User‑friendly extraction and multistage tandem mass spectrometry based analysis of lipid‑linked oligosaccharides in microalgae - Archive ouverte HAL
Article Dans Une Revue Plant Methods Année : 2018

User‑friendly extraction and multistage tandem mass spectrometry based analysis of lipid‑linked oligosaccharides in microalgae

Résumé

Background: Protein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipidlinkedoligosaccharides (LLO) precursor. This precursor is then transferred en bloc on neo-synthesized proteins throughthe action of the oligosaccharyltransferase giving birth to glycoproteins. The N-linked glycans bore by the glycoproteinsare then processed into oligomannosides prior to the exit of the glycoproteins from the endoplasmic reticulumand its entrance into the Golgi apparatus. In this compartment, the N-linked glycans are further maturated in complextype N-glycans. This process has been well studied in a lot of eukaryotes including higher plants. In contrast, littleinformation regarding the LLO precursor and synthesis of N-linked glycans is available in microalgae.Methods: In this report, a user-friendly extraction method combining microsomal enrichment and solvent extractionsfollowed by purification steps is described. This strategy is aiming to extract LLO precursor from microalgae.Then, the oligosaccharide moiety released from the extracted LLO were analyzed by multistage tandem massspectrometry in two models of microalgae namely the green microalgae, Chlamydomonas reinhardtii and the diatom,Phaeodactylum tricornutum.Results: The validity of the developed method was confirmed by the analysis of the oligosaccharide structuresreleased from the LLO of two xylosyltransferase mutants of C. reinhardtii confirming that this green microalga synthesizesa linear Glc3Man5GlcNAc2identical to the one of the wild-type cells. In contrast, the analysis of the oligosaccharidereleased from the LLO of the diatom P. tricornutum demonstrated for the first time a Glc2Man9GlcNAc2structure.Conclusion: The method described in this article allows the fast, non-radioactive and reliable multistage tandemmass spectrometry characterization of oligosaccharides released from LLO of microalgae including the ones belongingto the Phaeodactylaceae and Chlorophyceae classes, respectively. The method is fully adaptable for extracting andcharacterizing the LLO oligosaccharide moiety from microalgae belonging to other phyla.
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Dates et versions

hal-01945734 , version 1 (05-12-2018)

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Pierre-Louis Lucas, Rodolphe Dumontier, Corinne Loutelier-Bourhis, Alain A. Mareck, Carlos Afonso, et al.. User‑friendly extraction and multistage tandem mass spectrometry based analysis of lipid‑linked oligosaccharides in microalgae. Plant Methods, 2018, 14 (1), pp.e107. ⟨10.1186/s13007-018-0374-8⟩. ⟨hal-01945734⟩
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