Evaluating the challenges associated with time-of-fight secondary ion mass spectrometry for metabolomics using pure and mixed metabolites

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is potentially well placed to contribute to metabolomic analysis while bringing the added benefit of high resolution, label free imaging. The focused ion beams used to desorb species from the sample can be focused below 1 μm allowing chemical imaging on a sub-cellular scale. In this study we test the capability of ToF-SIMS to generate mass spectrometry and MSMS spectra from a set of standard metabolites that can be compared with open access metabolite databases containing ESI-CID MSMS spectra. The influence of the chemical environment, the matrix effect, on the observed mass spectra is assessed using a mixed metabolite sample and the data discussed in terms of compound identification and quantification. Radical ions and small fragment ions seem to be less sensitive to ion suppression or enhancement and may provide a route to quantification. Understanding such parameters will be key for the successful application of the technique for in situ metabolomics with ToF-SIMS.     

Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.     

Microbial transformation of primaquine by Candida tropicalis.

The microbial metabolism of primaquine, a 6-methoxy-8-aminoquinoline antimalarial agent, was investigated. The yeast Candida tropicalis was found to convert primaquine to the previously reported N-acetylated derivative. On continued incubation of C. tropicalis in the presence of the N-acetylated derivative, a minor dimeric metabolite was formed. The proposed structure of the metabolite was based primarily on the analysis of its spectroscopic properties (1H and 13C nuclear magnetic resonance spectra and field-desorption mass spectrum). The structure of the metabolite was proven by direct comparison with an authentic sample of the minor dimeric metabolite prepared by treatment of the N-acetylated derivative with formaldehyde in the presence of formic acid in methanol.     

Microbial transformation of primaquine by Candida tropicalis

The microbial metabolism of primaquine, a 6-methoxy-8-aminoquinoline antimalarial agent, was investigated. The yeast Candida tropicalis was found to convert primaquine to the previously reported N-acetylated derivative. On continued incubation of C. tropicalis in the presence of the N-acetylated derivative, a minor dimeric metabolite was formed. The proposed structure of the metabolite was based primarily on the analysis of its spectroscopic properties (1H and 13C nuclear magnetic resonance spectra and field-desorption mass spectrum). The structure of the metabolite was proven by direct comparison with an authentic sample of the minor dimeric metabolite prepared by treatment of the N-acetylated derivative with formaldehyde in the presence of formic acid in methanol.     

Production of a novel dimeric metabolite of primaquine by Streptomyces rimosus.

Primaquine, an 8-amino-6-methoxyquinoline antimalarial agent, was subjected to metabolic studies with microorganisms. Streptomyces rimosus converted primaquine to the previously reported N-acetyl derivative. Continued incubation of S. rimosus resulted in the formation of a minor dimeric metabolite. The structure of the minor dimeric metabolite was proposed based primarily on its spectral data (1H and 13C nuclear magnetic resonance spectra and mass spectrum). The proposed structure of the metabolite was confirmed by synthesis of the dimer by treatment of primaquine-N-acetate with potassium ferricyanide in a biphasic chloroform-aqueous sodium bicarbonate system with a phase-transfer catalyst. Since (+/-)-primaquine was used for both the microbial transformation and synthesis, a diastereomeric mixture of symmetrical dimers was formed in each case. The metabolite sample was identical to the synthetic sample, as shown by direct comparison (thin-layer chromatography, co-thin-layer chromatography, high-pressure liquid chromatography, co-high-pressure liquid chromatography, 1H nuclear magnetic resonance spectra, and mass spectrum).     

Production of a novel dimeric metabolite of primaquine by Streptomyces rimosus

Primaquine, an 8-amino-6-methoxyquinoline antimalarial agent, was subjected to metabolic studies with microorganisms. Streptomyces rimosus converted primaquine to the previously reported N-acetyl derivative. Continued incubation of S. rimosus resulted in the formation of a minor dimeric metabolite. The structure of the minor dimeric metabolite was proposed based primarily on its spectral data (1H and 13C nuclear magnetic resonance spectra and mass spectrum). The proposed structure of the metabolite was confirmed by synthesis of the dimer by treatment of primaquine-N-acetate with potassium ferricyanide in a biphasic chloroform-aqueous sodium bicarbonate system with a phase-transfer catalyst. Since (+/-)-primaquine was used for both the microbial transformation and synthesis, a diastereomeric mixture of symmetrical dimers was formed in each case. The metabolite sample was identical to the synthetic sample, as shown by direct comparison (thin-layer chromatography, co-thin-layer chromatography, high-pressure liquid chromatography, co-high-pressure liquid chromatography, 1H nuclear magnetic resonance spectra, and mass spectrum).     

MALDI-imaging mass spectrometry - An emerging technique in plant biology

Recent advances in instrumentation and sample preparation have facilitated the mass spectrometric (MS) imaging of a large variety of biological molecules from small metabolites to large proteins. The technique can be applied at both the tissue and the single-cell level, and provides information regarding the spatial distribution of specific molecules. Nevertheless, the use of MS imaging in plant science remains far from routine, and there is still a need to adapt protocols to suit specific tissues. We present an overview of MALDI-imaging MS (MSI) technology and its use for the analysis of plant tissue. Recent methodological developments have been summarized, and the major challenges involved in using MALDI-MSI, including sample preparation, the analysis of metabolites and peptides, and strategies for data evaluation are all discussed. Some attention is given to the identification of differentially distributed compounds. To date, the use of MALDI-MSI in plant research has been limited. Examples include leaf surface metabolite maps, the characterization of soluble metabolite translocation in planta, and the profiling of protein/metabolite patterns in cereal grain cross-sections. Improvements to both sample preparation strategies and analytical platforms (aimed at both spectrum acquisition and post-acquisition analysis) will enhance the relevance of MALDI-MSI technology in plant research.     

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.     

Metabolomic Method: UPLC-q-ToF Polar and Non-Polar Metabolites in the Healthy Rat Cerebellum Using an In-Vial Dual Extraction

Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.     

Meta-analysis of untargeted metabolomic data from multiple profiling experiments

metaXCMS is a software program for the analysis of liquid chromatography/mass spectrometry-based untargeted metabolomic data. It is designed to identify the differences between metabolic profiles across multiple sample groups (e.g., 'healthy' versus 'active disease' versus 'inactive disease'). Although performing pairwise comparisons alone can provide physiologically relevant data, these experiments often result in hundreds of differences, and comparison with additional biologically meaningful sample groups can allow for substantial data reduction. By performing second-order (meta-) analysis, metaXCMS facilitates the prioritization of interesting metabolite features from large untargeted metabolomic data sets before the rate-limiting step of structural identification. Here we provide a detailed step-by-step protocol for going from raw mass spectrometry data to metaXCMS results, visualized as Venn diagrams and exported Microsoft Excel spreadsheets. There is no upper limit to the number of sample groups or individual samples that can be compared with the software, and data from most commercial mass spectrometers are supported. The speed of the analysis depends on computational resources and data volume, but will generally be less than 1 d for most users. metaXCMS is freely available at http://metlin.scripps.edu/metaxcms/.     

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.     

The application of rapid evaporative ionization mass spectrometry in the analysis of Drosophila species—a potential new tool in entomology

There is increasing emphasis on the use of new analytical approaches in subject analysis and classification, particularly in respect to minimal sample preparation. Here, we demonstrate that rapid evaporative ionization mass spectrometry (REIMS), a method that captures metabolite mass spectra after rapid combustive degradation of an intact biological specimen, generates informative mass spectra from several arthropods, and more specifically, is capable of discerning differences between species and sex of several adult Drosophila species. A model including five Drosophila species, built using pattern recognition, achieves high correct classification rates (over 90%) using test datasets and is able to resolve closely related species. The ease of discrimination of male and female specimens also demonstrates that sex-specific differences reside in the REIMS metabolite patterns, whether analysed across all five species or specifically for D. melanogaster. Further, the same approach can correctly discriminate and assign Drosophila species at the larval stage, where these are morphologically highly similar or identical. REIMS offers a novel approach to insect typing and analysis, requiring a few seconds of data acquisition per sample and has considerable potential as a new tool for the field biologist.     

Validation of a field‐ready handheld meter for plasma β‐hydroxybutyrate analysis

Plasma metabolite analysis is frequently used to assess the energetic state and energy intake of birds. Plasma β‐hydroxybutyrate (BUTY) is a common metabolite used in these studies, and is correlated with fasting and mass loss. BUTY is typically quantified in laboratory assays that are costly, time‐consuming, and prone to human error. We tested the accuracy and precision of a field‐ready handheld BUTY meter. We compared BUTY concentration values obtained in the laboratory and with the handheld meter in plasma samples from 19 Grasshopper Sparrows (Ammodramus savannarum), and assessed precision with repeat analysis of a single sample. The handheld meter reported BUTY concentrations in < 2 min, was highly precise, and as accurate as the laboratory assay method—all ideal for field conditions. Collecting blood samples for laboratory analysis, particularly from remote field sites, involves a series of risks and challenges for permitting or the logistics of storage and shipping, as well as the associated costs. Analyzing samples on site, whether with the unit we tested or other similar handheld meters, makes plasma metabolite analysis practical and possible in field conditions and for taxa where this technique has been underused due to permitting, transport, or other logistical constraints of laboratory analysis methods. The cost of analysis is similar on a per sample basis, but, without the need to store and transport samples, using handheld meters in the field may be cheaper.     

Imaging of metabolites using secondary ion mass spectrometry

This article provides an overview of the technique of secondary ion mass spectrometry imaging and highlights some current and future areas of application relevant to the field of metabolomics. The approach benefits from label-free analysis of molecular species up to ~1500 Da with minimal sample preparation. Offering the highest spatial resolution of current mass spectrometry imaging methodologies, the technique is well-suited to metabolite imaging in both biological tissue and cells, in both 2D and 3D.     

An approach towards method development for untargeted urinary metabolite profiling in metabonomic research using UPLC/QToF MS

The application of LC-MS for untargeted urinary metabolite profiling in metabonomic research has gained much interest in recent years. However, the effects of varying sample pre-treatments and LC conditions on generic metabolite profiling have not been studied. We aimed to evaluate the effects of varying experimental conditions on data acquisition in untargeted urinary metabolite profiling using UPLC/QToF MS. In-house QC sample clustering was used to monitor the performance of the analytical platform. In terms of sample pre-treatment, results showed that untreated filtered urine yielded the highest number of features but dilution with methanol provided a more homogenous urinary metabolic profile with less variation in number of features and feature intensities. An increased cycle time with a lower flow rate (400mul/min vs 600mul/min) also resulted in a higher number of features with less variability. The step elution gradient yielded the highest number of features and the best chromatographic resolution among three different elution gradients tested. The maximum retention time and mass shift were only 0.03min and 0.0015Da respectively over 600 injections. The analytical platform also showed excellent robustness as evident by tight QC sample clustering. To conclude, we have investigated LC conditions by studying variability and repeatability of LC-MS data for untargeted urinary metabolite profiling.     

Imaging of metabolites using secondary ion mass spectrometry

This article provides an overview of the technique of secondary ion mass spectrometry imaging and highlights some current and future areas of application relevant to the field of metabolomics. The approach benefits from label-free analysis of molecular species up to ~1500 Da with minimal sample preparation. Offering the highest spatial resolution of current mass spectrometry imaging methodologies, the technique is well-suited to metabolite imaging in both biological tissue and cells, in both 2D and 3D.

     

Isolation and characterisation of Nocardioides sp. SP12, an atrazine-degrading bacterial strain possessing the gene trzN from bulk- and maize rhizosphere soil

We report the characterisation of Nocardioides sp. SP12, an atrazine-degrading bacteria isolated from atrazine-treated bulk- and maize rhizosphere soil. Based on 16S rDNA alignment, strain SP12 showed close phylogenic relationships with Nocardioides sp. C157 and Nocardioides simplex. Internal transcribed spacer (ITS) sequences of strain SP12 were longer than those of other Nocardioides sp. and present Ala- and Ile-tRNA unlike Actinomycetales. Nocardioides sp. SP12 presents a novel atrazine catabolic pathway combining trzN with atzB and atzC. Atrazine biodegradation ends in a metabolite that co-eluted in HPLC with cyanuric acid. This metabolite shows an absorption spectrum identical to that of cyanuric acid with a maximal absorption at 214.6 nm. The mass of the atrazine metabolite is in concordance with that of cyanuric acid according to mass spectrometry analysis. Quantitative PCR revealed that the ITS sequence of Nocardioides sp. SP12 was at a lower number than the one of trzN in atrazine-treated soil samples. It suggests that trzN could also be present in other atrazine degrading bacteria. The numbers of trzN and ITS sequences of Nocardioides sp. SP12 were higher in the maize rhizosphere than in bulk soil.     

Spatio-Temporal Metabolite Profiling of the Barley Germination Process by MALDI MS Imaging

MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals’ germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest.     

Purification and mass spectrometry identification of leukotriene D4 synthesized by human alveolar macrophages

Human AM obtained by BAL from normal subjects and asthmatic patients converted [1-14C]-AA into a polar labeled metabolite. The structure of this metabolite, after two successive purifications on TLC (silicagel plates then reversed phase plates) and mass spectrometric analysis was shown to be identical to an authentic sample of LTD4. The amount of LTD4 recovered in the culture medium of AM was attempted to be related to pathological lung profile. In our experimental conditions AM from allergic asthmatics synthetized more LTD4 than cells from healthy subjects and from aspirin sensitive asthmatic patients.