Jet stream electrospray ionization source

Enzymatic hydrolysis was performed for extracted DNA and RNA, as described by Noyon et al. [13] (link). Briefly, DNA and RNA were digested into nucleosides in the presence of internal standards (labeled 15N5-dGua and 5-fluorocytidine) using nuclease P1, PDE II, PDE I, alkaline phosphatase, and appropriated buffers. Thereafter, all samples were dried by vacuum centrifuge, dissolved in 50 μl aqueous mobile phase, and 10 μl was injected into LC/MSMS (in dynamic MRM positive mode) for the analysis of chloro(deoxy)cytidine (Cl-(d) Cyt) and 8-oxo(deoxy)guanosine (oxo-(d)Gua). Briefly, the analyses were performed using a LC/MS system from Agilent Technologies (Santa Clara, CA, USA): Agilent 1290 Infinity Binary -UHPLC system fitted to a mass spectrometer Agilent Jet Stream electrospray ionization source (AJS) -Triple Quadrupole (QqQ) 6490 series. Nucleoside separation was performed at 4 °C on Poroshell 120, EC-C18, 2.1 × 100 mm, 2.7-μm column, preceded by a Poroshell 120, EC-C18, 2.1 × 5 mm, 2.7-μm guard column, using an ammonium acetate 10 mM in water pH 5/methanol gradient. All these LC and MS parameters were detailed and validated in a previous article [13] (link). The results are expressed as the ratio Cl-dCyt/dCyt, Cl-Cyt/ Cyt, oxo-dGua/dGua, and oxo-Gua/Gua.

Chromatography was performed on a 1260 Infinity liquid chromatography system. 15 μL of sample is injected on to a Zorbax SB-C18 column (4.6 x 250mm, 5 μm) held at 35 °C. The elution program was as follows: 0 min, 5% B; 110 min, 47% B; 120-125 min, 90% B; 126-135 min, 5% B. The flow rate was 1 mL/min, where A was 0.1% formic acid in water and B was 0.1% formic acid in acetonitrile. The LC system was coupled to an Agilent 6400 series Triple Quadrupole mass spectrometer equipped with an Agilent Jet Stream electrospray ionization source (AJS ESI), operating with 3.5 kV capillary voltage in both positive and negative modes (sheath gas temperature 250 °C, sheath gas flow rate 11 L/min, drying gas temperature 300°C, drying gas flow rate 5 L/min and nebulizer pressure 45 psi in both positive and negative mode). The fragmentor voltage is 135V. Positive and negative ionization mode mass spectra were simultaneously collected across the mass range of 100-1500 m/z. For MS/MS acquisition mode, the parameters were the same except that collision energy settings of 15V was used.

Enzymatic hydrolysis was performed for extracted DNA and RNA and dephosphorylation for free nucleotides from cytoplasm, as described in [22] (link).
Briefly, DNA and RNA were digested into nucleosides in the presence of internal standards (Labeled dGua ( 15 N5) and 5-fluorocytidine) using nuclease P1, PDE II, PDE I, alkaline phosphatase and appropriated buffers. Free nucleotides from cytoplasmic pool were dephosphorylated using alkaline phosphatase and appropriated buffers.
Thereafter, all samples were dried by vacuum centrifuge, dissolved in 50 µL aqueous mobile phase and 10 µL were injected into LC/MSMS (in dynamic MRM positive mode). Briefly, the analyses were performed using a LC/MS system from Agilent Technologies (Santa Clara, CA, USA): an Agilent 1290 Infinity Binary -UHPLC system fitted to a mass spectrometer Agilent Jet Stream electrospray ionization source (AJS) -Triple Quadrupole (QqQ) 6490 series. Nucleoside separation were performed at 4°C on Poroshell 120, EC-C18, 2.1 x 100 mm, 2.7 µm column, preceded by a Poroshell 120, EC-C18, 2.1 x 5 mm, 2.7 µm guard column, using an ammonium acetate 10 mM in water pH 5/methanol gradient. All these LC and MS parameters were detailed and validated in a previous article [22] (link).