Dynamic range and mass accuracy of wide-scan direct infusion nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method

Direct infusion nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (DI nESI FT-ICR MS)offers high mass accuracy and resolution for analyzing complex metabolite mixtures. High dynamic range across a wide mass range, however, can only be achieved at the expense of mass accuracy, since the large numbers of ions entering the ICR detector induce adverse spacecharge effects. Here we report an optimized strategy for wide-scan DI nESI FT-ICR MS that increases dynamic range but maintains high mass accuracy. It comprises the collection if multiple adjacent selected ion monitoring (SIM) windows that are stitched together using novel algorithms. The final SIM-stitching method, derived from several optimization experiments, comprises 21 adjoining SIM windows each of width m/z 30 (from m/z 70 to 500; adjacent windows overlap by m/z 10) with an automated gain control (AGC) target of 1 105 charges. SIMstitching and wide-scan range (WSR; Thermo Electron)were compared using a defined standard to assess mass accuracy and a liver extract to assess peak count and dynamic range. SIM-stitching decreased the maximum mass error by 1.3- and 4.3-fold, and increased the peak count by 5.3- and 1.8-fold, versus WSR (AGC targets of 1 x 105 and 5 x 105, respectively). SIM-stitching achieved an rms mass error of 0.18 ppm and detected over 3000 peaks in liver extract. This novel approach increases metabolome coverage, has very high mass accuracy, and at 5.5 min/sample is conducive for high- throughput metabolomics

The metabolic response of marine copepods to environmental warming and ocean acidification in the absence of food

Marine copepods are central to the productivity and biogeochemistry of marine ecosystems. Nevertheless, the direct and indirect effects of climate change on their metabolic functioning remain poorly understood. Here, we use metabolomics, the unbiased study of multiple low molecular weight organic metabolites, to examine how the physiology of Calanus spp. is affected by end-of-century global warming and ocean acidification scenarios. We report that the physiological stresses associated with incubation without food over a 5-day period greatly exceed those caused directly by seawater temperature or pH perturbations. This highlights the need to contextualise the results of climate change experiments by comparison to other, naturally occurring stressors such as food deprivation, which is being exacerbated by global warming. Protein and lipid metabolism were up-regulated in the food-deprived animals, with a novel class of taurine-containing lipids and the essential polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid and docosahexaenoic acid, changing significantly over the duration of our experiment. Copepods derive these PUFAs by ingesting diatoms and flagellated microplankton respectively. Climate-driven changes in the productivity, phenology and composition of microplankton communities, and hence the availability of these fatty acids, therefore have the potential to influence the ability of copepods to survive starvation and other environmental stressors

Metabolomics discovers early-response metabolic biomarkers that can predict chronic reproductive fitness in individual daphnia magna

Chemical risk assessment remains entrenched in chronic toxicity tests that set safety thresholds based on animal pathology or fitness. Chronic tests are resource expensive and lack mechanistic insight. Discovering a chemical’s mode-of-action can in principle provide predictive molecular biomarkers for a toxicity endpoint. Furthermore, since molecular perturbations precede pathology, early-response molecular biomarkers may enable shorter, more resource efficient testing that can predict chronic animal fitness. This study applied untargeted metabolomics to attempt to discover early-response metabolic biomarkers that can predict reproductive fitness of Daphnia magna, an internationally-recognized test species. First, we measured the reproductive toxicities of cadmium, 2,4-dinitrophenol and propranolol to individual Daphnia in 21-day OECD toxicity tests, then measured the metabolic profiles of these animals using mass spectrometry. Multivariate regression successfully discovered putative metabolic biomarkers that strongly predict reproductive impairment by each chemical, and for all chemicals combined. The non-chemical-specific metabolic biomarkers were then applied to metabolite data from Daphnia 24-h acute toxicity tests and correctly predicted that significant decreases in reproductive fitness would occur if these animals were exposed to cadmium, 2,4-dinitrophenol or propranolol for 21 days. While the applicability of these findings is limited to three chemicals, they provide proof-of-principle that early-response metabolic biomarkers of chronic animal fitness can be discovered for regulatory toxicity testing

Characterisation of the dynamic nature of lipids throughout the lifespan of genetically identical female and male Daphnia magna

Lipids play a significant role in regulation of health and disease. To enhance our understanding of the role of lipids in regulation of lifespan and healthspan additional studies are required. Here, UHPLC-MS/MS lipidomics was used to measure dynamic changes in lipid composition as a function of age and gender in genetically identical male and female Daphnia magna with different average lifespans. We demonstrate statistically significant age-related changes in triglycerides (TG), diglycerides (DG), phosphatidylcholine, phosphatidylethanolamine, ceramide and sphingomyelin lipid groups, for example, in males, 17.04% of TG lipid species decline with age whilst 37.86% increase in relative intensity with age. In females, 23.16% decrease and 25.31% increase in relative intensity with age. Most interestingly, the rate and direction of change can differ between genetically identical female and male Daphnia magna, which could be the cause and/or the consequence of the different average lifespans between the two genetically identical genders. This study provides a benchmark dataset to understand how lipids alter as a function of age in genetically identical female and male species with different average lifespan and ageing rate.Peer reviewe

A Systems Biology Approach Reveals a Calcium-Dependent Mechanism for Basal Toxicity in Daphnia magna.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Environmental Science & Technology, copyright © American Chemical Society after peer review and technical editing by the publisher.The expanding diversity and ever increasing amounts of man-made chemicals discharged to the environment pose largely unknown hazards to ecosystem and human health. The concept of adverse outcome pathways (AOPs) emerged as a comprehensive framework for risk assessment. However, the limited mechanistic information available for most chemicals and a lack of biological pathway annotation in many species represent significant challenges to effective implementation of this approach. Here, a systems level, multistep modeling strategy demonstrates how to integrate information on chemical structure with mechanistic insight from genomic studies, and phenotypic effects to define a putative adverse outcome pathway. Results indicated that transcriptional changes indicative of intracellular calcium mobilization were significantly overrepresented in Daphnia magna (DM) exposed to sublethal doses of presumed narcotic chemicals with log Kow ≥ 1.8. Treatment of DM with a calcium ATPase pump inhibitor substantially recapitulated the common transcriptional changes. We hypothesize that calcium mobilization is a potential key molecular initiating event in DM basal (narcosis) toxicity. Heart beat rate analysis and metabolome analysis indicated sublethal effects consistent with perturbations of calcium preceding overt acute toxicity. Together, the results indicate that altered calcium homeostasis may be a key early event in basal toxicity or narcosis induced by lipophilic compounds

Defining the baseline and oxidant perturbed lipidomic profiles of Daphnia magna

Recent technological advancement has enabled the emergence of lipidomics as an important tool for assessing molecular stress, one which has yet to be assessed fully as an approach in an environmental toxicological context. Here we have applied a high-resolution, non-targeted, nanoelectrospray ionisation (nESI) direct infusion mass spectrometry (DIMS) technique to assess the effects of oxidative stress to Daphnia magna both in vitro (air exposure of daphniid extracts) and in vivo (Cu2+ exposure). Multivariate and univariate statistical analyses were used to distinguish any perturbations including oxidation to the D. magna baseline lipidome. This approach enabled the putative annotation of the baseline lipidome of D. magna with 65% of the lipid species discovered previously not reported. In vitro exposure of lipid extracts to air, primarily to test the methodology, revealed a significant perturbation to this baseline lipidome with detectable oxidation of peaks, in most cases attributed to single oxygen addition. Exposure of D. magna to Cu2+ in vivo also caused a significant perturbation to the lipidome at an environmentally relevant concentration of 20 µg/L. This nESI DIMS approach has successfully identified perturbations and oxidative modifications to the D. magna lipidome in a high-throughput manner, highlighting its suitability for environmental lipidomic studies

How close are we to complete annotation of metabolomes?

The metabolome describes the full complement of the tens to hundreds of thousands of low molecular weight metabolites present within a biological system. Identification of the metabolome is critical for discovering the maximum amount of biochemical knowledge from metabolomics datasets. Yet no exhaustive experimental characterisation of any organismal metabolome has been reported to date, dramatically contrasting with the genome sequencing of thousands of plants, animals and microbes. Here we review the status of metabolome annotation and describe advances in the analytical methodologies being applied. In part through new international coordination, we conclude that we are now entering a new era of metabolome annotation

Metabolomics reveals an involvement of pantothenate for male production responding to the short-day stimulus in the water flea, Daphnia pulex

Under favorable conditions, the micro-crustacean Daphnia pulex produces female offspring by parthenogenesis, whereas under unfavorable conditions, they produce male offspring to induce sexual reproduction (environmental sex determination: ESD). We recently established a suitable system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be regulated by alterations in day-length; long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we have already demonstrated that methyl farnesoate (MF) synthesis is necessary for male offspring production, and identified ionotropic glutamate receptors as an upstream regulator of MF signaling. Despite these findings, the molecular mechanisms associated with MF signaling have not yet been well elucidated. In this study, we analyzed the whole metabolic profiles of mother daphnids reared under long-day (female-producing) and short-day (male-producing) conditions, and discovered that pantothenate (vitamin B5), a known precursor to coenzyme A, was significantly accumulated in response to the short-day condition. To confirm the innate role of pantothenate in D. pulex, this metabolite was administered to mother daphnids resulting in a significantly increased proportion of male offspring producing mothers. This study provides novel insights of the metabolic mechanisms of the ESD system in D. pulex