Analysis And Application Of Chiral Amino Acids Based On Chlorine-labeled Chiral Aldehyde Probe

Amino acids are an important research target of metabolomics as well as other amine compounds.More and more studies have shown that D-and L-amino acids have different sources,metabolic pathways,biological activities and toxicities and play different roles in the diagnosis of various diseases.Due to chiral amino acids（？）type variety,large polarity,chromophore deficiency,low D-configuration content,easily being interfered by endogenous substances and chiral resolution difficulty,the screening of chiral amino acid-based biomarkers is extremely challenging.Here,we synthesized a chemical isotope labeling probe L-/D-BPCl that can be used for the separation and detection of chiral and achiral amino acids in biological samples and established a targeted and untargeted method by high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）.This method can overcome the matrix effect of complex biological samples and has been successfully applied to the targeted and untargeted analysis of amine compounds in urine,plasma,saliva,and cell samples;it was further applied to urine samples of gastric cancer（GC）patients and gastric cancer cells for amine compounds analysis.Urine and cell metabolomics in combination with statistical methods were used to identify the characteristic biomarkers of gastric cancer.The main researches and results are as follows:1.The synthesis of the chiral aldehyde probe BPCl and the method establishment of chiral amino acid analysis.Based on the previous work,we synthesize and purified a stable CIL probe（L-/D-BPCl）（purity>99.9%）for chiral amino acid derivatization.L-and D-BPCl have the stereoselectivity towards the mass spectrum response of L-and D-amino acids,respectively,and the chiral selectivity（Cs）of D-BPCl for D-amino acids against L-amino acids was calculated to be in 3.31-14.37.Based on Cs as well as the characteristic product ions at m/z 218 and 105 and the characteristic ³⁵Cl:³⁷Cl ratio（3:1）,an HPLC-MS/MS method was established for separating and detecting 29 chiral and achiral amino acids.2.The targeted and untargeted analysis of amine compounds in urine,plasma and saliva samples using the BPCl probe.A D-BPCl-based targeted analytical method for amine compounds in urine,plasma and saliva was developed.This method showed good analytical performance in terms of selectivity,sensitivity,linearity,matrix effect,standard addition recovery,precision and accuracy,stability,etc.It successfully detected 29,29,and 26 targeted amino acids in urine,plasma and saliva samples,respectively.The average concentrations of achiral glycine and L-amino acid in urine,plasma,and saliva samples were 2.77-144.16,10.58-364.86,and 0.8-29.17μmol/L,respectively,and the D-amino acid contents were 0.06-31.29,0.02-0.39,and 0.02-5.11μmol/L,respectively.Additionally,an untargeted analytical method based on D-and L-BPCl was developed and 165,110,and 47 amine metabolites were detected in urine,plasma and saliva samples,respectively,including 52,39,and 27 carboxyl-containing and 35,20,and 7 achiral amine metabolites,respectively.3.Application of the D-BPCl probe in GC urine amine metabolomics.A pseudotargeted,D-BPCl-based,high-throughput HPLC-MS/MS method was established.The method was applied to the urine analysis of 84 GC patients and 80 healthy volunteers to screen for amino-containing biomarkers suitable for GC diagnosis.Among the 29targeted amino acids,the average contents of all the L-and D-amino acids in urine were0.29-83.31（containing achiral glycine）and 0.014-21.93μmol/mmol urinary creatinine,respectively.Multivariate and univariate statistical analysis revealed 18 variables as the differential variables of GC patients against healthy controls.Nine unknown metabolites were compound-matched in HMDB using the high-resolution MS data,and two were identified as enantiomers（β-（pyrazol-1-yl）-L-alanine andβ-（pyrazol-1-yl）-D-alanine）.A GC diagnostic equation with high prediction accuracy（88.9%）,using age,D-isoleucine,D-serine andβ-（pyrazol-1-yl）-L-alanine as variables,was then established and evaluated through binary logistic regression analysis.4.Application of D-BPCl in GC cell amine metabolomics.HGC27 cell and gastric epithelial cell GES-1 were selected.An analytical pipeline for cell metabolomics including cell culturing,cell metabolite extraction,D-BPCl derivatization,and HPLC-MS/MS detection was constructed.A total of 95 chiral and achiral amine metabolites were detected in pooled cell extracts.By use of multivariate and univariate statistical methods,a total of 20 differential variables were found.The contents of 19 differential variables decreased significantly in HGC27 against GES-1,and only L-citrulline（？）s content increased.The 6 main metabolic pathways involved were drawn and analyzed.