Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

1. Cloning and Purification of Untagged Human TET2 Dioxygenase

1. Clone human TET2 dioxygenase (TET2 1129-1936, Δ1481-1843) into the pDEST14 destination vector using the site-specific recombination technique as previously described²².
   NOTE: Previous studies have demonstrated that the C-terminal TET2 dioxygenase (TET2 1129-1936, Δ1481-1843) domain is the minimal catalytically active domain^21,23. In order to express the untagged TET2 dioxygenase domain using the pDONR221 vector, Shine Dalgarno and Kozak sequences were incorporated in the forward primer during PCR (Table 1).
2. For bacterial transformation, add 1 µL of recombinant pDEST14 expression vector containing untagged human TET2 dioxygenase (TET2 1129-1936, Δ1481-1843) to 100 µL of chemically competent E. coli BL21 (DE3) cells in a 1.7 mL tube. Keep the mixture on ice for at least 15 minutes followed by heat shock at 42 °C for 30 s in a water bath.
   1. Immediately after heat shock, keep the cells back on ice for minimum of 2 min. Following this, add 250 µL of super optimal broth with catabolite repression (S.O.C media) to cells. Incubate the bacterial cells for 1 h at 37 °C in a shaker.
   2. After incubation, spin down the cells by centrifuging the tube at 9,000 x g for 1 min. Discard 70% supernatant by pipetting and dissolve the pellet in left over media.
   3. Spread the cell suspension on a Luria broth (LB) agar plate containing 100 µg/mL ampicillin. Incubate the plate for 16 h at 37 °C.
3. Select one isolated colony and inoculate it into 10 mL of LB-ampicillin media in a 50 mL tube. Incubate the tube at 37 °C in a shaker for overnight. Following this, use 100 µL  of culture from the 50 mL tube to inoculate 100 mL of LB-ampicillin media. Incubate the flask at 37 °C on a shaker at 180 rpm and use it as primary culture. Next day, use 6 mL each of primary culture to inoculate 15 flasks, each containing 600 mL LB-ampicillin media. Now, incubate 15 flasks at 37 °C on a shaker at 180 rpm.
   NOTE: For verifying transformed clones, perform DNA sequencing or restriction digestion with the isolated plasmid DNA.
4. To check the density of bacterial culture, measure its OD₆₀₀ using a spectrophotometer. After the culture reaches a density of 0.8 at OD₆₀₀, induce the expression of TET2 protein with 300 µL of 1 M (final concentration of 0.5 mM in 600 mL) IPTG in each flask and further grow the culture for 16 h at 17 °C.
5. After 16 h, transfer the bacterial culture to centrifuge bottles. Centrifuge the bacterial culture expressing the TET2 enzyme at 5,250 x g for 45 min. Use the bacterial pellet for TET2 purification.
   NOTE: Perform all remaining protein purification steps either on the ice or at 4 °C.
6. Resuspend the bacterial pellet in 100 mL of 50 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 6 and sonicate for 5 × 30 s at power 20 with 60 s cooling intervals.
7. Spin the lysate at 5,250 x g for 45 min. Collect the supernatant containing the soluble TET2 enzyme and pass through 0.45-µm filters before loading on an FPLC system.
   NOTE: In these experiments, the TET2 enzyme was very stable, but if needed a protease inhibitor cocktail containing 1 mM benzamidine-HCl, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.5 mM 1,10-o-phenanthroline can be added to the cell lysate to prevent degradation of TET2 enzyme. Avoid using EDTA or EGTA in the inhibitor cocktail as these may interfere with the following cation-exchange chromatography.
8. Pack 30 mL of a strong cation exchange resin into a FPLC column. Equilibrate the column with 10 bed volumes of wash buffer (50 mM MES buffer, pH 6) at the constant flow rate of 0.3 mL/min using a FPLC system.
9. Load the clarified lysate onto the pre-equilibrated column and wash with ∼10 bed volumes of wash buffer until the flow through becomes clear.
10. Elute TET2 using a 0-100% gradient from the wash buffer to the elution buffer (50 mM MES buffer, pH 6, 1 M NaCl) in 15 bed volumes followed by holding at 100% elution buffer for two-bed volumes.
    NOTE: Collect samples (100 µL each) of cell lysate before and after column loading along with all the elution fractions and analyze on 10% resolving SDS-PAGE.
11. Pool the fractions containing TET2 protein, freeze dry, dissolve enzyme pallet in 10 mL water and store at -80 °C.

2. 5mC Oxidation by TET2 Dioxygenase

1. Perform all demethylation reactions in triplicate with 3 µg of substrate (25-mer double stranded DNA, Table 2).
   1. Add 100 µg of purified TET2 enzyme in 50 µL of total reaction buffer containing 50 mM HEPES (pH 8.0), 200 µM FeSO₄, 2 mM 2OG (2-oxoglutarate/α-ketoglutarate), and 2 mM ascorbate and incubate at 37 °C for 1 h²¹.
   2. Quench TET2 catalyzed oxidation reactions with 5 µL of 500 mM EDTA.
2. After quenching TET2 reactions, prepare samples for LC-MS/MS analysis by separating the DNA from TET2 reaction mixture using oligo purification columns.
   1. Add 100 µL of oligo binding buffer to 55 µL of quenched reaction.
   2. Following this, add 400 µL of 100% ethanol to the mixture. Pass this mixture through an oligo binding column.
   3. Wash bound DNA with 750 µL wash buffer and elute in 20 µL water.
3. Digest the isolated DNA with 2 units of DNase I and 60 units of S1 nuclease at 37 °C for 12 h to produce individual nucleoside-monophosphates.
4. Following digestion, add 2 units of calf intestinal alkaline phosphatase (CIAP) in the samples and incubate for addition 12 h at 37 °C to remove the terminal phosphate groups from nucleoside-monophosphates to obtain nucleosides.
5. Quantify all nucleosides, especially modified cytosines, using the LC-MS/MS method described below.

3. Quantitative LC-MS/MS-based Assay Development

1. Prepare 100 µM stock solution of all modified cytosine nucleosides [5-methyl-2′-deoxycytidine (5mdC), 5-hydroxymethyl-2′-deoxycytidine (5hmdC), 5-formyl-2′-deoxycytidine (5fdC), and 5-carboxy-2′-deoxycytidine (5cadC)] and normal DNA bases (adenine, thymine, cytosine, and guanine) in HPLC-grade water for the development of the LC-MS/MS method.
2. Optimize nucleoside-dependent MS/MS parameters by infusing stock solutions, one at a time, in mass spectrometer at a flow rate of 10 µL/min in EMS scan mode. Optimize following parameters: Declustering Potential (DP), Entrance Potential (EP), Collision Cell Entrance Potential (CEP), Collision Energy (CE), and Collision Cell Exit Potential (CXP) for each DNA nucleoside using automated quantitative optimization feature of the software.
3. Optimize source-dependent MS/MS parameters by injecting 10 µL of stock solution using a gradient with 25% solvent B at a flow rate of 0.3 mL/min where solvent A is 10 mM ammonium acetate (pH 4.0) and solvent B is 20% acetonitrile with 10 mM ammonium acetate (pH 4.0). Optimize following parameters: Curtain Gas (CUR): 10-50, Temperature: 0-600 °C, Gas Flow 1 (GS1): 0-50, Gas Flow 2 (GS2): 0-50, Collisionally Activated Dissociation (CAD): Low-Medium-High, Ion spray Voltage (IS): 4000-5500 for each DNA nucleoside using manual quantitative optimization feature of the software in FIA (Flow Injection Analysis) mode.
4. To separate all eight DNA nucleosides, perform liquid chromatography using the following gradient: 0% solvent B (0-2 min), 0-20% solvent B (2-5 min), 20-60% solvent B (5-9 min), 60-0% solvent B (9-10 min) and then equilibrate with solvent A for 5 min at a flow rate of 0.3 mL/min on a C18 column (Particle Size: 5 µm, Pore Size: 120 Å).
5. Using the optimum MS/MS parameters (step 3.2 and 3.3) coupled with the above-mentioned liquid chromatography gradient (step 3.4), determine the response linearity, limit of detection (LOD), and lower limit of quantification (LLOQ) using a two-fold serial dilution of a 100 µM standard mixture containing all eight nucleosides. Draw standard curves for all eight DNA nucleosides.
6. Detect and quantify all nucleosides, especially modified cytosines, produced in step 2.4 using the LC-MS/MS method and standard curves.