Api 3000 triple quadrupole mass spectrometer

Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All of the other reagents and solvents were purchased as the highest grade available and used without further purification.
Thin layer chromatography (TLC) was performed using pre-coated silica gel plates (Yantai dexin Bio-Technology Co., Ltd., Yantai, China). Column chromatography was performed using silica gel (200–300 mesh; Yantai Chemical Industry Research Institute, Yantai, China). NMR spectra were recorded on a JNM-ECA-400 400 MHz spectrometer (JEOL Ltd., Tokyo, Japan) using CDCl3 and DMSO-d6 as solvent. Chemical shifts are expressed in (ppm), with tetramethylsilane (TMS) functioning as the internal reference. MS was performed on an API 3000 triple-quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada) equipped with a Turbo Ion Spray electrospray ionization source (AB Sciex, Concord, ON, Canada) that was used for mass analysis and detection. Analyst 1.4 software (AB Sciex, Concord, ON, Canada) was used for data acquisition.

All of the reagents and solvents were purchased from Innochem (Beijing, China), Aladdin (Shanghai, China), Energy Chemical (Shanghai, China), TCI (Tokyo, Japan), Ark Pharm (Libertyville, IL, USA), and used without additional purification. Anhydrous solvents were stored in sure-seal bottles under dry nitrogen. Analytical thin-layer chromatography was conducted on pre-coated silica gel plates (Yantai Dexin Biotechnology Co., Ltd., Yantai, China). Visualization was accomplished with 254 nm and 365 nm UV light. Column chromatography was performed using silica gel (200–300 mesh; Qingdao Ocean Chemical Engineering Co., Ltd., Qingdao, China).
Nuclear magnetic resonance (NMR) spectra were obtained on a JNM-ECA-400 400 MHz spectrometer (JEOL Ltd., Tokyo, Japan). Chemical shifts were reported in ppm and TMS was used as the internal standard. Coupling constants (J) are given in Hertz. Spin multiplicities were reported as the following abbreviations: s (singulet), d (doublet), dd (doublet doublet), t (triplet), q (quadruplet), m (multiplet). The mass spectrometry (MS) systems were the API 3000 triple-quadrupole mass spectrometer equipped with a Turbo Ion Spray electrospray ionization (ESI) source (AB Sciex, Concord, ON, Canada). HPLC analysis was performed using an Agilent 1260 Series (California, CA, USA).

IDMF (1 mM) was dissolved in DMSO (2%) and ethylene glycol (2%) in 20 mM HEPES (pH 7.4). We added 20 μL of enzyme (1 mg/mL) and the solution was incubated at 37 °C for 15, 30, 60, and 120 min. The reaction was stopped using Methanol. IDMF and MMF were quantified using a UV detector at a wavelength of 210 nm. HPLC was performed with 0.1% formic acid in water as the weak solvent and methanol as the strong solvent. The analysis was performed using a Luna C18 4.6 mm × 100 mm column with a flow rate of 1.0 mL/min (Phenomenex, Torrance, CA, USA).
The molecular weight of IMMF was determined using the API3000 triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) and electrospray ionization (ESI). The Q1 scan was performed from 100–1500 Da with negative mode. MS analysis conditions were as follows: curtain gas (10 arbitrary units), nebulizer gas (14 arbitrary units), ion spray voltage (−4500 V), channel electron multiplier (CEM) at 2800 V. Declustering and entrance potentials were −130 V and −10 V, respectively.

Proteinase K, phenol, and chloroform digestions were used for DNA extraction. The levels of 5mdC were quantified using high-performance liquid chromatography (Agilent 1260VL, Agilent Technology, Santa Clara, CA, USA) equipped with an API 3000™ triple quadrupole mass spectrometer (AB SCIEX, Concord, ON, Canada). The detailed protocol of the analysis has been described previously [18 (link),19 (link)]. The 5mdC level present in the DNA sample was expressed as a percentage of the total cytosine content (methylated and non-methylated). The DNA methylation level was expressed as either (5mdC)/(5mdC + dC) without internal standard adjustments or (5mdC)/(dG) with adjustment to the respective ¹⁵N-labeled internal standards, where dG is an internal standard that allows a simpler and accurate determination of dC methylation and avoids the use of expensive isotope-labeled internal standards [18 (link),19 (link)]. Figure 1 displays the chromatogram of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detecting reference compounds of 5mdC.