【作者】 黄荣荣；

【导师】 孙翠荣；

【作者基本信息】 浙江大学 ， 药学， 2021， 博士

【摘要】 氨基酸属于胺类化合物,是代谢组学的重要组成部分。越来越多的研究表明,手性氨基酸的不同构型涉及不同来源、代谢途径、生物活性和毒性,并且在各种疾病的诊断中发挥不同的指示作用。由于手性氨基酸存在种类多、极性大、发色团缺乏、D构型含量低、内源性干扰物多、手性拆分难等问题,基于手性氨基酸分离与检测的生物标志物的筛选极具挑战。本文通过合成一种可用于生物样品中手性和非手性氨基酸分离与检测的化学同位素标记探针L-/D-BPCl,建立基于探针和高效液相色谱-串联质谱联用技术的靶向和非靶向分析方法。该方法能够克服复杂生物样本的基质效应,成功应用于尿液、血浆、唾液、细胞样品中胺类化合物的靶向和非靶向分析;并进一步应用于胃癌及健康人群尿液样本、胃癌细胞与胃正常细胞中手性氨基酸的分离与定量,综合利用尿液和细胞代谢组学的相关技术与统计学方法筛选出胃癌的特征生物标志物。主要研究内容及结果如下:1.手性醛探针BPCl的合成及其手性氨基酸检测方法的建立:基于课题组前期研究,设计并合成、纯化、鉴定了一种可用于手性氨基酸衍生的稳定性好的L-/D-BPCl探针,其手性纯度大于99.9%。L和D构型BPCl对相同构型的氨基酸具有立体选择性质谱响应,其中D-BPCl对D型氨基酸的手性选择性是L型的3.31-14.37倍。基于上述特征,并结合特征性产物离子m/z 218负离子和m/z 105正离子以及Cl原子天然同位素的识别功能(³⁵Cl:³⁷Cl为3:1),建立了能同时分离与检测29个手性和非手性氨基酸的高效液相色谱-串联质谱联用方法。2.BPCl探针对尿液、血浆和唾液中胺类化合物的靶向和非靶向分析:发展了基于D-BPCl的尿液、血浆和唾液中胺类化合物的靶向分析方法,该方法显示出良好的选择性、灵敏度、线性、基质效应、加标回收率、精密度和准确度、稳定性等分析效能,并成功检测到尿液、血浆和唾液样本中29、29、26个目标氨基酸,非手性甘氨酸和L-氨基酸在以上三种样品中的平均浓度分别为2.77-144.16、10.58-364.86、0.8-29.17μmol/L;D-氨基酸的平均浓度分别为0.06-31.29、0.02-0.39、0.02-5.11μmol/L。此外,基于D-BPCl和L-BPCl的非靶向分析方法在尿液、血浆和唾液中分别检测到165、110、47个胺类代谢物,从中归属了52、39、27个含羧基的和35、20、7个非手性的胺类代谢物。3.D-BPCl探针在胃癌尿液手性胺类代谢组学中的应用:建立了一种基于D-BPCl的高效液相色谱-串联质谱联用的拟靶向高通量检测方法,将其应用于84位胃癌患者和80位健康志愿者尿液样本的分析中以筛选适用于胃癌诊断的胺类生物标志物。29个目标氨基酸中,甘氨酸和L型氨基酸在尿液中的平均含量为0.29-83.31μmol/mmol尿肌酐,D型氨基酸的平均含量为0.014-21.93μmol/mmol尿肌酐。基于多变量和单变量统计分析方法,筛选出胃癌的18个差异变量。根据高分辨数据对其中9个未知代谢物进行数据库的检索和匹配,成功鉴定出β-（吡唑-1-基）-L-丙氨酸和β-（吡唑-1-基）-D-丙氨酸这对手性对映异构体。随后以年龄、D-异亮氨酸、D-丝氨酸和β-（吡唑-1-基）-L-丙氨酸为变量通过二元逻辑回归分析方法建立胃癌诊断方程,对其区分度和准确度进行评估,该方程的预测准确率高（88.9%）,可用于临床诊断。4.D-BPCl探针在胃癌细胞手性胺类代谢组学中的应用:以人胃癌细胞HGC27和人胃上皮细胞GES-1为研究对象,构建了一套包含细胞培养、细胞代谢物提取、D-BPCl衍生、高效液相色谱-串联质谱联用检测的细胞代谢组学研究方法。将方法应用于细胞提取物中手性和非手性胺类代谢物的分析中,共检测到95个胺类代谢物,并对甘氨酸、14个L型氨基酸、11个D型氨基酸进行绝对定量。结合多变量和单变量统计方法,共找到20个差异变量,其中19个变量的含量在HGC27中显著下调,只有L-瓜氨酸含量增加,绘制并分析了受影响的6条主要代谢通路。更多还原

【Abstract】 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.更多还原