Establishment And Application Of Analytical Methods For Nucleosides And Nucleotides Based On LC-MS/MS

Nucleotides are precursors to the synthesis of deoxyribonucleic acid(DNA)and ribonucleic acid(RNA),which are involved in almost all cellular processes and play an important role in structure,metabolism,energy and regulatory functions.In humans and animals,normal unmodified nucleosides can be phospholipated by enzymes to form new RNA,or degraded into uric acid and?-alanine.The modified nucleoside does not have a specific enzyme system to degrade or reuse it,and can only be excreted with urine,so the content of modified nucleosides in the urine can reflect the metabolic rate of the body cells.Tumor cells proliferate faster than normal cells,and when converted from one growth phase to another,they are usually accompanied by changes in nucleotide,nucleoside,and base turnover rates.Therefore,nucleosides and nucleotides have often been proposed as tumor markers for diagnosing cancer in recent years.Gastric cancer is one of the most common malignancies in the world,and its incidence is high and found was later period,leading patients to miss the best treatment opportunity.The 5-year prognosis survival rate of patients with advanced gastric cancer is less than 30%.Finding a tumor marker for diagnosing gastric cancer is an urgent problem to be solved.In this experiment,targeted metabolomics techniques were used to quantitatively analyze nucleosides and nucleotides in cells and plasma to obtain differential metabolites,in order to obtain tumor markers that can be used to diagnose gastric cancer.The research contents are as follows:(1)The liquid phase conditions and mass spectrometry conditions for the detection of 35 nucleic acid species were determined.Chromatographic conditions:ACQUITY UPLC HSS T3(1.8?m,2.1 x 100 mm)as Chromatographic column,2 mmol/L ammonium acetate(pH 5.0±0.05)-methanol solution as mobile phase,at a flow rate of0.2 mL/min and the injection volume was 1?L.Mass spectrometry conditions:via electrospray ionization(ESI),multiple reaction monitoring(MRM)scanning mode in positive ion monitoring mode for quantitative analysis of 35 nucleosides and nucleotides.(2)Optimized the conditions of cell sample collection,quenching,solvent extraction,reconstitution solvent,etc.The cell sample pretreatment method was determined,and Specificity,calibration curve and linear range,precision,accuracy,stability,and matrix effects were examined.The results showed that the method was sensitive,rapid and reproducible,and suitable for the absolute quantitative analysis of35 kinds of nucleosides and nucleotides in cells.(3)The established LC-MS/MS analysis method and cell pretreatment method were applied to the gastric cancer cell,which contained highly differentiated cell NCI-N87,the moderately differentiated cell SGC-7901,the poorly differentiated cell MGC-803,and the gastric mucosal epithelial cell GES-01.Principal component analysis and t-test analysis showed that there were 15 differential metabolites in highly differentiated cells NCI-N87 and medium differentiated cells SGC-7901 compared with GES-01 in gastric mucosal epithelial cells;there were 17 differential metabolites in differentiated cells MGC-803 and gastric mucosal cells GES-01.Among them,the comparison of the differential metabolites between high,medium and low-differentiated cells and gastric mucosal cells found that N~6-methyladenosine and 1,-methyladenosine were higher in differentiated cells than in gastric mucosa cells;Uridine,cytidine,2,-oxy-methylcytidine,5,-methyluridine,and dCMP are lower in differentiated cells than gastric mucosal cells.(4)The J2 compound is an anti-tumor drug which is designed and modified by oridonin as a lead compound,and has a good proliferation inhibition effect on gastric cancer cell MGC-803.The established analysis method was used to quantitatively analyze the MGC-803 cells stimulated by J2 compound for 24 h and 48 h.The principal component analysis score showed that the MGC-803 cells stimulated by J2 compound for 24 h and 48 h were different from the control group.The results of t-test showed that 10 kinds of nucleosides and nucleotides were down-regulated in MGC-803 cells stimulated by J2 compounds for 24 h;11 kinds of nucleosides and nucleotides were down-regulated in MGC-803 cells stimulated by J2 compounds for 48 h.(5)Optimized the pretreatment conditions such as extraction solvent and reconstituted solvent of plasma samples,and the plasma sample pretreatment method was determined,and the specificity,calibration curve and linear range,three-day precision,and the stability of three freeze-thaw cycles and matrix effects were examined.The results showed that the method was stable,reliable and reproducible,and was suitable for the absolute quantitative analysis of 35 kinds of nucleosides and nucleotides in plasma.(6)Quantitative analysis of plasma samples from 80 gastric cancer patients and20 healthy volunteers by LC-MS/MS analysis and plasma pretreatment.The principal component analysis score showed that the two groups of samples were poorly separated.When the two groups of samples were analyzed by OPLS-DA showed that the two samples were separated.The permutation test showed that the model had no overfitting,and 12 differential metabolisms were screened by VIP analysis.The t-test results showed that there were 11 differential metabolites(P<0.05).ROC curve analysis was performed on differential metabolites with VIP>1 and P<0.05.There are four differential metabolites that the AUC?0.75 are expected to become tumor markers,namely:2,-oxy-methyluridine,2,-Oxygen-methylcytidine,AMP,allantoin.