonic development, reproduction, breakdown of extracellular matrix in normal physiological metalloproteinase (MMP) family are involved in the matrix metalloproteinase MMP9. Proteins of the This application note describes the assay conditions for fluorescence over time reveals the extent of reaction. detected via laser-induced fluorescence. The signature of the cleaved product are separated by electrophoresis and sipper onto the chip, where peptide substrate and from a microtiter plate well, is introduced through capillary peptide substrate to a cleaved product. The reaction mixture, microfluidic chip to measure the conversion of a fluorescent The off-chip incubation, mobility shift assay uses a I. Introduction jjmVW=j~íêáñ=jÉí~ääçéêçíÉáå~ëÉ=V=^ëë~ó MMP9 Assay Conditions weight of 688 and a net charge -2.85. LabChip Assay: Off-Chip Incubation, Mobility Shift - C P L

G FITC-AHA L

K FL-AHA- A R -CONH (Final in reaction) - C C P P 2 L L

G -COOH has a molecular L K A R -CONH 2

) Table 1: MMP9 Assay Parameters for Screening Separation Conditions 500 nM MMP9 Inhibitor 1 40 mM EDTA 0.1% Coating Reagent 3 0.05% Brij-35 10 mM MgCl 100 mM HEPES, pH 7.5 Stop Solution 0.1% Coating Reagent 3 20 mM EDTA 0.05% Brij-35 10 mM CaCl 100 mM HEPES, pH 7.5 Chip/Trough Buffer 3.2% DMSO 0.05% Brij-35 10 mM CaCl 50 mM HEPES, pH 7.5 Reaction Buffer apeSpTm sc 0.2 -2.8 20 -1700 -500 Post-Sample Buffer Sip Time (sec) Sample Sip Time (sec) Downstream Voltage (V) Upstream Voltage (V) Pressure (psi) 2 2 2 (Termination Buffer) (Final concentration in reaction) Application Note 203 4-Sipper 203 MMP9 Matrix Metalloproteinase Assay MMP9 Matrix Metalloproteinase Assay 203

The plate was immediately placed on the LabChip 3000 III. Results Time vs Product Formed 100 min MMP9 DMSO Tolerence system and samples introduced onto a 4-sipper chip every Substrate/Product Peak Separation 130 0.3 Figure 1 shows the separation of product and substrate 5 minutes for 2 hours. Temperature and humidity in the re- 120 PeptidePeptide ((µμM)M) on a 4-sipper chip using the parameters shown in Table 1 action chamber were maintained at 20 °C and 50% respec- 110 100 891.0 and a 60 second post-sample buffer sip. Marker Dye is tively. Peptide and cleaved product were separated and 90 297.0 0.2 sipped between rows to enable well assignments by the detected using the LabChip 3000 system. The data repre- 80 99.0 70 33.0 sents the linear regression of the average ± std error of 4 P/P+S data analysis software (HTSWA). Peptide substrate and 60 11.0 0.1 the cleaved product are separated on the chip and ap- replicate wells over 83 minutes. Linearity was lost after 83 50 3.7 1.2 pear as separate peaks. The HTSWA software deter- minutes. 40 30 0.41 mines peak heights from which ratios of the product to Product Formed (pmoles) 0.0 20 0 5 10 15 20 the peak sums P/(P+S) are calculated. The P/(P+S) x MMP9 10 DMSO (%) Enzyme Concentration (nM) 0 100 = % product formed. 1.25 0 25 50 75 100 125 250 nM Time (min) Figure 4. DMSO Tolerance: Increasing amounts of DMSO 1.00 50 nM in the reaction inhibits MMP9 activity 10 nM Panel A 0.75 2 nM Dye Marker MMP9 Peptide Km Inhibitor IC50 P/P+S 0.50 Known matrix metalloproteinase inhibitors, four small mol- Michaelis-Menton Plot 0.25 VMAX 22.36 ecule and one peptide, were selected for analysis. 31 μL KM 91.52 14 Substrate 0.00 reactions containing 1.5 μM peptide substrate and 5 nM 0 25 50 75 100 13 12 MMP9 enzyme were incubated for 1 hour in the presence Product Time (min) 11 of increasing concentrations of MMP inhibitors. The reac- 10 tions were stopped by the addition of 20 mM EDTA and Lin Reg of of MMP9 Timecourse, 20 cycles (83 min) 9 8 0.5 μM MMP9 Inhibitor 1. Peptide and cleaved product 0.2 7 90

g/min) Lineweaver-Burk Plot n 6 80 were separated and detected using the LabChip 3000 sys- 70 v ( 5 60 50 tem. Inhibition curves are shown in Figure 5. IC values 4 1/v 40 50 30 3 20 were calculated using non-linear regression analysis and 2 10 0.1 0 Figure 1. Caliper LabChip 3000 Data Signature; Representa- 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 are shown in Table 2. As expected from the known litera- 1

amounts of peptide substrate and 5 nM MMP9 enzyme. formed [P/(P+S) = 0.3] in one hour (see MMP9 assay condi- Product Formed (P/P+S) using the LabChip 3000 system. The enzyme concentration -7.5 -5.0 -2.5 0.0 2.5 The plate was immediately placed on the LabChip 3000 tions in Methods). The effect of DMSO on MMP9 activity resulting in 30% product formed after 60 minutes incuba- Log [Inhibitor Conc (μM)] system and samples introduced into the chip every 5 min- was then determined by incubating increasing amounts of tion was extrapolated (5 nM) and chosen for further assay utes for 2 hours. Temperature and humidity in the reaction 100% DMSO with 15 μL each of 3.0 μM peptide substrate Figure 5. Inhibitor Panel IC50 Determinations development studies. chamber were maintained at 20 °C and 50% respectively. and 10 nM MMP9 enzyme. The reaction was incubated for Compound IC (nM) Peptide and cleaved product were separated and detected 1 hour at room temperature followed by the addition of 50 Reaction Linearity using the LabChip 3000 system. Initial rates V(ng/min) were 30 μL Stop Buffer containing 20 mM EDTA and 0.5 μM NNGH 1.058 Reaction linearity using real-time kinetic reads at a final determined by calculating the slopes at each peptide con- MMP9 Inhibitor 1. Peptide and cleaved product were sep- GM6001 0.164 concentration of 5 nM MMP9 was found to be approxi- centration of cleaved product formed in the first 100 min of arated and detected using the LabChip 3000 system. Figure SB-3CT 107.0 mately 80 minutes and is shown in Figure 2B. 60 μL reac- the reaction over time (Figure 3 Panel A). K was deter- 4 shows the effect of increasing DMSO concentrations on tions containing 1.5 μM peptide substrate and 5 nM MMP9 m CTT-Gelatinase 1.02 x 107 mined by plotting V (ng/min) vs. [S ] and non-linear the enzyme activity. MMP9 enzyme activity is sensitive to enzyme were assembled in wells A1, A5, E1, and E5. peptide MMP9 Inhibitor 1 30.17 regression analysis using the Michaelis-Menton equation the presence of DMSO in the reaction. Typically the DMSO Table 2: IC Values Determined for Inhibitors (Figure 3 Panel B). The peptide Km was found to be approx- concentration is 3.2% in a 31 μL reaction containing 1 μL 50 imately 91.5 μM. Vmax was extrapolated from the data. The DMSO. At this concentration the enzyme activity is re- inset shows the Lineweaver-Burke plot of the kinetic data. duced approximately 25%.

2 3 203 MMP9 Matrix Metalloproteinase Assay MMP9 Matrix Metalloproteinase Assay 203

P/P+S 1/[S] tive peaks from 5 sips: Electropherogram illustrating the 0 ture values, the 4 small molecule inhibitors exhibit IC in 0 25 50 75 100 125 150 50 fluorescent signal of a single channel from a 4-sipper chip the nM range. The peptide requires mM concentrations to μ detected by the LabChip 3000 system. [S] ( mol/L) achieve inhibition under the experimental conditions used. 0.0 0 1000 2000 3000 4000 5000 Panel B Time (sec) rate linear regression for the increasing concentrations of Enzyme Titration Figure 3. Substrate Km: Real Time Kinetics Panel A - initial The initial enzyme titration is shown in Figure 2A. 60 μL re- peptide. Panel B- Michaelis-Menton non-linear regression Figure 2A and 2B. MMP9 Time Course: Real Time Kinetics MMP9 Inhibitor Panel actions containing 1.5 μM peptide substrate and 4 different analysis. Inset shows the Lineweaver-Burke plot of the ki- Figure 2A: MMP9 enzyme titration. Figure 2B: Linear re- MMP9 enzyme concentrations were assembled in wells netic data. Inhibitor gression of the product formed during reactions at A1, A5, E1, and E5 on a 384-well microtiter plate. The plate NNGH 5 nM MMP9. was placed immediately onto the LabChip 3000 system and GM6001 DMSO Tolerance samples introduced onto a 4-sipper chip every 5 minutes SB-3CT K Determinations From the assay development parameters determined for 80 minutes. Temperature and humidity in the reaction m CTT-Gelatinase Peptide K was determined using real-time kinetic initial above, the final assay conditions were selected using the MMP9 Inhibitor 1 chamber were maintained at 20 °C and 50% respectively. m rates by assembling 60 μL reactions containing increasing concentration that provides approximately 30% product Peptide and cleaved product were separated and detected amounts of peptide substrate and 5 nM MMP9 enzyme. formed [P/(P+S) = 0.3] in one hour (see MMP9 assay condi- Product Formed (P/P+S) using the LabChip 3000 system. The enzyme concentration -7.5 -5.0 -2.5 0.0 2.5 The plate was immediately placed on the LabChip 3000 tions in Methods). The effect of DMSO on MMP9 activity resulting in 30% product formed after 60 minutes incuba- Log [Inhibitor Conc (μM)] system and samples introduced into the chip every 5 min- was then determined by incubating increasing amounts of tion was extrapolated (5 nM) and chosen for further assay utes for 2 hours. Temperature and humidity in the reaction 100% DMSO with 15 μL each of 3.0 μM peptide substrate Figure 5. Inhibitor Panel IC50 Determinations development studies. chamber were maintained at 20 °C and 50% respectively. and 10 nM MMP9 enzyme. The reaction was incubated for Compound IC (nM) Peptide and cleaved product were separated and detected 1 hour at room temperature followed by the addition of 50 Reaction Linearity using the LabChip 3000 system. Initial rates V(ng/min) were 30 μL Stop Buffer containing 20 mM EDTA and 0.5 μM NNGH 1.058 Reaction linearity using real-time kinetic reads at a final determined by calculating the slopes at each peptide con- MMP9 Inhibitor 1. Peptide and cleaved product were sep- GM6001 0.164 concentration of 5 nM MMP9 was found to be approxi- centration of cleaved product formed in the first 100 min of arated and detected using the LabChip 3000 system. Figure SB-3CT 107.0 mately 80 minutes and is shown in Figure 2B. 60 μL reac- the reaction over time (Figure 3 Panel A). K was deter- 4 shows the effect of increasing DMSO concentrations on tions containing 1.5 μM peptide substrate and 5 nM MMP9 m CTT-Gelatinase 1.02 x 107 mined by plotting V (ng/min) vs. [S ] and non-linear the enzyme activity. MMP9 enzyme activity is sensitive to enzyme were assembled in wells A1, A5, E1, and E5. peptide MMP9 Inhibitor 1 30.17 regression analysis using the Michaelis-Menton equation the presence of DMSO in the reaction. Typically the DMSO Table 2: IC Values Determined for Inhibitors (Figure 3 Panel B). The peptide Km was found to be approx- concentration is 3.2% in a 31 μL reaction containing 1 μL 50 imately 91.5 μM. Vmax was extrapolated from the data. The DMSO. At this concentration the enzyme activity is re- inset shows the Lineweaver-Burke plot of the kinetic data. duced approximately 25%.

2 3 4 V. Materials TM TMNM AUATRRCATALOG # MANUFACTURER Assay Components Caliper Life Sciences Discovery System ITEM NAME Components LabChip 3000 Drug Microfluidic System ITEM niiosMP9IhbtrIClice 444278 Calbiochem MMP-9 Inhibitor I Inhibitors MOBrikadJcsnBJ081-1 Burdick and Jackson B 4184 E7889 760050 Caliper Life Sciences Sigma 1-1309 18 M 391333 Sigma DMSO 391338 EDTA, disodium salt, 0.5 M solution J T Baker Coating Reagent 3 Calbiochem Calbiochem Brij-35 Solution Calcium Chloride, Anhydrous HEPES, Sodium Salt ULTROL HEPES, Free Acid ULTROL M9CtltcDmi BOO nentoa SE-244 BIOMOL International Substrate: FITC-AHA-CPLGLKAR-NH 760137-0372R Specific Activity: 92.31U/μg Lot: T4744 Caliper Life Sciences (human, recombinant) MMP9 Catalytic Domain 761043-0266R Caliper Life Sciences Caliper Life Sciences 12-Sipper, with Coating-3 Reagent Off-chip Mobility Shift Chip, Caliper Life Sciences 4-Sipper, with Coating-3 Reagent Off-chip Mobility Shift Chip, Caliper Chip Module FS Caliper Chip Module TC B3TBoo EI 325-0001 PI 136-0001 EI 300-0001 PI 115-0005 BioMol BioMol BioMol BioMol CTT-Gelatinase SB-3CT GM6001 NNGH Ω Water 2 Tufts University Analytical Core Facility Email: cust.support Fax: 1-508-435-3439 www.caliperLS.com 1-508-435-9500 Hopkinton, MA 01748-1668 68 Elm Street Corporate Headquarters Caliper Life Sciences Custom @