INVESTIGATIONS OF URINARY METABOLIC PROFILING USING GAS CHROMATOGRAPHY/MASS SPECTROMETRY: A PRETREATMENT METHOD INVESTIGATRION

As one of the most widely adopted techniques in metabonomics, gas chromatography/mass spectrometry (GC/MS) combines the advantages of GC and MS, which includes high-efficiency separation and resolution, as well as informative mass spectral of reference compounds for metabolites identification, providing the basis for gaining multitudinous metabolites. In the meantime, attentions have to be paid to the stability and reproducibility of GC/MS based metabonomics as well, which has a great impact on the accuracy and validation of metabonomic investigations. Furthermore, urine is frequently applied in metabonomic research. Accordingly, stability and reproducibility of urinary metabolic profiling were studied using gas chromatography/time of flight mass spectrometry (GC/TOFMS) in this work. It was demonstrated that the activity of urease dissolved in ultrapure water could be maintained for at least 15 days, however, urease prepared in phosphate buffer (pH=7.0) remained active only for 5 days under the same condition at -20℃. Ethylmalonic acid, glycerol, 1,2,3-butantriol, L-isoleucine, L-threonine, glycine, succinic acid, which were overlaid by urea in the urine samples without urease treatment, could be effectively isolated and identified after urease treatment. Nevertheless, the amount of detected metabolites and the contents of majority of the metabolites all decreased with urea degradation. Additionally, metabolites in urine with urea treatment for 15 min are more stable compared to 30 min and 60 min according to the results of relative standard deviation (RSD) distribution of ion peak areas. On the other hand, it is clear from Fig.1. that metabolites in two-step derivatized samples are more stable than those in one-step samples. The average RSD of peak areas of ions in two-step derivatized samples is 13.10±14.62, significantly lower than that in one-step samples. And the number of ion peaks (RSD of peak areas<20%) from two-step derivatization samples is 1509, obviously larger than that from one-step samples. Besides, the volume ratio of methanol to urinary incubation solutions was studied. It was found that repeatability and stability became higher with the increase of the ratio of methanol to urinary solutions. In conclusion, high repeatability and stability of urinary metabolic profiling based on GC/MS techniques could be obtained by employing the following preprocessing conditions. Urease is prepared in ultrapure water; the time of urease treatment is 15 min; ratio of methanol to urinary incubation solutions is 4:1; and two-step derivatization is employed.As one of the most widely adopted techniques in metabonomics, gas chromatography/mass spectrometry (GC/MS) combines the advantages of GC and MS, which includes high-efficiency separation and resolution, as well as informative mass spectral of reference compounds for metabolites identification, providing the basis for gaining multitudinous metabolites. In the meantime, attentions have to be paid to the stability and reproducibility of GC/MS based metabonomics as well, which has a great impact on the accuracy and validation of metabonomic investigations. Furthermore, urine is frequently applied in metabonomic research. Accordingly, stability and reproducibility of urinary metabolic profiling were studied using gas chromatography/time of flight mass spectrometry (GC/TOFMS) in this work. It was demonstrated that the activity of urease dissolved in ultrapure water could be maintained for at least 15 days, however, urease prepared in phosphate buffer (pH=7.0) remained active only for 5 days under the same condition at -20℃. Ethylmalonic acid, glycerol, 1,2,3-butantriol, L-isoleucine, L-threonine, glycine, succinic acid, which were overlaid by urea in the urine samples without urease treatment, could be effectively isolated and identified after urease treatment. Nevertheless, the amount of detected metabolites and the contents of majority of the metabolites all decreased with urea degradation. Additionally, metabolites in urine with urea treatment for 15 min are more stable compared to 30 min and 60 min according to the results of relative standard deviation (RSD) distribution of ion peak areas. On the other hand, it is clear from Fig.1. that metabolites in two-step derivatized samples are more stable than those in one-step samples. The average RSD of peak areas of ions in two-step derivatized samples is 13.10±14.62, significantly lower than that in one-step samples. And the number of ion peaks (RSD of peak areas<20%) from two-step derivatization samples is 1509, obviously larger than that from one-step samples. Besides, the volume ratio of methanol to urinary incubation solutions was studied. It was found that repeatability and stability became higher with the increase of the ratio of methanol to urinary solutions. In conclusion, high repeatability and stability of urinary metabolic profiling based on GC/MS techniques could be obtained by employing the following preprocessing conditions. Urease is prepared in ultrapure water; the time of urease treatment is 15 min; ratio of methanol to urinary incubation solutions is 4:1; and two-step derivatization is employed

基于质谱基础上的个性化癌症医学代谢组学研究

Towards Personalised Medicine of Cancer based on MS-based Metabolomic

通过基于质谱的代谢组学和脂类组学鉴定前列腺癌SPOP相关代谢途径

Identification of SPOP Related Metabolic Pathways in Prostate Cancer by Mass Spectrometry-Based Metabolomics and Lipidomic

Method for liver tissue metabolic profiling based on liquid chromatography

Hepatocellular carcinoma (HCC) is one of the most frequent malignancy worldwide. Evaluation of tissue metabolites is with significant value in HCC study which can provide more direct information of metabolic disorder compared with biofluids. A protocol for the metabolic profiling of liver tissue was developed based on ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS). According to the design of experiment (DOE), methanol/water (4:1, v:v) was selected as the optimal extraction solvent. The established method was validated with a linearity over the 10–5000 ng/mL for internal standards (IS) and got an average correlation coefficient of 0.9986. The intra-day and inter-day RSD for most endogenous compounds were below 15% and the recovery of IS was from 84.8% to 109.1%. After method validation, this method was applied to HCC study. Liver tissue samples were collected from HCC patients and each sample group involved carcinoma tissue, adjacent noncancerous tissue and distal noncancerous tissue, respectively. The data demonstrated that noncancerous tissues from the adjacent and distal were nearly identical, but were greatly different from the carcinoma tissue. After the removal of missing values, totally 880 significantly changed ions between the carcinoma tissue and distal noncancerous tissue group were filtered out. 44 metabolites in the ESI positive mode and 65 in the negative mode were identified by databases and some of them were further confirmed by authentic standard samples. Several important metabolic pathways were clarified. The result proved that the established method was adequate for liver and other tissues metabolic profiling analysis.Hepatocellular carcinoma (HCC) is one of the most frequent malignancy worldwide. Evaluation of tissue metabolites is with significant value in HCC study which can provide more direct information of metabolic disorder compared with biofluids. A protocol for the metabolic profiling of liver tissue was developed based on ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS). According to the design of experiment (DOE), methanol/water (4:1, v:v) was selected as the optimal extraction solvent. The established method was validated with a linearity over the 10–5000 ng/mL for internal standards (IS) and got an average correlation coefficient of 0.9986. The intra-day and inter-day RSD for most endogenous compounds were below 15% and the recovery of IS was from 84.8% to 109.1%. After method validation, this method was applied to HCC study. Liver tissue samples were collected from HCC patients and each sample group involved carcinoma tissue, adjacent noncancerous tissue and distal noncancerous tissue, respectively. The data demonstrated that noncancerous tissues from the adjacent and distal were nearly identical, but were greatly different from the carcinoma tissue. After the removal of missing values, totally 880 significantly changed ions between the carcinoma tissue and distal noncancerous tissue group were filtered out. 44 metabolites in the ESI positive mode and 65 in the negative mode were identified by databases and some of them were further confirmed by authentic standard samples. Several important metabolic pathways were clarified. The result proved that the established method was adequate for liver and other tissues metabolic profiling analysis

一种基于HPLC-MS的代谢组学新分析方法

Novel Analytical Methods of HPLC-MS Based Metabolomic

农业资源高效利用与管理技术

该课题建立了区域农业资源利用效率评价指标体系与评价模型,首次编制了中国农业生态区划方案,建立了农业资源高效利用决策支持系统,在农业资源高效利用研究的理论与方法方面取得了重要进展。系统分析了中国的农业资源态势和发展前景,提出了以县为单元的商品粮基地建设构想,综合评估了中国不同时期、不同生态区的农业资源综合生产能力和人口承载能力,总结提出了适宜不同类型区的14种农业资源高效利用优化模式与技术体系集成方案和21项节水、节地、节肥、节粮、节本的高效利用农业资源的技术创新组合方案,具有一定的推广应用价值;研究提出的藏粮于土、提高农业资源综合生产能力的相关结论和建议,对国家和地区农业资源高效利用与区域可持..