The yeast metabolome addressed by electrospray ionization mass spectrometry: Initiation of a mass spectral library and its applications for metabolic footprinting by direct infusion mass spectrometry

Mass spectrometry (MS) has been a major driver for metabolomics, and gas chromatography (GC)-MS has been one of the primary techniques used for microbial metabolomics. The use of liquid chromatography (LC)-MS has however been limited, but electrospray ionization (ESI) is very well suited for ionization of microbial metabolites without any previous derivatization needed. To address the capabilities of ESI-MS in detecting the metabolome of Saccharomyces cerevisiae, the in silico metabolome of this organism was used as a template to present a theoretical metabolome. This showed that in combination with the specificity of MS up to 84% of the metabolites can be identified in a high mass accuracy ESI-spectrum. A total of 66 metabolites were systematically analyzed by positive and negative ESI-MS/MS with the aim of initiating a spectral library for ESI of microbial metabolites. This systematic analysis gave insight into the ionization and fragmentation characteristics of the different metabolites. With this insight, a small study of metabolic footprinting with ESI-MS demonstrated that biological information can be extracted from footprinting spectra. Statistical analysis of the footprinting data revealed discriminating ions, which could be assigned using the in silico metabolome. By this approach metabolic footprinting can advance from a classification method that is used to derive biological information based on guilt-by-association, to a tool for extraction of metabolic differences, which can guide new targeted biological experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Widely Targeted Metabolomics Based on Large-Scale MS/MS Data for Elucidating Metabolite Accumulation Patterns in Plants

Metabolomics is an ‘omics’ approach that aims toanalyze all metabolites in a biological sample comprehensively.The detailed metabolite profiling of thousands of plant sampleshas great potential for directly elucidating plant metabolicprocesses. However, both a comprehensive analysis and a highthroughput are difficult to achieve at the same time due tothe wide diversity of metabolites in plants. Here, we have establisheda novel and practical metabolomics methodology for quantifyinghundreds of targeted metabolites in a high-throughput manner.Multiple reaction monitoring (MRM) using tandem quadrupole massspectrometry (TQMS), which monitors both the specific precursorions and product ions of each metabolite, is a standard techniquein targeted metabolomics, as it enables high sensitivity, reproducibilityand a broad dynamic range. In this study, we optimized the MRMconditions for specific compounds by performing automated flowinjection analyses with TQMS. Based on a total of 61,920 spectrafor 860 authentic compounds, the MRM conditions of 497 compoundswere successfully optimized. These were applied to high-throughputautomated analysis of biological samples using TQMS coupledwith ultra performance liquid chromatography (UPLC). By thisanalysis, approximately 100 metabolites were quantified in eachof 14 plant accessions from Brassicaceae, Gramineae and Fabaceae.A hierarchical cluster analysis based on the metabolite accumulationpatterns clearly showed differences among the plant families,and family-specific metabolites could be predicted using a batch-learningself-organizing map analysis. Thus, the automated widely targetedmetabolomics approach established here should pave the way forlarge-scale metabolite profiling and comparative metabolomics.     

Liquid chromatography-tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat

A rapid, sensitive and specific liquid chromatography-electrospray ionization (ESI) tandem mass spectrometry (LC-MS-MS) method has been developed for the identification of nitazoxanide metabolites in goat plasma and urine. The purified samples was separated using an XTerra MS C8 column with the mobile phase consisted of acetonitrile and 10-mM ammonium acetate buffer (pH 2.5) followed a linear gradient elution, and detected by MS-MS. Identification and structural elucidation of the metabolites were performed by comparing their retention-times, full scan, product ion scan, precursor ion scan and neutral loss scan MS-MS spectra with those of the parent drug or other available standard. Four metabolites (tizoxanide, tizoxanide glucuronide, tizoxanide sulfate and hydroxylated tizoxanide sulfate) were found and identified in goat after single oral administration of 200mg/kg dose of nitazoxanide. In addition, the possible metabolic pathway was proposed for the first time. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active metabolites responsible for pharmacological effects of nitazoxanide and to better understand its in vivo metabolism.     

Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.     

Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-(13)C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.     

Metabolomic profiling from formalin-fixed, paraffin-embedded tumor tissue using targeted LC/MS/MS: application in sarcoma

The relatively new field of onco-metabolomics attempts to identify relationships between various cancer phenotypes and global metabolite content. Previous metabolomics studies utilized either nuclear magnetic resonance spectroscopy or gas chromatography/mass spectrometry, and analyzed metabolites present in urine and serum. However, direct metabolomic assessment of tumor tissues is important for determining altered metabolism in cancers. In this respect, the ability to obtain reliable data from archival specimens is desirable and has not been reported to date. In this feasibility study, we demonstrate the analysis of polar metabolites extracted directly from ten formalin-fixed, paraffin-embedded (FFPE) specimens, including five soft tissue sarcomas and five paired normal samples. Using targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) via selected reaction monitoring (SRM), we detect an average of 106 metabolites across the samples with excellent reproducibility and correlation between different sections of the same specimen. Unsupervised hierarchical clustering and principal components analysis reliably recovers a priori known tumor and normal tissue phenotypes, and supervised analysis identifies candidate metabolic markers supported by the literature. In addition, we find that diverse biochemical processes are well-represented in the list of detected metabolites. Our study supports the notion that reliable and broadly informative metabolomic data may be acquired from FFPE soft tissue sarcoma specimens, a finding that is likely to be extended to other malignancies.     

Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS

Metabolic changes in response to histidine starvation were observed in histidine-auxotrophic Escherichia coli using a capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics technique. Prior to the analysis, we prepared an E. coli metabolome list of 727 metabolites reported in the literature. An improved method for metabolite extraction was developed, which resulted in higher extraction efficiency in phosphate-rich metabolites, e.g., ATP and GTP. Based on the results, 375 charged, hydrophilic intermediates in primary metabolisms were analysed simultaneously, providing quantitative data of 198 metabolites. We confirmed that the intracellular levels of intermediates in histidine biosynthesis are rapidly accumulated in response to a drop in histidine level under histidine-starved conditions. Simultaneously, disciplined responses were observed in the glycolysis, tricarboxylic acid cycle, and amino acid and nucleotide biosynthesis pathways as regulated by amino acid starvation.     

Patterns of time since last meal revealed by sparse PCA in an observational LC–MS based metabolomics study

In metabolomics studies, liquid chromatography mass spectrometry (LC–MS) provides comprehensive information on biological samples. However, extraction of few relevant metabolites from this large and complex data is cumbersome. To resolve this issue, we have employed sparse principal component analysis (SPCA) to capture the underlying patterns and select relevant metabolites from LC–MS plasma profiles. The study involves a small pilot cohort with 270 subjects where each subject’s time since last meal (TSLM) has been recorded prior to plasma sampling. Our results have demonstrated that both PCA and SPCA can capture the TSLM patterns. Nevertheless, SPCA provides more easily interpretable loadings in terms of selection of relevant metabolites, which are identified as amino acids and lyso-lipids. This study demonstrates the utility of SPCA as a pattern recognition and variable selection tool in metabolomics. Furthermore, amino acids and lyso-lipids are determined as dominating compounds in response to TSLM.     

Differences between the metabolic profiles of decompensated and compensated cirrhosis patients with Hepatitis B virus infections under high-performance liquid chromatography-mass spectrometry

To improve the grading and staging of liver cirrhosis among patients with HBV infection noninvasively, a high-performance liquid chromatography with mass spectrometry metabolomics method was used to investigate the potential metabolic biomarkers in the serum of patients with different degrees of hepatic cirrhosis. The results demonstrate that lysophosphatidyl choline (LPC) from positive electrospray ionization (ESI) mode, and fatty acids and bile acids from negative ESI mode play important roles in distinguishing decompensated from compensated cirrhosis. A total of 21 differential metabolites were found from the two groups of patients. LPCs, fatty acids, and taurocholic acid (TCA) 3-sulfate decreased in patients with decompensated cirrhosis, whereas other bile acids increased significantly. The levels of TCA 3-sulfate, LPC 16:0, and LPC 18:0 were significantly correlated with the stages of the decompensated cirrhosis, and they may serve as potential biomarkers for the stage assessment of liver cirrhosis in patients with HBV infections.     

Granger causality in integrated GC–MS and LC–MS metabolomics data reveals the interface of primary and secondary metabolism

Metabolomics has emerged as a key technique of modern life sciences in recent years. Two major techniques for metabolomics in the last 10 years are gas chromatography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). Each platform has a specific performance detecting subsets of metabolites. GC–MS in combination with derivatisation has a preference for small polar metabolites covering primary metabolism. In contrast, reversed phase LC–MS covers large hydrophobic metabolites predominant in secondary metabolism. Here, we present an integrative metabolomics platform providing a mean to reveal the interaction of primary and secondary metabolism in plants and other organisms. The strategy combines GC–MS and LC–MS analysis of the same sample, a novel alignment tool MetMAX and a statistical toolbox COVAIN for data integration and linkage of Granger Causality with metabolic modelling. For metabolic modelling we have implemented the combined GC–LC–MS metabolomics data covariance matrix and a stoichiometric matrix of the underlying biochemical reaction network. The changes in biochemical regulation are expressed as differential Jacobian matrices. Applying the Granger causality, a subset of secondary metabolites was detected with significant correlations to primary metabolites such as sugars and amino acids. These metabolic subsets were compiled into a stoichiometric matrix N. Using N the inverse calculation of a differential Jacobian J from metabolomics data was possible. Key points of regulation at the interface of primary and secondary metabolism were identified.     

A Strategy for Sensitive,Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.  Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.     

Pharmacokinetics of ergosterol in rats using rapid resolution liquid chromatography-atmospheric pressure chemical ionization multi-stage tandem mass spectrometry and rapid resolution liquid chromatography/tandem mass spectrometry

Rapid resolution liquid chromatography/tandem multi-stage mass spectrometry (RRLC-MS(n)) and rapid resolution liquid chromatography/tandem mass spectrometry (RRLC/MS/MS) methods were developed for the identification and quantification of ergosterol and its metabolites from rat plasma, urine and faeces. Two metabolites (ERG1 and ERG2) were identified by RRLC/MS(n). The concentrations of the ergosterol were determined by RRLC/MS/MS. The separation was performed on an Agilent Zorbax SB-C18 with the mobile phase consisting of methanol and water (containing 0.1% formic acid). The detection was carried out by means of atmospheric pressure chemical ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 7-2000, 6-2000 and 8-7500 ng/mL for plasma, urine and faecal homogenate, respectively. The intra- and inter-day precision values (RSD) were below 10%. The method was applied to the pharmacokinetic properties and elimination pathway of ergosterol in rats.     

Drug analysis by direct liquid introduction micro liquid chromatography mass spectrometry

The analytical capabilities of a micro high performance liquid chromatograph interfaced to an unchanged quadrupole mass spectrometer are presented. Continuous monitoring of the total micro liquid chromatographic effluent allows full scan chemical ionization mass spectra of from one to five nanograms of drugs and their metabolites to be recorded. The interface is a simple, inexpensive device which can be assembled from commercially available components. An eight microliter per minute flow rate of the micro liquid chromatographic eluant allows separation and identification of biologically important substances not amenable to gas chromatography mass spectrometry techniques. The sensitivity of micro liquid chromatography mass spectrometry performed as described is comparable with gas chromatography mass spectrometry and is achieved by introducing the total micro liquid chromatographic effluent into the chemical ionization ion source of the mass spectrometer. Selected ion monitoring provides 20 pg detection limits of phenothiazine tranquilizers injected on column.     

Metabolic fingerprinting of rat urine by LC/MS Part 1. Analysis by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry

Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part 1 of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C(18) column and a ZIC-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography.     

Key taste components in two wild edible Boletus mushrooms using widely targeted metabolomics

Compounds from wild edible mushrooms has been reported to exert biological activities and contribute to the different flavors of mushrooms in our diet. Wild edible Boletus mushrooms are popular in Southwest China. In this study, we performed ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) based on widely targeted metabolomics analysis to identify key components. A total of 194 metabolites (113 upregulated, 81 downregulated) divided into 11 groups (49 metabolites in group lipids, 34 in amino acids and derivatives, 30 in organic acids, 18 in phenolic acids, 16 in nucleotides and derivatives, 13 in alkaloids, 6 in flavonoids, 3 in lignans and coumarins, 3 in tannins, 2 in terpenoids, and 20 in others) were found among Boletus bainiugan compared with B. subsplendidus. Through clustering analysis, principal component analysis (PCA), and orthogonal signal correction and partial least squares-discriminant analysis (OPLS-DA), different metabolites from fruiting bodies were clearly identified. Significant differences were observed in the metabolites between Boletus bainiugan and B. subsplendidus. These metabolites are involved in important biological functions. Our results provide new insights into important metabolites and theoretical basis for the taste difference of two wild edible Boletus mushrooms.     

Hyphenated liquid chromatographic techniques in forensic toxicology

The prerequisite of applicability of hyphenated methods in forensic analysis is the achievement of a stage of “final maturity”. In the field of liquid chromatography, HPLC coupled with diode array detection (DAD) seems to fulfill this criterion, whilst the combination with atmospheric pressure ionization mass spectrometry (HPLC–API-MS) is still in a development stage. HPLC–DAD is broadly used as identification tool in forensic and in emergency toxicology. Two main approaches were observed; development of retention index scales for intra-laboratory exchange of data and establishing of databases only for intra-laboratory use. Using these approaches, several databases were established for toxicological relevant substances (illicit and therapeutic drugs and their metabolites, environmental poisons etc.) in biological fluids. Also, complete HPLC–DAD identification systems are commercially available. Further possibility of progress depends on the on-line combination (“triple hyphenation”) with other detection methods, preferably API-MS. HPLC–API-MS, both in electrospray (ESI) and atmospheric pressure chemical ionization (APCI) options, underwent dramatic development in the last decade and is reaching its final shape. The method was broadly applied for various groups of toxicologically relevant substances, a lot of them unaccessible for other techniques, including GC–MS. Particularly important was application of HPLC–API-MS for detection and quantitation of active, polar metabolites of various drugs and for analysis of macromolecules. APCI seems to be more useful for analysis of less polar compounds, whereas ESI is particularly valuable for determination of polar, large molecules (e.g., toxic peptides, polar metabolites etc.) Up to now, HPLC–API-MS has been mainly applied for dedicated analyses, but the introduction of APCI or ESI in systematic toxicological screening may be expected in the near future.     

Simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application in pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the positive/negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma. After addition of internal standards diazepam (for asperosaponin VI) and glycyrrhetic acid (for hederagenin), the plasma sample was deproteinized with acetonitrile, and separated on a reversed phase C18 column with a mobile phase of methanol (solvent A)-0.05% glacial acetic acid containing 10 mM ammonium acetate and 30 μM sodium acetate (solvent B) using gradient elution. The detection of target compounds was done in multiple reaction monitoring (MRM) mode using a tandem mass spectrometry equipped with positive/negative ion-switching ESI source. At the first segment, the MRM detection was operated in the positive ESI mode using the transitions of m/z 951.5 ([M+Na](+))→347.1 for asperosaponin VI and m/z 285.1 ([M+H](+))→193.1 for diazepam for 4 min, then switched to the negative ESI mode using the transitions of m/z 471.3 ([M-H](-))→471.3 for hederagenin and m/z 469.4 ([M-H](-))→425.4 for glycyrrhetic acid, respectively. The sodiated molecular ion [M+Na](+) at m/z 951.5 was selected as the precursor ion for asperosaponin VI, since it provided better sensitivity compared to the deprotonated and protonated molecular ions. Sodium acetate was added to the mobile phase to make sure that abundant amount of the sodiated molecular ion of asperosaponin VI could be produced, and more stable and intensive mass response of the product ion could be obtained. For the detection of hederagenin, since all of the mass responses of the fragment ions were very weak, the deprotonated molecular ion [M-H](-)m/z 471.3 was employed as both the precursor ion and the product ion. But the collision energy was still used for the MRM, in order to eliminate the influences induced by the interference substances from the rat plasma. The validated method was successfully applied to study the pharmacokinetics of asperosaponin VI and its active metabolite hederagenin in rat plasma after oral administration of asperosaponin VI at a dose of 90 mg/kg.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.