Establishment And Application Of Cell Metabolomics Method Based On UHPLC-MS/MS

Gastric cancer is one of the most common malignant tumors in the world,China is a high-risk area for gastric cancer.Due to its large population base,the number of gastric cancer population ranks first in the world.Males have a higher incidence and faster progress than females.However,early diagnosis of gastric cancer is at a low level,so that the patients did not get timely treatment and lose their life.Therefore,it is of great significance to find a new diagnosis method for early gastric cancer.The occurrence of gastric cancer is associated with metabolic disorders such as amino acid metabolism,glycolysis pathway,and tricarboxylic acid cycle.Based on UHPLC-MS/MS technology,the metabolic analysis of amino acid,and glycolysis tricarboxylic acid cycle between MGC-803 cells and hormone-stimulated MGC-803 cells was carried out.In addition,quantification of 24 amino acids in cell samples was also studied.I.UHPLC-MS/MS metabolic analysis for amino acids,sugars and organic acids in MGC-803 cells was established.The method of cell sample pretreatment was established,and the method of cell pretreatment was determined through the optimization of cell collection,quenching,washing solvent,solvent extraction and crushing method.(2)Liquid chromatography and mass spectrometry conditions were established and optimized.UPLC BEH Amide(1.7 μm,2.1×100 mm,Waters)column was used with a gradient elution for a run time of 45 min.Mass spectrometry conditions:Electrospray ionization(ESI),positive and negative ion detection mode,and multiple reaction monitoring(MRM)scanning mode.95 MRM ion pairs were used for analysis.(3)Multivariate statistical analysis showed that the MGC-803 cells stimulated by DHT were compared with normal MGC-803 cells,and 27 different metabolites(P<0.05)were obtained.The MGC-803 cells stimulated by E2 were compared withnormal MGC-803 cells.23 different metabolites were derived(P<0.05).II.UHPLC-MS/MS quantitative analysis method for 24 kinds of amino acids was established(1)Using amino acid standards to optimize chromatographic and mass spectrometry conditions.UPLC BEH Amide(1.7 μm,2.1×100 mm,Waters)column was used with a gradient elution for a run time of 8 min.Mass spectrometry conditions: electrospray ionization(ESI),positive ion detection mode,and multiple reaction monitoring(MRM)scanning mode.(2)Method validation was performed.Specificity,precision,accuracy,calibration curve and linear range,recovery rate,and stability were examined.The results showed that the developed method was fast,sensitive and reproducible,and suitable for quantitative analysis of 24 kinds of amino acids.(3)Compound J3 is a brand-new compound which is designed and modified by oridonin as a lead compound.Compound J3 was found to have good inhibition on the proliferation of gastric cancer cell MGC-803.This study used this method to perform quantitative analysis of amino acids in MGC-803 cells and J3-stimulated MGC-803 cells.Statistical analysis showed that proline,alanine,glycine,hydroxyproline,methionine,arginine,asparagine,and tyrosine in J3-stimulated MGC-803 cells was significantly increased,and glutamine,phenylalanine,serine,valine,homoserine,and tryptophan were significantly reduced(P<0.05).The concentration of the other amino acids remained basically unchanged.