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Multiplexed assay for protein quantitation in the invertebrate Gammarus fossarum by liquid chromatography coupled to tandem mass spectrometry

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Abstract

A highly multiplexed liquid chromatography mass spectrometry–selected reaction monitoring (SRM)-based assay for determination of 40 potential protein biomarkers from Gammarus fossarum, an ecotoxicological relevant species, was described. The assay relies on 71 stable isotope-labeled reported peptide standards for the quantitation of proteins of interest in relation to essential physiological functions such as reproductive cycle, defense mechanism, and enzymes involved in homeostasis process and in energy. A direct linear relationship between the spiked peptide concentration and the area under the peak was clearly demonstrated in biological extracts. Precision and accuracy were determined to be between 1.1 and 21% and between 79 and 120%, respectively, depending on the selected protein in a few samples after optimization of digestion conditions. The validity of the assay was documented for several biomarkers linked with reproduction and the molting process was performed with the assessment of protein levels throughout contrasted physiological process (sex, reproductive status). This assay is easy to use, robust, sensitive, and has high-throughput capabilities. The proposed strategy may be extended to any non-model organisms relevant in environmental science.

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Acknowledgments

We thank the French “Ministère de la recherche et de l’enseignement supérieur” for the fellowship of Aurore Charnot. We also thank the ANR program “ProteoGam” (ANR-14-CE21-0006-02) and the Regional Water Agency Rhône-Méditerranée-Corse for financial support.

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Authors and Affiliations

  1. Univ Lyon, CNRS, Université Claude Bernard Lyon 1, ENS de Lyon, Institut des Sciences Analytiques, UMR 5280, 69100, Villeurbanne, France

    Aurore Charnot, Jérôme Lemoine & Arnaud Salvador

  2. Laboratoire d’écotoxicologie, IRSTEA, Unité de recherché MALY, CS70077, 69626, Villeurbanne, France

    Duarte Gouveia, Arnaud Chaumot & Olivier Geffard

  3. CEA-Marcoule, DRF/IBITEC-S/SPI/Li2D, Laboratory “Innovative Technologies for Detection and Diagnostics”, BP 17171, 30200, Bagnols-sur-Cèze, France

    Jean Armengaud & Christine Almunia

Authors
  1. Aurore Charnot
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  2. Duarte Gouveia
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  3. Jean Armengaud
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  4. Christine Almunia
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  5. Arnaud Chaumot
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  6. Jérôme Lemoine
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  7. Olivier Geffard
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  8. Arnaud Salvador
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Correspondence to Arnaud Salvador.

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Charnot, A., Gouveia, D., Armengaud, J. et al. Multiplexed assay for protein quantitation in the invertebrate Gammarus fossarum by liquid chromatography coupled to tandem mass spectrometry. Anal Bioanal Chem 409, 3969–3991 (2017). https://doi.org/10.1007/s00216-017-0348-0

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  • DOI: https://doi.org/10.1007/s00216-017-0348-0

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