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MMP9: Matrix Metalloproteinase 9 Assay

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MMP9: Matrix Metalloproteinase 9 Assay Application Note 203 V. Materials Worldwide Offices ITEM ITEM NAME MANUFACTURER CATALOG # LabChip Assay: Off-Chip Incubation, Mobility Shift Benelux Microfluidic System LabChip 3000 Drug Caliper Life Sciences Caliper Life Sciences N.V. Components Discovery System Klapstraat 13 Caliper Chip Module TC Caliper Life Sciences B-1790Teralfene, Belgium Telephone: +32-53-66-26-70 Caliper Chip Module FS Caliper Life Sciences Fax: +32-53-66-27-32 jjmVW=j~íêáñ=jÉí~ääçéêçíÉáå~ëÉ=V=^ëë~ó Off-chip Mobility Shift Chip, Caliper Life Sciences 761043-0266R France 4-Sipper, with Coating-3 Reagent Caliper Life Sciences S.A. ZAC PARIS-NORD II Off-chip Mobility Shift Chip, Caliper Life Sciences 760137-0372R 13 rue de la Perdrix 12-Sipper, with Coating-3 Reagent BP 48016Tremblay en France I. Introduction Reaction Buffer (Final concentration in reaction) Assay Components MMP9 Catalytic Domain BIOMOL International SE-244 95911 Roissy CDG Cedex, France The off-chip incubation, mobility shift assay uses a 50 mM HEPES, pH 7.5 (human, recombinant) Telephone: +33-1-48-63-71-35 microfluidic chip to measure the conversion of a fluorescent 10 mM CaCl2 Specific Activity: 92.31U/μg Lot: T4744 Fax: +33-1-48-63-71-53 peptide substrate to a cleaved product. The reaction mixture, 0.05% Brij-35 Germany Substrate: FITC-AHA-CPLGLKAR-NH2 Analytical Core Facility Custom from a microtiter plate well, is introduced through a capillary 3.2% DMSO Tufts University Caliper Life Sciences GmbH Eisenstrasse 9c sipper onto the chip, where the peptide substrate and the Chip/Trough Buffer HEPES, Free Acid ULTROL Calbiochem 391338 DE-65428 Rüsselsheim, Germany cleaved product are separated by electrophoresis and 100 mM HEPES, pH 7.5 Telephone: +49-6142-834-93-0 detected via laser-induced fluorescence. The signature of the HEPES, Sodium Salt ULTROL Calbiochem 391333 10 mM CaCl2 Fax: +49-6142-162-821 fluorescence over time reveals the extent of the reaction. Calcium Chloride, Anhydrous J T Baker 1-1309 0.05% Brij-35 Japan This application note describes the assay conditions for the 20 mM EDTA Caliper Life Sciences Japan Brij-35 Solution Sigma B 4184 matrix metalloproteinase MMP9. Proteins of the matrix 0.1% Coating Reagent 3 Saito-Bldg. 2F metalloproteinase (MMP) family are involved in the Coating Reagent 3 Caliper Life Sciences 760050 Yushima 2-17-15, Bunkyo-ku breakdown of extracellular matrix in normal physiological Stop Solution (Termination Buffer) Tokyo 113-0034, Japan EDTA, disodium salt, 0.5 M solution Sigma E7889 100 mM HEPES, pH 7.5 Telephone: +81-3-5840-6551 processes, such as embryonic development, reproduction, DMSO Burdick and Jackson BJ081-1 Fax: +81-3-5840-6554 and tissue remodeling, as well as in disease processes, such 10 mM MgCl2 0.05% Brij-35 18 MΩ Water as arthritis and metastasis. Switzerland 0.1% Coating Reagent 3 Inhibitors MMP-9 Inhibitor I Calbiochem 444278 Caliper Life Sciences AG Nordstrasse 17 II. Methods 40 mM EDTA NNGH BioMol PI 115-0005 CH-4665 Oftringen, Switzerland Substrate 500 nM MMP9 Inhibitor 1 Telephone: +41-62-788-7000 GM6001 BioMol EI 300-0001 FITC-AHA-CPLGLKAR-CONH2 Separation Conditions Fax: +41-62-788-7017 This peptide substrate has a molecular weight of 1215 and a SB-3CT BioMol EI 325-0001 United Kingdom net charge of 0.15 at pH 7.5. Upon cleavage the fluorescent- 4-Sipper CTT-Gelatinase BioMol PI 136-0001 Caliper Life Sciences Ltd. tagged product FITC-AHA-CPL -COOH has a molecular Pressure (psi) -2.8 1 Wellfield weight of 688 and a net charge of -2.85. Preston Brook Upstream Voltage (V) -500 Runcorn, Cheshire WA7 3AZ MMP9 Assay Conditions (Final in reaction) Downstream Voltage (V) -1700 United Kingdom 5 nM MMP9 enzyme Telephone: +44-1928-711448 1 μL Compound in 100% DMSO Sample Sip Time (sec) 0.2 Fax: +44-1928-791228 1.5 μM Substrate (FL-AHA-CPLGLKAR-CONH2) Post-Sample Buffer Sip Time (sec) 20 Caliper Life Sciences has 31 μL Reaction Table 1: MMP9 Assay Parameters for Screening representative offices worldwide. 1 μL Compound, 15 mL Enzyme Please visit www.caliperLS.com for locations and contact 60 minute pre-incubation with compound at ambient temperature information. 15 μL Substrate 60 minute incubation at ambient temperature 30 μL Stop Solution Caliper Life Sciences Corporate Headquarters ©2005 Caliper Life Sciences, Inc. 68 Elm Street All rights reserved. Hopkinton, MA 01748-1668 Caliper and LabChip are registered trade- 1-508-435-9500 marks, the Caliper Logo and the phrase www.caliperLS.com "Working Innovation" are trademarks of Caliper Life Sciences, Inc. Fax: 1-508-435-3439 Email: [email protected] LC3000-AP-203 07/05 4 203 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 Lineweaver-Burk Plot g/min) 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 Figure 1. Caliper LabChip 3000 Data Signature; Representa- 0.1 0 1 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- 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) Enzyme Titration Figure 3. Substrate Km: Real Time Kinetics Panel A - initial The initial enzyme titration is shown in Figure 2A. 60 μL re- rate linear regression for the increasing concentrations of Figure 2A and 2B. MMP9 Time Course: Real Time Kinetics MMP9 Inhibitor Panel actions containing 1.5 μM peptide substrate and 4 different peptide. Panel B- Michaelis-Menton non-linear regression Figure 2A: MMP9 enzyme titration. Figure 2B: Linear re- MMP9 enzyme concentrations were assembled in wells analysis. Inset shows the Lineweaver-Burke plot of the ki- Inhibitor gression of the product formed during reactions at A1, A5, E1, and E5 on a 384-well microtiter plate. The plate netic data. 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.
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