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Poster communications

ICM-MS and Top down analysis to characterize biomarker

Abstract : Intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) is a well established differential and quantitative approach to display biomarkers on prokaryotic or eukaryotic cells. ICM-MS is a fast, reproducible and sensitive method for peptidomic and proteomic studies in 2-30 kDa mass range. However, by this approach the biomarker of interest remain to be identified. Here we demonstrate the application of ICM-MS profiling coupled to Top-down analysis to characterize three avian β-defensins : AvBD1, AvBD2 and a newly isolated β-defensin AvBD7. β-defensins are important components of chicken innate immunity in mucosal tissue, a major entry site for pathogens that may cause food poisoning. Chicken inbred lines diverge phenotypically with respect to levels of pathogens intestinal carriage and to level of gene expression of two β-defensin (AvBD1, AvBD2). In order to detect the known mature β-defensins and to discover other molecular species with a potential antimicrobial activity, ICM-MS was performed on intact heterophil granulocytes, which are known to infiltrate intestinal tissue early after pathogen colonization. The in silico predicted AvBD1 and AvBD2 sequences, taking into account the three characteristic β-defensins disulfide bonds, were compared to experimental peaks. Despite the lack of precision mass in linear MALDI-TOF spectra, two peaks may correspond to AvBD1 and AvBD2. To characterize finely molecular species observed on ICM-MS profile, we purified them by chromatography from bone marrow extract, in which defensin genes have been shown to be highly expressed and that contains the heterophils precursors. All size exclusion chromatography fractions were analysed by classic bottom-up proteomic in order to identify molecular species. AvBD1 and AVBD2 and a newly isolated AvBD7 were identified. To obtain structural information, a Top-down proteomic approach was applied on RP-HPLC purified intact molecular species. By a top-down MS strategy using nano-ESI–Q-TOF MS, the AvBDs were measured with a mass accuracy of 100 ppm. By comparison with the theoretical monoisotopic mass (with 3 disulfide bonds), bone marrow AvBD2 exhibited the expected mass but AVBD1 and AvBD7 presented respectively a delta mass = -1 Da and -17 Da. Through the topdown MS/MS strategy, we confirmed respectively the C and N terminal sequences for AvBD1 and AvBD7 and characterized post-translational modifications which correspond to an amidated C-terminal and to a Gln N-terminal cyclized as a pyroglutamic acid residue. In this study, a hybrid strategy combining «ICM-MS», «bottom-up» and «top-down» proteomic approaches enabled, for the first time, characterization of AvBD1, AvBD2, and AvBD7 as well as isoforms of these defensins. The use of complementary ICM-MS – Top-down technologies allowed us to follow these structures in their biological (or cellular) context.
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Poster communications
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https://hal.inrae.fr/hal-02750200
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Submitted on : Wednesday, June 3, 2020 - 4:47:39 PM
Last modification on : Thursday, August 5, 2021 - 3:42:30 AM

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  • HAL Id : hal-02750200, version 1
  • PRODINRA : 49087

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Valérie Labas, Grégoire Harichaux, Chrystelle Derache, Thierry Magallon, Ana-Paula Teixeira-Gomes, et al.. ICM-MS and Top down analysis to characterize biomarker. SMAP 2011, Sep 2011, Avignon, France. pp.110, 2011, Spectométrie de Masse et Analyse Protéomique. ⟨hal-02750200⟩

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