The Qiagen Blood & Cell Culture Maxi Kit was purchased from Qiagen (Valencia, CA). Nuclease P1, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), 2-N-(morpholino)ethanesulfonic acid (MES), α-cyano-4-hydroxy-cinnamic acid (CCA), ammonium citrate (dibasic), triethylammonium acetate buffer (TEAA, 1M), 5-hydroxymethyl-2′-deoxyuridine, thymidine-5′-monophosphate (TMP) and triethylammonium bicarbonate buffer (1M) were from Sigma (St. Louis, MO). Phosphodiesterase I was from Worthington (Lakewood, NJ). BIOMAX-5 Ultrafree MC centrifugal filter devices were from Millipore (Billerica, MA). Propanesulfonic acid silica was from J. T. Baker (Phillipsburg, NJ). Microcentrifuge tubes, pipetter tips, and HPLC grade acetonitrile (ACN) were from Fisher Scientific (Pittsburgh, PA). All materials were used as received. Human placenta from a smoker was obtained from the National Disease Research Interchange (Philadelphia, PA). Benzoylhistamine (BH) and d4-benzoylhistamine (d4-BH) were synthesized as described.12 ThymineGlycolThymineDimer (0801215) was a gift from ZeptoMetrix (Buffalo, NY). CS Chem3D Pro software was from CambridgeSoft Corporation (Cambridge, MA).
DNA was isolated using a Qiagen Blood & Cell Culture Maxi Kit. Digestion of 25 μg of DNA to nucleotides was done with nuclease P1 at pH 5.5 followed with phosphodiesterase I at pH 9, 3 hours at 45 °C for both. The DNA digest was filtered with a Ultrafree-MC Biomax-5 Membrane 5 kDa filter.
Digested DNA (3 μg aliquot) was dried, and redissolved in 20 μL of 80 mM triethylammonium bicarbonate buffer for HPLC separation, using an XBridge C18 column (1×150 mm, 3.5 μm). The gradient flow rate was 30 μL/min, from 1% to 30 % ACN in 30 min, with 20 mM triethylammonium bicarbonate buffer at pH 8.5. Based on monitoring by absorbance, fractions were collected between the peaks for the four major normal nucleotides, and evaporated in a Speed Vac (Savant Instruments, Nassau-Suffolk, NY).
The labeling reaction was performed by adding 3.5 μL of 12 mM BH (do-BH) or d4-BH (6 mM BH and 6 mM d4-BH when isotopologue labeling with tag mixture was done), and 3.5 μL of 80 mM EDC, in 0.01 M MES buffer at pH 6, to the dried HPLC collection vial, and then, after mixing, the sample was kept at room temperature for 3 hours.
The residual BH tag and EDC were removed with an UltraMicroSpin Column (The Nest Group, Southborough, MA) packed with 55 μL of propyl sulfonic acid silica, followed by evaporation and then redissolving in 5 μL of TEAA buffer (0.02 M at pH 7, 3% methanol, 3% ACN). The sample was injected into a trapping column (PepMap C18, 300 μm ID × 5 mm) on a column switching module of a Micro-LC system (Dionex, Sunnyvale, CA) at a flow rate of 7 μL/min with 20 mM TEAA buffer containing 1% ACN for 4 min, then 2 min at 14 μL/min. Switching was then done onto a capillary column (180 μm I.D × 150 mm), which was gradient eluted from 3% to 60 % ACN in 40 min at 2.2 μL/min. A droplet was collected onto a MALDI plate every 20 sec with a Probot Micro Fraction Collector (Dionex, Sunnyvale, CA).
CCA matrix (0.5 μl, 5 mg/mL in ACN: water, 50:50, v/v, with 2.5 mM of ammonium citrate, dibasic) was deposited on each dried spot, followed by air-drying for 5 min. For calibration, a reference compound (e.g. 1,N6-etheno dAMP subjected to BH-labeling) can be included in the CCA matrix solution. Analysis was done on a Voyager DE STR MALDI-TOF-MS or a Model 5800 MALDI-TOF/TOF-MS (AB SCIEX, Foster City, CA) in a negative, reflectron mode with a delay time of 150 ns. Each sample well was surveyed to find “sweet spot”, and then 400 laser pulses were averaged to generate a spectrum. MS/MS was performed with a medium pressure of air and a mass resolution window of 400 with the metastable-suppresor on.
DNA was isolated using a Qiagen Blood & Cell Culture Maxi Kit. Digestion of 25 μg of DNA to nucleotides was done with nuclease P1 at pH 5.5 followed with phosphodiesterase I at pH 9, 3 hours at 45 °C for both. The DNA digest was filtered with a Ultrafree-MC Biomax-5 Membrane 5 kDa filter.
Digested DNA (3 μg aliquot) was dried, and redissolved in 20 μL of 80 mM triethylammonium bicarbonate buffer for HPLC separation, using an XBridge C18 column (1×150 mm, 3.5 μm). The gradient flow rate was 30 μL/min, from 1% to 30 % ACN in 30 min, with 20 mM triethylammonium bicarbonate buffer at pH 8.5. Based on monitoring by absorbance, fractions were collected between the peaks for the four major normal nucleotides, and evaporated in a Speed Vac (Savant Instruments, Nassau-Suffolk, NY).
The labeling reaction was performed by adding 3.5 μL of 12 mM BH (do-BH) or d4-BH (6 mM BH and 6 mM d4-BH when isotopologue labeling with tag mixture was done), and 3.5 μL of 80 mM EDC, in 0.01 M MES buffer at pH 6, to the dried HPLC collection vial, and then, after mixing, the sample was kept at room temperature for 3 hours.
The residual BH tag and EDC were removed with an UltraMicroSpin Column (The Nest Group, Southborough, MA) packed with 55 μL of propyl sulfonic acid silica, followed by evaporation and then redissolving in 5 μL of TEAA buffer (0.02 M at pH 7, 3% methanol, 3% ACN). The sample was injected into a trapping column (PepMap C18, 300 μm ID × 5 mm) on a column switching module of a Micro-LC system (Dionex, Sunnyvale, CA) at a flow rate of 7 μL/min with 20 mM TEAA buffer containing 1% ACN for 4 min, then 2 min at 14 μL/min. Switching was then done onto a capillary column (180 μm I.D × 150 mm), which was gradient eluted from 3% to 60 % ACN in 40 min at 2.2 μL/min. A droplet was collected onto a MALDI plate every 20 sec with a Probot Micro Fraction Collector (Dionex, Sunnyvale, CA).
CCA matrix (0.5 μl, 5 mg/mL in ACN: water, 50:50, v/v, with 2.5 mM of ammonium citrate, dibasic) was deposited on each dried spot, followed by air-drying for 5 min. For calibration, a reference compound (e.g. 1,N6-etheno dAMP subjected to BH-labeling) can be included in the CCA matrix solution. Analysis was done on a Voyager DE STR MALDI-TOF-MS or a Model 5800 MALDI-TOF/TOF-MS (AB SCIEX, Foster City, CA) in a negative, reflectron mode with a delay time of 150 ns. Each sample well was surveyed to find “sweet spot”, and then 400 laser pulses were averaged to generate a spectrum. MS/MS was performed with a medium pressure of air and a mass resolution window of 400 with the metastable-suppresor on.
