Material and methods

2.1. Material

The expression vector pet21a including the sequence G₁₀₆ to N₂₆₈ catalytic domain plus the initiator M (via NdeI / NotI) of MMP-12 CD was obtained from GENEART (Regensburg, Germany).

Ammonium bicarbonate (AB), dimethyl sulfoxide (DMSO), chloroform, dithiothreitol (DTT), formic acid (FA), calcium chloride, ammonium persulfate, sodium acetate, and iodoacetamide (IAA) were purchased from Sigma Aldrich. Trypsin (sequencing grade, cat. n° V5111) was from Promega, NaCl, glacial acetic acid, sodium thiosulfate, and sodium carbonate were obtained from Merck. Methanol and acetonitrile (ACN) were purchased from Biosolve and formaldehyde was from JT Baker. Sodium dodecyl sulfate (SDS), tris, glycine, ethylenediaminetetraacetic acid (EDTA), and silver nitrate were purchased from Duchefa, Brij-35 was from Janssen Chemica, and glycerol from Genfarma BV. 30%

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acrylamide/bis-acrylamide (29:1) solution, bromophenol blue, and tetramethylethylenediamine (TEMED) were from BioRad. Monomeric Avidin agarose, trifluoroacetic acid (TFA) and Zeba Spin desalting columns (cut-off: 7000 Da) were purchased from Pierce (Pierce, cat. n° 89882). Ultrapure water (resistivity of 18.2 MΩ·cm) was obtained from a Sartorius Stedim water purification system (model 611 VF). Ultra low molecular weight marker was purchased from Sigma (M3546), Precision Plus protein standards dual color from BioRad (161-0374), and the BenchMark protein ladder from Invitrogen (10747-012).

2.2. Synthesis of the probes

Probe 1 and Probe 2 (see Figures 1A and 1B for chemical structures) including a succinyl hydroxamate motif and a trifluoromethylphenyldiazirine photoreactive group were synthetized as previously reported ¹⁶.

Figure 1. Structure of the photoactivatable inhibitors containing a photoactivatable trifluoromethyldiazirine group in the P1’ position targeting the S1’ pocket of MMP-12 CD. A) probe with a biotin moiety serving as tag to enrich the labeled protein via monomeric avidin beads (Probe 1); B) probe without biotin moiety used for the study of the stoichiometry ratio of the photolabeling (Probe 2).

2.3. Expression of recombinant MMP-12 CD

The catalytic domain of recombinant human MMP-12 was expressed and purified according the protocol of Parkar et al.¹⁷. The expression vector pet21a including the sequence G₁₀₆ to N₂₆₈ (via NdeI / NotI) of MMP-12 CD (with a Methionine residue at the N-terminal extremity) was obtained from GENEART (Regensburg, Germany). The 163-residue catalytic domain of MMP-12 CD was expressed in E. coli BL21 cells. The vector sequence was verified (Macrogen; http://dna.macrogen.com/eng/) from two different colonies after transformation.

Cells were cultured in baffled shake flasks at 37°C in Luria broth medium supplemented with ampicillin. Protein expression was induced at an optical density (OD600) of 0.5 by the addition of isopropyl beta-D-thiogalactoside (IPTG) to a final concentration of 1 mM. Cells were harvested by centrifugation and resuspended in 160 mL (for 2 liters of culture) of 100 mM Tris-HCl, pH8.0, supplemented by 4 tablets of protease inhibitors (EDTA-free; Roche). Deoxycholate was added to 0.1 % and the resuspended cells were incubated for 10 min on ice. After adding magnesium chloride and DNAse I to 40 mM and 5 µg/mL, respectively, the cells were lysed by sonication (3-times for 1 min; with a 1 min pause in between). The homogenized cell lysate was centrifuged and the insoluble material was washed as follows : 160 mL 0.1 % (v/v) Triton X-100, followed by 160 mL of demineralized water and finally with 10 mL of butan-1-ol. The inclusion bodies were resuspended in 10 mL (for 2 L of culture) of 8 M guanidinium-HCl, 50 mM Tris-HCl pH 8, 30 mM DTT (1h at 37ºC).

The resolubilized, filtered inclusion bodies (1.2mL / 2.1 mg) were loaded on a size exclusion chromatography column (HiLoad Superdex 200 16/60 from GE Healthcare; 120mL column volume) that was equilibrated with 6 M guanidinium–HCl, 20 mM Tris-HCl, pH 8.0 at a flow rate of 1 mL/

min (detection at 280nm) to remove large aggregates and interfering substances (ÄKTAprime plus system).

Fractions containing MMP-12 CD (verified by SDS-PAGE and MALDI-TOF-MS analysis) were subjected to refolding in two steps. Initially fractions were dialyzed (48 h at 4°C against 8 volume equivalents) of A) 3 M urea, 50 mM Hepes, 10 mM CaCl₂, 30 mM NaCl, 0.1 mM Zinc (II) acetate, pH 7.2 and then of B) 3 M urea, 10 mM Hepes, 2 mM CaCl₂, pH 7.0 (three buffer changes, 24 h each). MMP-12 CD autodegradation fragments were removed from the samples during the second refolding step on a strong cation exchange resin (SP-Sepharose fast flow, GE Healthcare).

The resin (2 mL) was first washed with 3 M urea, 10 mM Hepes, 2 mM CaCl₂, pH 7.0 before being incubated for 1 h at room temperature with 20 mL of the dialyzed protein at 0.1 mg/mL in 3 M urea, 10 mM Hepes, 2 mM CaCl₂, pH 7.0. After incubation with the samples, the resin was packed manually into a P10 column. Final refolding of MMP-12 CD was completed by gradually reducing the urea concentration in the following steps: 3 washes (one wash corresponds to 10 column volumes) with 2 M urea, 10 mM Hepes, 2 mM CaCl₂, pH 7.0, one wash with 1 M urea, 10 mM Hepes, 2 mM CaCl₂, pH 7.0, two washes with 10 mM Hepes, 2 mM CaCl₂, pH 7.0. Active MMP-12 was eluted with 10 mM Hepes, 2 mM CaCl₂, 250 mM NaCl, pH 7.0.

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2.4. Labeling of MMP-12 CD

1.5 μg (~75 pmol) MMP-12 CD were incubated for 10 min in the dark at 4º C with a 2-fold molar excess of the Probe 1 or Probe 2 (unless otherwise mentioned) in 30 μL of 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 5 mM CaCl₂, 0.005 % Brij 35 and labeled by irradiation at 366 nm for 30 min (Camag universal UV lamp (20 W), distance to the plate of 4 cm in 96-well plates (Costar White)) at room temperature. Salt and detergent were removed prior to mass spectrometric analysis by methanol-chloroform precipitation according to the protocol of Wessel and Flugge ¹⁸. After photoaffinity labeling the protein solution was mixed with 600 μL methanol, 150 μL chloroform and 450 μL water by vortexing and centrifuged at 13600 g for 5 min. The pellet was washed with 450 μL of methanol and centrifuged as above. The final pellet was dried in a vacuum concentrator centrifuge prior to further analysis.

2.5. Analysis by liquid chromatography – mass spectrometry (LC-MS)

Photolabeled MMP-12 CD was analyzed by nanoLC-MS using a microfluidics (chip-cube) interface (Agilent, cat. n° G4240A) on a C8 custom-made chip (Agilent, cat. n º G4240-63001 batch SPQ105) with a 40 nL enrichment column (Zorbax 300SB C-8, 5 μm) and a 75 μm × 43 mm separation column packed with the same chromatographic material. The interface contained a nanoelectrospray tip (2 mm length with conical shape: 100 μm OD × 6 μm ID) that was coupled on-line to an ion-trap mass spectrometer (MSD Trap-SL, Agilent). Photolabeled MMP-12 CD (1.5 μg) was dissolved in 0.1

% aqueous formic acid at a concentration of 0.1 μg/μL and 3 μL were loaded on the trap column in 0.1 % aqueous formic acid at 3 μL/min via the autosampler (Agilent, cat. no. G1389A) equipped with an injection loop of 8 μL and a thermostated cooler maintaining the samples in the autosampler at 4°C. The interface was connected to an Agilent 1100 series HPLC system containing the following additional modules: nanopump (cat. no. G2226A), capillary pump (cat. no. G1376A) and solvent degasser. The samples were enriched for 5 min followed by washing in the forward flush mode for another 5 min (0.1% aq. formic acid, 3% acetonitrile, 3 μL/min) after which the trapping column was switched in-line with the analytical column. MMP-12 CD was eluted with eluents A (0.1% aq.

formic acid) and B (0.1% formic acid in acetonitrile) at a flow-rate of 0.25 μL/min using the following program: 5 min with 3% eluent A, a linear gradient from 3 to 60% eluent B at 0.9%/min followed by a gradient from 60 to 90% eluent B at 10%/min. Eluent B (90%) was maintained for 2 min to clean the column. Before each injection, the in-line trap and the analytical column were equilibrated with eluent A for 10 min at 3 and 0.25 μL/ min, respectively.

2.6. Analysis by MALDI-TOF (MS)

The protein pellet (~2 µg) of the methanol-chloroform precipitation was resuspended with 10 µL of Sinapinic Acid (SA) (10 mg/mL in 50% ACN 0.1% TFA). 2 µL were spotted on the target plate for

MALDI-TOF-MS analysis (Voyager DE-PRO, Applied Biosystems). The instrument was calibrated with a protein calibrant mix from Bruker Daltonics (No. 206355).

The MALDI-TOF-MS mass spectrometer was operated in the positive linear mode with an accelerating voltage of 25000V. Proteins masses were screened from 5000 – 45000 Da with a low mass gate of 5000. Data were acquired with 500 shots/ spectrum and summed up for the final analysis.

2.7. Enrichment of photolabeled MMP-12 CD on monomeric avidin beads

High affinity biotin-binding sites (oligomeric avidin groups) of monomeric avidin (Pierce) were first blocked with a biotin-containing buffer. To this purpose, 50 μL of beads were washed twice with 1 mL of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl buffer for 10 min before blocking oligomeric avidin binding sites by incubation for 1h with 500 μL of the above buffer containing 2 mM biotin.

Biotin was removed from the lower affinity monomeric binding sites by elution (2 washes for 10 min each) with 1 mL of regeneration buffer (0.1 M glycine in 50 mM Tris-HCl, pH 2.8, 150 mM NaCl) and two final washes (10 min each) with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl according to the manufacturer’s instructions. All steps were performed at room temperature.

For enrichment of the labeled protein, 10μL of monomeric avidin beads diluted to 100 μL with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 2 µg of the labeled protein were incubated for 1h at room temperature. After 4 washes with 300 μL of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl (for 10 min each at room temperature) proteins were eluted with 3-times 10 μL of 8 M guanidinium-HCl, pH 1.8 at 65°C for 2 min.

2.8. SDS PAGE analysis

Discontinuous, reducing SDS-PAGE was performed according on 1 mm thick gels in a Mini protean III cell assembly (BioRad). Experimental conditions were as follows: stacking gel (1 cm), 0.1%

(w/v) SDS, 0.1% (w/v) APS, 0.2% (v/v) TEMED, 125 mM Tris-HCl, pH 6.8; separating gel (6 cm), 0.1%

(w/v) SDS, 0.1% (w/v) APS, 0.04% (v/v) TEMED, 375 mM Tris-HCl pH 8.8; running buffer, 25 mM Tris base, 200 mM glycine, pH 8.3, 0.1% (w/v) SDS. One volume of loading buffer (10% SDS (w/v), 10 mM DTT, 20% glycerol (v/v), 200 mM Tris-Hcl pH 6.8, 0.05% bromophenol blue (w/v)) was added to four volumes of samples. Samples were heated for 5 min at 95°C and loaded on the gel directly after heating. Running conditions: 10 min at ~120 V (stacking) and 50 min at ~160 V until migration of the bromophenol blue to the end of the gel. After electrophoresis, gels were stained according to Yan et al. ¹⁹.

2.9. Tryptic digestion

Tryptic digestion was performed on labeled and non-labeled protein after exchanging the labeling medium against 50 mM of ammonium bicarbonate by ultrafiltration at 7 kDa (Zeba Spin

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Desalting Columns (Pierce, cat. n° 89882)) according to the manufacturer’s instructions. Alternatively, proteins were denatured by methanol-chloroform precipitation (see 2.4. Labeling of MMP-12 CD).

Samples were then digested for 4 hours with trypsin (1:30 enzyme to protein ratio) in a final volume of 10 µL of 50 mM of ammonium bicarbonate. Tryptic digest were mixed with the loading buffer for Tricine-SDS-PAGE analysis.

2.10. Tricine-SDS PAGE and Western blotting

Electrophoresis was performed according the protocol of Schägger (18% Tricine-SDS-PAGE, 1 mm thickness containing a 10% stacking gel (1 cm)) ²⁰. The proteins were transferred to an Immun-Blot PVDF (polyvinylidene fluoride) membrane by wet blotting in a mini Trans-blot cell at 350 mA for 60 min in 25 mM Tris, 190 mM glycine with 20% v/v methanol (BioRad). Membranes were blocked overnight at 4°C in TBST (25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% v/v Tween-20), supplemented with 5% w/v non-fat dried milk (Protifar Plus, Nutricia, Zoetermeer, The Netherlands) and incubated for 1 h in a 1:1500 dilution of streptavidin-alkaline phosphatase (0.67 μg/mL) in TBST (25 mM Tris buffer pH 7.5 containing 150 mM NaCl, 0.05% v/v Tween-20) supplemented with 1% non-fat dried milk. Biotinylated proteins were visualized by staining with a BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium chloride) tablet (Sigma Aldrich) in 10 mL of ultrapure water.

2.11. Fluorescent measurements

Activity assays were performed in 96-well plates (Costar-white) on a Fluostar Optima plate reader (BMG, Labtech) with 100 μL of MMP assay buffer. The enzyme activity (MMP-12 CD or ^15NMMP-12 CD spiked at 10 ng/100 μL) was followed by monitoring (for 15 min) conversion of the fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (BACHEM) spiked at 2µM (λex, λem=327, 420 nm).