Amsha Nahid

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.

Central carbon metabolism of Leishmania parasites.

Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.

1 H-NMR analysis of the human urinary metabolome in response to an 18-month multi-component exercise program and calcium-vitamin-D 3 supplementation in older men

The musculoskeletal benefits of calcium and vitamin-D3 supplementation and exercise have been extensively studied, but the effect on metabolism remains contentious. Urine samples were analyzed by (1)H-NMR spectroscopy from participants recruited for an 18-month, randomized controlled trial of a multi-component exercise program and calcium and vitamin-D3 fortified milk consumption. It was shown previously that no increase in musculoskeletal composition was observed for participants assigned to the calcium and vitamin-D3 intervention, but exercise resulted in increased bone mineral density, total lean body mass, and muscle strength. Retrospective metabolomics analysis of urine samples from patients involved in this study revealed no distinct changes in the urinary metabolome in response to the calcium and vitamin-D3 intervention, but significant changes followed the exercise intervention, notably a reduction in creatinine and an increase in choline, guanidinoacetate, and hypoxanthine (p < 0.001, fold change > 1.5). These metabolites are intrinsically involved in anaerobic ATP synthesis, intracellular buffering, and methyl-balance regulation. The exercise intervention had a marked effect on the urine metabolome and markers of muscle turnover but none of these metabolites were obvious markers of bone turnover. Measurement of specific urinary exercise biomarkers may provide a basis for monitoring performance and metabolic response to exercise regimes.

Cross-Platform Urine Metabolomics of Experimental Hyperglycemia in Type 2 Diabetes

Hyperglycemia causes diabetic nephropathy, a condition for which there are no specific diagnostic markers that predict progression to renal failure. Here we describe a multiplatform metabolomic analysis of urine from individuals with type 2 diabetes, collected before and immediately following experimental hyperglycemia. We used targeted nuclear magnetic resonance spectroscopy (NMR), liquid chromatography - mass spectrometry (LC-MS) and gas chromatography - MS (GC-MS) to identify markers of hyperglycemia. Following optimization of data normalisation and statistical analysis, we identified a reproducible NMR and LC-MS based urine signature of hyperglycemia. Significant increases of alanine, alloisoleucine, isoleucine, leucine, N-isovaleroylglycine, valine, choline, lactate and taurine and decreases of arginine, gamma-aminobutyric acid, hippurate, suberate and N-acetylglutamate were observed. GC-MS analysis identified a number of metabolites differentially present in post-glucose versus baseline urine, but these could not be identified using current metabolite libraries. This analysis is an important first step towards identifying biomarkers of early-stage diabetic nephropathy.

Plasmodium falciparum is dependent on de novo myo-inositol biosynthesis for assembly of GPI glycolipids and infectivity

Intra‐erythrocytic stages of the malaria parasite, Plasmodium falciparum, are thought to be dependent on de novo synthesis of phosphatidylinositol, as red blood cells (RBC) lack the capacity to synthesize this phospholipid. The myo‐inositol headgroup of PI can either be synthesized de novo or scavenged from the RBC. An untargeted metabolite profiling of P. falciparum infected RBC showed that trophozoite and schizont stages accumulate high levels of myo‐inositol‐3‐phosphate, indicating increased de novo biosynthesis of myo‐inositol from glucose 6‐phosphate. Metabolic labelling studies with 13C‐U‐glucose in the presence and absence of exogenous inositol confirmed that de novo myo‐inositol synthesis occurs in parallel with myo‐inositol salvage pathways. Unexpectedly, while both endogenous and scavenged myo‐inositol was used to synthesize bulk PI, only de novo‐synthesized myo‐inositol was incorporated into GPI glycolipids. Moreover, gene disruption studies suggested that the INO1 gene, encoding myo‐inositol 3‐phosphate synthase, is essential in asexual parasite stages. Together these findings suggest that P. falciparum asexual stages are critically dependent on de novo myo‐inositol biosynthesis for assembly of a sub‐pool of PI species and GPI biosynthesis. These findings highlight unexpected complexity in phospholipid biosynthesis in P. falciparum and a lack of redundancy in some nutrient salvage versus endogenous biosynthesis pathways.

Metabolomics – The combination of analytical biochemistry, biology, and informatics

The combination of analytical biochemistry to measure the metabolic complement with sophisticated informatics, bioinformatics, and statistics makes up the newest of the ‘omics fields called metabolomics. Metabolites are characterized by a diverse chemistry and therefore require the application of numerous analytical approaches for their extraction, separation, detection, and quantification. In the past decade, the technologies have improved substantially allowing the analysis of thousands of compounds simultaneously. However, this has led to the current bottleneck in metabolomics that is how to extract information from raw data of many different analytical platforms and the subsequent appropriate analysis in a biological context. Before any statistical analysis can be performed on the resulting high-density data sets, a number of preprocessing steps including peak finding, integration, filtering, normalization, and transformation are required. In this article, we aim to summarize the state of the art of metabolomics technologies from both an analytical and a bioinformatics point of view. We present the challenges currently faced by metabolomics researchers and provide the readers with potential approaches to address those challenges.

MASTR-MS: a web-based collaborative laboratory information management system (LIMS) for metabolomics

BackgroundAn increasing number of research laboratories and core analytical facilities around the world are developing high throughput metabolomic analytical and data processing pipelines that are capable of handling hundreds to thousands of individual samples per year, often over multiple projects, collaborations and sample types. At present, there are no Laboratory Information Management Systems (LIMS) that are specifically tailored for metabolomics laboratories that are capable of tracking samples and associated metadata from the beginning to the end of an experiment, including data processing and archiving, and which are also suitable for use in large institutional core facilities or multi-laboratory consortia as well as single laboratory environments.ResultsHere we present MASTR-MS, a downloadable and installable LIMS solution that can be deployed either within a single laboratory or used to link workflows across a multisite network. It comprises a Node Management System that can be used to link and manage projects across one or multiple collaborating laboratories; a User Management System which defines different user groups and privileges of users; a Quote Management System where client quotes are managed; a Project Management System in which metadata is stored and all aspects of project management, including experimental setup, sample tracking and instrument analysis, are defined, and a Data Management System that allows the automatic capture and storage of raw and processed data from the analytical instruments to the LIMS.ConclusionMASTR-MS is a comprehensive LIMS solution specifically designed for metabolomics. It captures the entire lifecycle of a sample starting from project and experiment design to sample analysis, data capture and storage. It acts as an electronic notebook, facilitating project management within a single laboratory or a multi-node collaborative environment. This software is being developed in close consultation with members of the metabolomics research community. It is freely available under the GNU GPL v3 licence and can be accessed from, https://muccg.github.io/mastr-ms/.

Physicochemical properties and trace organic compounds in a dairy processor's aerobic bioreactor.

Wastewater samples were taken from an aerobic bioreactor, operated by a dairy processor in southeastern Australia to reduce nutrient and pollutant loads. Samples were taken over a two-year period, to determine whether trace organic compounds or physicochemical analyses of the wastewater could be used to discriminate the water taken before, during and after processing of the wastewater in the bioreactor. Multivariate analyses of the physicochemical data suggested that nitrate, pH and total dissolved nitrogen best described the infeed wastewater entering the bioreactor, while organic and particulate phosphorus concentrations where predominantly responsible for describing the composition of the content of the bioreactor. Gas chromatography-mass spectrometry data of organic compounds within the wastewater samples were also analysed via multivariate analyses. The analyses found that the compound 4-nitrophenol was associated with ammonia concentrations and mixed liquor wastewater. Therefore, 4-nitrophenol may possibly be used to act as an indicator of anaerobicity in aerobic bioreactors.