Calf thymus DNA, 2´-deoxycytidine-5´-monophosphate, disodium salt (dCMP), hydroquinone,
p-benzoquinone, nuclease P1, ferric chloride, triethylammonium acetate buffer (TEAA, 1M), 2-N-(morpholino)ethanesulfonic acid (MES), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), α-cyano-4-hydroxy-cinnamic acid (CCA), and ammonium citrate (dibasic) were from Sigma-Aldrich (St. Louis, MO). 5-Methyl-2´-deoxycytidine-5´-monophosphate, disodium salt (mdCMP) was a gift from Affymetrix (Cleveland, Ohio). 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). The OASIS columns were from Waters (Milford, MA). All materials were used as received. Benzoylhistamine (BH), benzoylhistamine-d
4 (BH(d
4), where the ethylene moiety is tetradeuteriated), and
p-bromo-benzoylhistamine (Br-BH) were synthesized as described.
9 Hydroquinone was reacted in the presence of ferric chloride with calf thymus DNA as described.
10 (link) Our method for measuring DNA adducts, described before,
8 (link),9 is summarized here. The digestion of the modified 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 sample was purified with an OASIS column (186000383, Waters) and dried. The labeling reaction was performed by adding 3.5 μL of 12 mM BH (6 mM BH and 6 mM BH(d
4)), or 12 mM Br-BH, and 3.5 μL of 80 mM EDC in 0.01 M MES buffer at pH 6, to the dried OASIS collection vial, and then, after mixing, the sample was kept at room temperature in the dark for 3 hours. The sample was separated on a capillary HPLC column (PepMap 180 μm I.D×150 mm, Dionex, Sunnyvale, CA) using a gradient flow 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 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 onto each dried spot, followed by air-drying for 5 min. 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 ion mode with a delay time of 150 ns. Each sample well was surveyed to find a “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-ion suppressor on.
p-Benzoquinone was reacted with dCMP or mdCMP as described.
13 (link) The nucleotide (8 mg of dCMP or 8.3 mg of mdCMP, 0.023 mmol) and
p-benzoquinone (16 mg, 0.15 mmol) were allowed to react in 1 mL of 0.1 M sodium acetate buffer (pH 5) at 37 °C. After 17 h, the diluted reaction mixture (1:100 in water) was mixed with CCA matrix in a 1:5 ratio and subjected to MALDI-TOF-MS in a negative ion mode. Also, MS/MS in a positive ion mode was performed on the protonated modified nucleobase formed in MALDI ion source. Further, a combined sample (1 μL of each diluted reaction mixture) was labeled with Br-BH and subjected to MALDI analysis directly.
Wang P., Gao J., Li G., Shimelis O, & Giese R.W. (2012). Nontargeted Analysis of DNA Adducts by Mass-Tag MS: Reaction of p-Benzoquinone with DNA. Chemical research in toxicology, 25(12), 2737-2743.