Dataset Information

Optimization of a Top-Down Proteomics Platform for Closely-Related Pathogenic Bacteria Discrimination

ABSTRACT: The current technique used for microbial identification in hospitals is MALDI-TOF MS. However, it suffers from important limitations, in particular for closely-related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a high throughput LC-MS/MS top-down proteomics platform dedicated to intact bacterial protein analysis. Using Escherichia coli as a model, all steps of the workflow were optimized: protein extraction, on-line liquid chromatographic separation, MS/MS fragmentation and data analysis. Using the optimized parameters, about 220 proteins, corresponding to more than 500 proteoforms, could be identified in a single run. We then demonstrated the suitability of the developed platform for the characterization and discrimination of enterobacterial pathogens rather undistinguishable by MALDI-TOF although leading to very different clinical outcomes. For each pathogen, we identified specific proteoforms that could potentially be used as biomarkers. We also improved the characterization of poorly described bacterial strains. Our results highlight the advantage of targeting proteoforms vs peptides for accurate bacterial characterization, and qualify top-down proteomics as a promising tool in clinical microbiology.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium Salmonella Enterica Subsp. Enterica Serovar Muenchen Salmonella Enterica Subsp. Enterica Serovar Enteritidis Escherichia Coli O26:h11 Salmonella Enterica Subsp. Enterica Serovar Newport Shigella Flexneri Shigella Sonnei Escherichia Coli Escherichia Coli O157:h7

SUBMITTER: Magalie Duchateau

LAB HEAD: Julia Chamot-Rooke

PROVIDER: PXD019247 | Pride | 2020-09-24

REPOSITORIES: Pride

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Publications

Optimization of a Top-Down Proteomics Platform for Closely Related Pathogenic Bacterial Discrimination.

Dupré Mathieu M Duchateau Magalie M Malosse Christian C Borges-Lima Diogo D Calvaresi Valeria V Podglajen Isabelle I Clermont Dominique D Rey Martial M Chamot-Rooke Julia J

Journal of proteome research 20201001 1

The current technique used for microbial identification in hospitals is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, it suffers from important limitations, in particular, for closely related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a liquid chromatography (LC)-MS/MS top-down proteomics platform, which aims at discriminating closely related pathogenic bacteria through ...[more]

PMID: 32929970

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Project description:In mammals, the capacity of the female germ cell, the oocyte, to develop into embryo is acquired throughout meiotic maturation. Immature oocyte cannot be fertilized while mature oocyte is apt to accept spermatozoa and to develop an embryo. In a follicle, the oocyte is surrounded by mural granulosa cells (GC) and is physically and metabolically coupled with specialized granulosa cumulus cells (CC) which play an important role in oocyte maturation and fertilization. Factors expressing in GC and CC during maturation may reflect the oocyte quality, i.e. its capacity to be fertilized and assure early embryo development. However, the modifications of the content and the amount of peptide/proteins in the oocyte and the surrounding CC during oocyte maturation are mostly unknown and so there is not an accurate way of evaluating/monitoring how different in vitro maturation (IVM) protocols being in use in assisted reproduction technologies, can affect the process. In this context, Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) was applied to bovine follicular cells (bovine single oocytes, cumulus cells and granulosa cells) in order to characterize proteomic changes that occur in the follicle during female gamete development. In order to characterize finely endogenous molecular species observed on ICM-MS profiles and to identify markers of interest with their post-translational modifications, we carried out top-down proteomic on the different follicular cells from oocyte, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts. Prior to top-down MS using a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos, depending on the amount of available biological material, we employed three analytic strategies as a direct infusion, a mono-dimensional liquid chromatography (µLC-1D-MS/MS) and an off-line multi-dimensional liquid chromatography combining four fractionations (based on reverse phase or gel filtration LC) to µLC-MS/MS. Here, we deposited our dataset from µLC-1D-MS/MS (analyses of oocytes, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts) and MDLC-MS/MS (analyses of granulosa cells protein extract).

Dataset Information

Optimization of a Top-Down Proteomics Platform for Closely-Related Pathogenic Bacteria Discrimination

Publications

Optimization of a Top-Down Proteomics Platform for Closely Related Pathogenic Bacterial Discrimination.

Similar Datasets

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