Development of a multi-omics extraction method for ecotoxicology: investigation of the reproductive cycle of Gammarus fossarum

Faugere, Julien; Brunet, Thomas Alexandre; Clément, Yohann; Espeyte, Anabelle; Geffard, Olivier; Lemoine, Jérôme; Chaumot, Arnaud; Degli-Esposti, Davide; Ayciriex, Sophie; Salvador, Arnaud

doi:10.1016/j.talanta.2022.123806

Cited by 6 publications

(4 citation statements)

References 38 publications

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“…Chromatographic separation of lipid extracts, QCs and blanks were performed on an XSelect CSH C18 column (100 mm x 1 mm, particle size 3.5 µm) heated to 70°C (Waters, Milford, MA, USA) on an Exion LC system. The LC method was adapted from a previously described method [38]. Elution was performed at a flow rate of 200 µL/min with H 2 O:ACN (60:40, v/v ) containing 10 mM ammonium formate and 0.1% ( v/v ) formic acid for mobile phase A and IPA:ACN:H 2 O (90:9.5:0.5, v/v/v ) containing 10 mM ammonium formate and 0.1% ( v/v ) formic acid for mobile phase B, employing a linear gradient from 1% B to 100 % B in 13.6 min.…”

Section: Methodsmentioning

confidence: 99%

“…Next, we evaluated the benefits of the Zeno trapping on the intensity of the fragments generated in SWATH-DIA as well as their numbers and especially for low abundant compounds. We examined the lipidome by RPLC separation (apolar fraction) and the metabolome by HILIC (polar fraction) of female Gammarus fossarum, a freshwater amphipod routinely used in Ecotoxicology [37][38][39], at two distinct reproductive stages (C1 and D1). Multivariate data analysis encompassing an unsupervised approach (principal component analysis, PCA) and supervised modelling (partial least squares discriminant analysis, PLS-DA) was applied to visualize and explain data variability and to highlight specific molecular features involved in reproductive stage differentiation.…”

Section: Introductionmentioning

confidence: 99%

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Concomitant investigation of crustacean amphipods lipidome and metabolome during molting stage by Zeno SWATH Data-Independent Acquisition coupled with Electron Activated Dissociation and machine learning

Brunet,

Clément,

Calabrese

et al. 2023

Preprint

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BackgroundDIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) in metabolomics and lipidomics, for comprehensive profiling of molecules in biological samples. It provides unbiased coverage, improved reproducibility, and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipodGammarus fossarum, successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field.ResultsDIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to study molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified statistically significant features responsible for group segregation. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra DB annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness.SignificanceReproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing and EAD synergistically enhances detection sensitivity while providing orthogonal fragmentation information, thereby facilitating a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying biomarkers of significance in the field of environmental assessment.HighlightsAdvancements in Data-Independent-Acquisition methods for lipidomics and metabolomics.Zeno pulsing improves sensitivity in fragment detection and compound identification.Comprehensive structure elucidation of lipids and metabolites using EAD fragmentation.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Concomitant investigation of crustacean amphipods lipidome and metabolome during molting stage by Zeno SWATH Data-Independent Acquisition coupled with Electron Activated Dissociation and machine learning

Brunet,

Clément,

Calabrese

et al. 2023

Preprint

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“…Metabolite extraction was performed according to a slightly modified multi-omics sample extraction for G. fossarum by Faugere et al [68]. Entire frozen female gammarids (n = 6 for the C1 stage and n = 6 for the D1 stage) were individually homogenized in 2 mL tubes prefilled with beads (Precellys® CK28 Lysing Kit, Bertin) using 300 µL of a methanol/water mixture (80/20 v/v) on a homogenizer (Precellys® Evolution Touch, Bertin) at 5500 rpm during two cycles of 25 s each spaced by a 10 s interval.…”

Section: Chemical and Sample Preparationmentioning

confidence: 99%

Electron-activated dissociation (EAD) for the complementary annotation of metabolites and lipids through data-dependent acquisition analysis and feature-based molecular networking, applied to the sentinel amphipod Gammarus fossarum

Calabrese,

Brunet,

Degli-Esposti

et al. 2024

Anal Bioanal Chem

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The past decades have marked the rise of metabolomics and lipidomics as the -omics sciences which reflect the most phenotypes in living systems. Mass spectrometry-based approaches are acknowledged for both quantification and identification of molecular signatures, the latter relying primarily on fragmentation spectra interpretation. However, the high structural diversity of biological small molecules poses a considerable challenge in compound annotation. Feature-based molecular networking (FBMN) combined with database searches currently sets the gold standard for annotation of large datasets. Nevertheless, FBMN is usually based on collision-induced dissociation (CID) data, which may lead to unsatisfying information. The use of alternative fragmentation methods, such as electron-activated dissociation (EAD), is undergoing a re-evaluation for the annotation of small molecules, as it gives access to additional fragmentation routes. In this study, we apply the performances of data-dependent acquisition mass spectrometry (DDA-MS) under CID and EAD fragmentation along with FBMN construction, to perform extensive compound annotation in the crude extracts of the freshwater sentinel organism Gammarus fossarum. We discuss the analytical aspects of the use of the two fragmentation modes, perform a general comparison of the information delivered, and compare the CID and EAD fragmentation pathways for specific classes of compounds, including previously unstudied species. In addition, we discuss the potential use of FBMN constructed with EAD fragmentation spectra to improve lipid annotation, compared to the classic CID-based networks. Our approach has enabled higher confidence annotations and finer structure characterization of 823 features, including both metabolites and lipids detected in G. fossarum extracts.

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“…The past years of research have been mainly based on the use of single omics (proteomics, lipidomics, or metabolomics, to name a few) leading to a limited view of the investigated system. More recently, multi-omics approaches, based on the integration of multiple omics data on a same sample have been implemented in ecotoxicology studies leading to a more performant elucidation of complex processes (Nam et al, 2022;Faugere et al, 2023).…”

Section: Introductionmentioning

confidence: 99%

Challenges and perspectives in MS-based omics approaches for ecotoxicology studies: An insight on Gammarids sentinel amphipods

Calabrese

Salvador²,

Clément³

et al. 2023

Front. Anal. Sci.

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The aquatic environment is one of the most complex biosystems, as organism at all trophic levels may be exposed to a multitude of pollutants. As major goals, ecotoxicology typically investigates the impact of toxic pollutants on the ecosystems through the study of sentinel organisms. Over the past decades, Mass Spectrometry (MS)-based omics approaches have been extended to sentinel species both in laboratory and field exposure conditions. Single-omics approaches enable the discovery of biomarkers mirroring the health status of an organism. By covering a restricted set of the molecular cascade, they turn out to only partially satisfy the understanding of complex ecotoxicological effects. In contrast, a more complete understanding of the ecotoxicity pathways can be accessed through multi-omics approaches. In this perspective, we provide a state-of-the-art and a critical evaluation on further developments in MS-based single and multi-omics studies in aquatic ecotoxicology. As case example, literature regarding Gammarids freshwater amphipods, non-model sentinel organisms sensitive to pollutants and environmental changes and crucial species for downstream ecosystems, will be reviewed.

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Development of a multi-omics extraction method for ecotoxicology: investigation of the reproductive cycle of Gammarus fossarum

Cited by 6 publications

References 38 publications

Concomitant investigation of crustacean amphipods lipidome and metabolome during molting stage by Zeno SWATH Data-Independent Acquisition coupled with Electron Activated Dissociation and machine learning

Concomitant investigation of crustacean amphipods lipidome and metabolome during molting stage by Zeno SWATH Data-Independent Acquisition coupled with Electron Activated Dissociation and machine learning

Electron-activated dissociation (EAD) for the complementary annotation of metabolites and lipids through data-dependent acquisition analysis and feature-based molecular networking, applied to the sentinel amphipod Gammarus fossarum

Challenges and perspectives in MS-based omics approaches for ecotoxicology studies: An insight on Gammarids sentinel amphipods

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