Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.     

Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts

Metabolic profiling, metabolomic and metabonomic studies mainly involve the multicomponent analysis of biological fluids, tissue and cell extracts using NMR spectroscopy and/or mass spectrometry (MS). We summarize the main NMR spectroscopic applications in modern metabolic research, and provide detailed protocols for biofluid (urine, serum/plasma) and tissue sample collection and preparation, including the extraction of polar and lipophilic metabolites from tissues. 1H NMR spectroscopic techniques such as standard 1D spectroscopy, relaxation-edited, diffusion-edited and 2D J-resolved pulse sequences are widely used at the analysis stage to monitor different groups of metabolites and are described here. They are often followed by more detailed statistical analysis or additional 2D NMR analysis for biomarker discovery. The standard acquisition time per sample is 4-5 min for a simple 1D spectrum, and both preparation and analysis can be automated to allow application to high-throughput screening for clinical diagnostic and toxicological studies, as well as molecular phenotyping and functional genomics.     

One-step Metabolomics: Carbohydrates,Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.     

Identification and comparative oridonin metabolism in different species liver microsomes by using UPLC-Triple-TOF-MS/MS and PCA

Oridonin (ORI) is an active natural ent-kaurene diterpenoid ingredient with notable anti-cancer and anti-inflammation activities. Currently, a strategy was developed to identify metabolites and to assess the metabolic profiles of ORI in vitro using ultra-high-performance liquid chromatography-Triple/time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). Meanwhile, the metabolism differences of ORI in the liver microsomes of four different species were investigated using a principal component analysis (PCA) based on the metabolite absolute peak area values as the variables. Based on the proposed methods, 27 metabolites were structurally characterized. The results indicate that ORI is universally metabolized in vitro, and the metabolic pathway mainly includes dehydration, hydroxylation, di-hydroxylation, hydrogenation, decarboxylation, and ketone formation. Overall, there are obvious inter-species differences in types and amounts of ORI metabolites in the four species. These results will provide basic data for future pharmacological and toxicological studies of ORI and for other ent-kauranes diterpenoids. Meanwhile, studying the ORI metabolic differences helps to select the proper animal model for further pharmacology and toxicological assessment.     

Determination of oxcarbazepine and its metabolites in postmortem blood and hair by means of liquid chromatography with mass detection (HPLC/APCI/MS)

A typical use of hair analysis in forensic toxicology is the documentation of previous drug administration. This is illustrated in a suicidal death of a 58-year-old epileptic patient who was treated with oxcarbazepine and probably with levomepromazine. The toxicological analysis carried out by HPLC/APCI/MS included also the hair (6 cm length) besides postmortem blood. The method was validated for levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10-hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) in various biological matrices. The analysis of the postmortem blood indicated oxcarbazepine and its two main metabolites were present at therapeutic concentrations; levomepromazine was detected at a fatal concentration. In three 2-cm segments of hair, oxcarbazepine and its two metabolites were detected; however, levomepromazine was not detected in this specimen. As a result of complex chemical-toxicological investigation it was confirmed the information that the decedent. was an epileptic patient and was treated with oxcarbazepine for at least 6 months before death. In addition, he took a toxic dose of levomepromazine in order to commit suicide. The analysis revealed differences between the concentration levels of oxcarbazepine and its active metabolite CBZ-10OH in postmortem specimens and hair, suggesting different mechanisms of penetration of metabolites and their precursors into this matrix.     

A method for handling metabonomics data from liquid chromatography/mass spectrometry: combinational use of support vector machine recursive feature elimination, genetic algorithm and random forest for feature selection

Metabolic markers are the core of metabonomic surveys. Hence selection of differential metabolites is of great importance for either biological or clinical purpose. Here, a feature selection method was developed for complex metabonomic data set. As an effective tool for metabonomics data analysis, support vector machine (SVM) was employed as the basic classifier. To find out meaningful features effectively, support vector machine recursive feature elimination (SVM-RFE) was firstly applied. Then, genetic algorithm (GA) and random forest (RF) which consider the interaction among the metabolites and independent performance of each metabolite in all samples, respectively, were used to obtain more informative metabolic difference and avoid the risk of false positive. A data set from plasma metabonomics study of rat liver diseases developed from hepatitis, cirrhosis to hepatocellular carcinoma was applied for the validation of the method. Besides the good classification results for 3 kinds of liver diseases, 31 important metabolites including lysophosphatidylethanolamine (LPE) C16:0, palmitoylcarnitine, lysophosphatidylethanolamine (LPC) C18:0 were also selected for further studies. A better complementary effect of the three feature selection methods could be seen from the current results. The combinational method also represented more differential metabolites and provided more metabolic information for a “global” understanding of diseases than any single method. Further more, this method is also suitable for other complex biological data sets.     

Quality control and quality assessment of data from surface-enhanced laser desorption/ionization (SELDI) time-of flight (TOF) mass spectrometry (MS)

Background

Proteomic profiling of complex biological mixtures by the ProteinChip technology of surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) is one of the most promising approaches in toxicological, biological, and clinic research. The reliable identification of protein expression patterns and associated protein biomarkers that differentiate disease from health or that distinguish different stages of a disease depends on developing methods for assessing the quality of SELDI-TOF mass spectra. The use of SELDI data for biomarker identification requires application of rigorous procedures to detect and discard low quality spectra prior to data analysis.

Results

The systematic variability from plates, chips, and spot positions in SELDI experiments was evaluated using biological and technical replicates. Systematic biases on plates, chips, and spots were not found. The reproducibility of SELDI experiments was demonstrated by examining the resulting low coefficient of variances of five peaks presented in all 144 spectra from quality control samples that were loaded randomly on different spots in the chips of six bioprocessor plates. We developed a method to detect and discard low quality spectra prior to proteomic profiling data analysis, which uses a correlation matrix to measure the similarities among SELDI mass spectra obtained from similar biological samples. Application of the correlation matrix to our SELDI data for liver cancer and liver toxicity study and myeloma-associated lytic bone disease study confirmed this approach as an efficient and reliable method for detecting low quality spectra.

Conclusion

This report provides evidence that systematic variability between plates, chips, and spots on which the samples were assayed using SELDI based proteomic procedures did not exist. The reproducibility of experiments in our studies was demonstrated to be acceptable and the profiling data for subsequent data analysis are reliable. Correlation matrix was developed as a quality control tool to detect and discard low quality spectra prior to data analysis. It proved to be a reliable method to measure the similarities among SELDI mass spectra and can be used for quality control to decrease noise in proteomic profiling data prior to data analysis.

     

Mass spectrometry methods in metabolomics

The review deals with metabolomics, a new and rapidly growing area directed to the comprehensive analysis of metabolites of biological objects. Metabolites are characterized by various physical and chemical properties, traditionally studied by methods of analytical chemistry focused on certain groups of chemical substances. However, current progress in mass spectrometry has led to formation of rather unified methods, such as metabolic fingerprinting and metabolomic profiling, which allow defining thousands of metabolites in one biological sample and therefore draw “a modern portrait of metabolomics.” This review describes basic characteristics of these methods, ways of metabolite separation, and analysis of metabolites by mass spectrometry. The examples shown in this review, allow to estimate these methods and to compare their advantages and disadvantages. Besides that, we consider the methods, which are of the most frequent use in metabolomics; these include the methods for data processing and the required resources, such as software for mass spectra processing and metabolite search database. In the conclusion, general suggestions for successful metabolomic experiments are given.     

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.     

Toxicity testing of destruxins and crude extracts from the insect-pathogenic fungus Metarhizium anisopliae

Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae. Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxins A, B and E) or the complete crude extract from liquid cultures. Furthermore, crude extract was separated into fractions, which did not contain the main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic or adverse effects are present in the crude extract. The results indicate that identification and toxicological assessment of each individual metabolite produced by a BCA is not only time and cost-intensive, but also does not convey the whole picture. Testing of the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.     

Quantitative analysis of procarbazine, procarbazine metabolites and chemical degradation products with application to pharmacokinetic studies

Quantitative analytical methods are described for the analysis of the anticancer drug procarbazine and eight known metabolites including those known to have cytotoxic activity. A direct sample insertion mass spectrometric assay for procarbazine and the urinary excretion product, N-isopropyl-terephthalamic acid, has been developed. This method employs stable isotope labeled variants in a procedure that minimizes analytical errors that may be encountered in the quantitation of the chemically unstable parent drug. A liquid chromatographic method is described for the analysis of seven known procarbazine metabolites. Use of these methods is demonstrated by the analysis of procarbazine metabolism during incubation in a 9000-g rat liver homogenate preparation. Procarbazine disappearance and metabolite appearance are also monitored in rat plasma following intraperitoneal administration of a 150 mg/kg bolus dose. Applications to patient pharmacokinetics is demonstrated using the liquid chromatographic assay to follow the appearance of active procarbazine metabolites on the first and fourteenth day of an oral 250 mg/kg/day course of therapy of a patient being treated for cancer.     

Efficient analysis of hepatic glucose output and insulin action using a liver slice culture system.

BACKGROUND: Liver slices have been reported to retain histological integrity and metabolic capacity for over 24 hours in flask culture systems, and they have been used for pharmacological and toxicological studies before. However, whether this method is suitable to measure hepatic glucose output is unknown. METHODS: Precision-cut liver slices were prepared from fresh male rat liver. After high-glucose pre-incubation (11.2 mmol/l), medium was changed to low-glucose conditions (0.5 mmol/l). Glucose and lactate levels as well as aspartate aminotransferase activity were monitored for 50 minutes with or without addition of insulin (600 pmol/l) and/or epinephrine (0.5 micromol/l). Slice potassium content and histology were examined to prove liver viability. RESULTS: We observed a stable glucose production from the liver slices of 0.3-0.4 micromol/g liver/min. Epinephrine increased (by 82+/-30%) and insulin decreased (by 80+/-8%) liver slice glucose output. Significant signs of ischemia were not detected. CONCLUSIONS: Hepatic glucose release can be reliably measured in a liver slice culture system, and it is regulated by major hormone systems. This method may be helpful for further characterization of direct insulin action and resistance in a complex tissue as the liver; however, pharmacological applications such as the analysis of drug effects on hepatic glucose metabolism can also be envisioned.     

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.     

液相串联质谱法同时定量检测生物样本中鞘脂类化合物

鞘脂类中的主要活性分子鞘氨醇1-磷酸（S1P）,可通过介导细胞增殖、分化和迁移等发挥广泛的生物学功能|同时,S1P可在相关酶的作用下转变为其它形式.本文旨在建立一种简便的鞘脂类的制备方法,并结合液相串联质谱（LC-MS/MS）快速检测生物样本中纳克水平的鞘脂类化合物. 采用甲醇沉淀或经典的脂质提取方法获得鞘脂类化合物,再采用LC-MS/MS在多反应监测模式（MRM）下进行定量分析. 实验结果表明,甲醇沉淀法可简便快捷地获得血浆或脂蛋白中鞘氨醇类化合物; S1P、二氢鞘氨醇（DH-S1P）和鞘氨醇（SPH）的定量限分别为110.5、215.6和44.3 pg;人血浆中S1P、DH-S1P和SPH的含量分别为257.8±49.4 nmol/L、93.5±17.3 nmol/L和44.6± 7.4 nmol/L, 鞘脂类化合物在人血浆脂蛋白上的分布存在显著性差异|在ApoE-/-小鼠血浆中S1P、DH-S1P和SPH的含量分别为590.1±78.2 nmol/L、197.8±60.6 nmol/L和35.4±16.7 nmol/L|每1×106个人脐静脉内皮细胞（EA.hy926）中,S1P和SPH的含量分别为103.7±21.8 pg和16.3±5.3 ng.甲醇沉淀法结合LC-MS/MS可简便快捷地对血浆和脂蛋白中的鞘脂类化合物进行定量检测. 该方法特别适用于大量生物样本中鞘脂类的快速定量分析.     

微流控器件-质谱联用接口技术研究进展与应用

生物分析是生命科学研究中的重要环节,分析仪器的小型化是提高生物分析灵敏度、速度、通量和降低成本的有效途径之一.微流控技术能够方便地操纵微量样品,具有集成度高、样品耗量小、污染少等诸多其他常量流控技术难以具备的优点,适用于进行多通道样品处理和高通量分析.除广泛采用的光学和电化学检测手段外,质谱也被用作这些微流控器件的检测器,并逐渐形成了微流控器件-质谱联用技术专门研究领域,进一步促进了自动化程度好、灵敏度高、特异性强的高通量生物分析方法的迅速发展.在大量调研国内外文献的基础上,对微流控器件-质谱联用领域的研究背景和现状进行了综述,不但介绍了微流控器件的制造技术还着重介绍了微流控器件-质谱联用技术在蛋白质组学等生物质谱分析方面的应用和新近进展,评述了可能的发展趋势.     

Simultaneous quantification of major phytohormones and related compounds in crude plant extracts by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive method was developed for simultaneous quantification of multiple classes of phytohormones and some related metabolites in crude plant extracts without purification or derivatization. High-performance liquid chromatography and electrospray ionization–tandem mass spectrometry with multiple reaction monitoring were used to quantify auxins, cytokinins, abscisic acid, gibberellins, jasmonates, salicylates, and a number of related metabolites in crude plant extracts. The technology was applied to analyze biotic and abiotic stress-induced changes of phytohormones in Arabidopsis tissues, starting with 50–100 mg fresh tissue. Biotic and/or abiotic stresses were shown to differentially affect levels of salicylic acid, jasmonic acid, indole-3-acetic acid, and benzoic acid, in comparison to their methyl esters. Compared with previous methods, sample preparation time and amount of sample required for analysis of phytohormones are reduced, and more classes of hormones are quantitatively profiled. Structurally diverse compounds from complicated biological matrices are determined with high selectivity and sensitivity.