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From shotgun to targeted proteomics: rapid Scout-MRM assay development for monitoring potential immunomarkers in Dreissena polymorpha

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Abstract

A highly multiplexed liquid chromatography mass spectrometry–multiple reaction monitoring (MRM)-based assay has been developed for evaluating 107 candidate immune biomarkers in both hemocytes and plasma of the zebra mussel Dreissena polymorpha. The Scout-MRM strategy was employed for the first time, shortening the implementation of a targeted MRM bottom-up proteomics assay using selected immune protein-related peptides identified by shotgun discovery proteogenomics. This strategy relies on spiking scout peptides during the discovery phase and using them to build and deploy the MRM targeted proteomics method. It proved to be highly relevant, since about 90% of the targeted peptides and proteins were monitored and rapidly measured in both hemocyte and plasma samples. The sample preparation protocol was optimized by evaluating the digestion efficiency of tryptic peptides over time. The accuracy and precision of 50 stable isotope-labeled peptides were evaluated for use as internal standards. Finally, the specificity of the transitions was thoroughly assessed to ensure the reliable measurement of protein biomarkers. Several analytical and biological validation criteria were evaluated across hemocytes and plasma samples exposed ex vivo to biological contaminants, resulting in the validation of two Scout-MRM assays for the relative quantitation of 85 and 89 proteins in hemocytes and plasma, respectively.

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Acknowledgments

We thank the University of Reims Champagne-Ardenne for its financial support (project PROMETHEE). This work benefitted from the French GDR “Aquatic Ecotoxicology” framework which is aimed at fostering stimulating scientific discussions and collaborations for more integrative approaches.

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Authors and Affiliations

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

    Maxime Leprêtre, Julien Faugere, Jérôme Lemoine & Arnaud Salvador

  2. Université de Reims Champagne-Ardenne, UMR-I 02 INERIS-URCA-ULH SEBIO Unité Stress Environnementaux et BIOsurveillance des milieux aquatiques, UFR Sciences Exactes et Naturelles, Campus du Moulin de la Housse, BP 1039, 51687, Reims, Cedex, France

    Maxime Leprêtre, Mélissa Palos-Ladeiro & Alain Geffard

  3. Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI , 30200, Bagnols-sur-Cèze, France

    Christine Almunia & Jean Armengaud

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  1. Maxime Leprêtre
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Correspondence to Arnaud Salvador.

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The authors declare the following financial interests: Jerôme Lemoine filed a patent describing the Scout-MRM concept for targeted analysis by mass spectrometry. The authors declare that they have no conflict of interest.

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Leprêtre, M., Palos-Ladeiro, M., Faugere, J. et al. From shotgun to targeted proteomics: rapid Scout-MRM assay development for monitoring potential immunomarkers in Dreissena polymorpha. Anal Bioanal Chem 412, 7333–7347 (2020). https://doi.org/10.1007/s00216-020-02868-2

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