Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatography

Highly polar metabolites, such as sugars and most amino acids are not retained by conventional RP LC columns. Without sufficient retention low concentration compounds are not detected due ion suppression and structural isomers are not resolved. In contrast, hydrophilic interaction chromatography (HILIC) and aqueous normal phase chromatography (ANP) retain compounds based on their hydrophilicity and therefore provides a means of separating highly polar compounds. Here, an ANP method based on the diamond hydride stationary phase is presented for profiling biological small molecules by LC. A rapid separation system based upon a fast gradient that delivers reproducible chromatography is presented. Approximately 1000 compounds were reproducibly detected in human urine samples and clear differences between these samples were identified. This chromatography was also applied to xylem fluid from soyabean (Glycine max) plants to which 400 compounds were detected. This method greatly increases the metabolite coverage over RP-only metabolite profiling in biological samples. We show that both forms of chromatography are necessary for untargeted comprehensive metabolite profiling and that the diamond hydride stationary phase provides a good option for polar metabolite analysis

A quantitative profiling method of phytohormones and other metabolites applied to barley roots subjected to salinity stress

As integral parts of plant signaling networks, phytohormones are involved in the regulation of plant metabolism and growth under adverse environmental conditions, including salinity. Globally, salinity is one of the most severe abiotic stressors with an estimated 800 million hectares of arable land affected. Roots are the first plant organ to sense salinity in the soil, and are the initial site of sodium (Na + ) exposure. However, the quantification of phytohormones in roots is challenging, as they are often present at extremely low levels compared to other plant tissues. To overcome this challenge, we developed a high-throughput LC-MS method to quantify ten endogenous phytohormones and their metabolites of diverse chemical classes in roots of barley. This method was validated in a salinity stress experiment with six barley varieties grown hydroponically with and without salinity. In addition to phytohormones, we quantified 52 polar primary metabolites, including some phytohormone precursors, using established GC-MS and LC-MS methods. Phytohormone and metabolite data were correlated with physiological measurements including biomass, plant size and chlorophyll content. Root and leaf elemental analysis was performed to determine Na + exclusion and K + retention ability in the studied barley varieties. We identified distinct phytohormone and metabolite signatures as a response to salinity stress in different barley varieties. Abscisic acid increased in the roots of all varieties under salinity stress, and elevated root salicylic acid levels were associated with an increase in leaf chlorophyll content. Furthermore, the landrace Sahara maintained better growth, had lower Na + levels and maintained high levels of the salinity stress linked metabolite putrescine as well as the phytohormone metabolite cinnamic acid, which has been shown to increase putrescine concentrations in previous studies. This study highlights the importance of root phytohormones under salinity stress and the multi-variety analysis provides an important update to analytical methodology, and adds to the current knowledge of salinity stress responses in plants at the molecular level

Elemental imaging of leaves from the metal hyperaccumulating plant Noccaea caerulescens shows different spatial distribution of Ni, Zn and Cd

Elemental imaging using laser ablation inductively coupled plasma mass spectrometry was performed on whole leaves of the hyperaccumulating plant Noccaea caerulescens after treatments with either Ni, Zn or Cd. These detailed elemental images reveal differences in the spatial distribution of these three elements across the leaf and provide new insights in the metal ion homeostasis within hyperaccumulating plants. In the Zn treated plants, Zn accumulated in the leaf tip while Mn was co-localised with Zn suggesting similar storage mechanisms for these two metals. These data show a Zn concentration difference of up to 13-fold higher in the distal part of the leaf. Also, there was no correlation between the S and Zn concentrations providing further evidence against S-binding ligands. In contrast, Ni was more evenly distributed while a more heterogeneous distribution of Cd was present with some high levels on leaf edges, suggesting that different storage and transport mechanisms are used for the hyperaccumulation of these two metals. These results show the importance of correct sampling when carrying out subcellular localisation studies as the hyperaccumulated elements are not necessarily homogenously distributed over the entire leaf area. The results also have great implications for biotechnological applications of N. caerulescens showing that it may be possible to use the mechanisms employed by N. caerulescens to increase the Zn concentration in nutrient poor crops without increasing the risk of accumulating other toxic elements such as Ni and Cd

The use of metabolomics in the study of metals in biological systems

Metabolomics may be defined as the comprehensive quantitative and/or qualitative analysis of all metabolites present in a bio-fluid, cell, tissue, or organism. It is essentially the study of biochemical phenotypes (or metabotypes). Metabolic profiles are context dependent, and vary in response to a variety of factors including environment and environmental stimuli, health status, disease and a myriad of other factors; as such, metabolomics has been applied to a wide range of fields and has been increasingly utilised to the study of the roles played by metals in a range of biological systems as well as, encouragingly, in understanding the underlying biochemical mechanisms. The role of metals (and metalloids) in biological organisms is complex and the majority of studies in this area have been performed in plants but the fields of natural product chemistry, human health and even bacterial corrosion of water distribution systems have been investigated using this technique. In this review some of the novel approaches in which the metabolomics toolbox has been used to unravel the roles of metals and metalloids in a range of biological systems are discussed and suggestions made for future research

Elemental and metabolite profiling of nickel hyperaccumulators from New Caledonia

Leaf material from nine Ni hyperaccumulating species was collected in New Caledonia: Homalium kanaliense (Vieill.) Briq., Casearia silvana Schltr, Geissois hirsuta Brongn. & Gris, Hybanthus austrocaledonicusSeem, Psychotria douarrei (G. Beauvis.) Däniker, Pycnandra acuminata (Pierre ex Baill.) Swenson & Munzinger (syn Sebertia acuminata Pierre ex Baill.), Geissois pruinosa Brongn. & Gris, Homalium deplanchei (Viell) Warb. and Geissois bradfordii (H.C. Hopkins). The elemental concentration was determined by inductively-coupled plasma optical emission spectrometry (ICP-OES) and from these results it was foundthat the species contained Ni concentrations from to 250–28,000 mg/kg dry mass. Gas chromatography mass spectrometry (GC–MS)-based metabolite profiling was then used to analyse leaves of each species.The aim of this study was to target Ni-binding ligands through correlation analysis of the metabolite levels and leaf Ni concentration. Approximately 258 compounds were detected in each sample. As has been observed before, a correlation was found between the citric acid and Ni concentrations in the leaves for all species collected. However, the strongest Ni accumulator, P. douarrei, has been found to contain particularly high concentrations of malonic acid, suggesting an additional storage mechanism for Ni. A size exclusion chromatography separation protocol for the separation of Ni-complexes in P. acuminata sap was also applied to aqueous leaf extracts of each species. A number of metabolites were identified in complexes with Ni including Ni-malonate from P. douarrei. Furthermore, the levels for some metabolites were found to correlate with the leaf Ni concentration. These data show that Ni ions can be bound by a range of small molecules in Ni hyperaccumulation in plants

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

Liquid chromatography–mass spectrometry (LC–MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and non-polar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC–MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds

LC–MS and GC–MS metabolite profiling of nickel(II) complexes in the latex of the nickel-hyperaccumulating tree Sebertia acuminata and identification of methylated aldaric acid as a new nickel(II) ligand

Targeted liquid chromatography–mass spectrometry (LC–MS) technology using size exclusion chromatography and metabolite profiling based on gas chromatography–mass spectrometry (GC–MS) were used to study the nickel-rich latex of the hyperaccumulating tree Sebertia acuminata. More than 120 compounds were detected, 57 of these were subsequently identified. A methylated aldaric acid (2,4,5-trihydroxy-3-methoxy-1,6-hexan-dioic acid) was identified for the first time in biological extracts and its structure was confirmed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. After citric acid, it appears to be one of the most abundant small organic molecules present in the latex studied. Nickel(II) complexes of stoichiometry NiII:acid = 1:2 were detected for these two acids as well as for malic, itaconic, erythronic, galacturonic, tartaric, aconitic and saccharic acids. These results provide further evidence that organic acids may play an important role in the transport and possibly in the storage of metal ions in hyperaccumulating plants

Spatio-temporal metabolite and elemental profiling of salt stressed barley seeds during initial stages of germination by MALDI-MSI and µ-XRF spectrometry

Seed germination is the essential first step in crop establishment, and can be severely affected by salinity stress which can inhibit essential metabolic processes during the germination process. Salt stress during seed germination can trigger lipid-dependent signalling cascades that activate plant adaptation processes, lead to changes in membrane fluidity to help resist the stress, and cause secondary metabolite responses due to increased oxidative stress. In germinating barley (Hordeum vulgare), knowledge of the changes in spatial distribution of lipids and other small molecules at a cellular level in response to salt stress is limited. In this study, mass spectrometry imaging (MSI), liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS), inductively coupled plasma mass spectrometry (ICP-MS), and X-ray fluorescence (XRF) were used to determine the spatial distribution of metabolites, lipids and a range of elements, such as K+ and Na+, in seeds of two barley genotypes with contrasting germination phenology (Australian barley varieties Mundah and Keel). We detected and tentatively identified more than 200 lipid species belonging to seven major lipid classes (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, prenol lipids, sterol lipids, and polyketides) that differed in their spatial distribution based on genotype (Mundah or Keel), time post-imbibition (0 to 72 h), or treatment (control or salt). We found a tentative flavonoid was discriminant in post-imbibed Mundah embryos under saline conditions, and a delayed flavonoid response in Keel relative to Mundah. We further employed MSI-MS/MS and LC-QToF-MS/MS to explore the identity of the discriminant flavonoid and study the temporal pattern in five additional barley genotypes. ICP-MS was used to quantify the elemental composition of both Mundah and Keel seeds, showing a significant increase in Na+ in salt treated samples. Spatial mapping of elements using µ-XRF localized the elements within the seeds. This study integrates data obtained from three mass spectrometry platforms together with µ-XRF to yield information on the localization of lipids, metabolites and elements improving our understanding of the germination process under salt stress at a molecular level

Comprehensive profiling and quantitation of amine group containing metabolites

Primary and secondary amines, including amino acids, biogenic amines, hormones, neurotransmitters, and plant siderophores, are readily derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using easily performed experimental methodology. Complex mixtures of these amine derivatives can be fractionated and quantified using liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS). Upon collision induced dissociation (CID) in a quadrupole collision cell, all derivatized compounds lose the aminoquinoline tag. With the use of untargeted fragmentation scan functions, such as precursor ion scanning, the loss of the aminoquinoline tag (Amq) can be monitored to identify derivatized species; and the use of targeted fragmentation scans, such as multiple reaction monitoring, can be exploited to quantitate amine-containing molecules. Further, with the use of accurate mass, charge state, and retention time, identification of unknown amines is facilitated. The stability of derivatized amines was found to be variable with oxidatively labile derivatives rapidly degrading. With the inclusion of antioxidant and reducing agents, tris(2-carboxyethyl)-phosphine (TCEP) and ascorbic acid, into both extraction solvents and reaction buffers, degradation was significantly decreased, allowing reproducible identification and quantification of amine compounds in large sample sets