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Development of Top-Down Mass Spectrometry Approaches for the Analysis of Type IV Pili

Abstract : Top-down mass spectrometry (TDMS) is an alternative protein characterisation strategy to the more widespread bottom-up (BU) approach. TDMS has the unique ability to fully characterise the variety of protein products expressed by the cell (proteoforms), including those bearing posttranslational modifications (PTMs). In this thesis TDMS has been developed on both FT-ICR and Orbitrap mass spectrometers for the analysis of type IV pili (T4P). This includes the first T4P to be visualised in a Gram positive bacterium (Streptococcus pneumoniae). T4P are filamentous, extracellular organelles primarily composed of a single protein subunit or major pilin that can be highly posttranslationally modified. For the major pilin, PilE, of the human pathogen Neisseria meningitidis (Nm), TDMS was extensively optimised and the first complete characterisation of all proteoforms of PilE from a single Nm strain performed. A biological role has been proposed for the enigmatic phosphoglycerol PTM. The approach was extended and applied in the first large scale study of PTMs on PilE from uncharacterised, pathogenic strains of Nm. Comparison of the TD and BU methodologies revealed both their complementarity and the inherent weakness of the BU approach for full proteoform characterisation. TDMS was combined with other structural techniques to reveal that pilins from the previously unstudied class II isolates of Nm are extensively glycosylated and that glycosylation is both driven by the primary structure of PilE and has a profound effect on pilus surface topology. These observations have been used to offer the first explanation of how T4P expressed by class II isolates of Nm avoid immune detection.
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Contributor : Joseph Gault Connect in order to contact the contributor
Submitted on : Monday, May 5, 2014 - 12:17:10 PM
Last modification on : Thursday, April 7, 2022 - 10:10:30 AM
Long-term archiving on: : Monday, April 10, 2017 - 7:09:14 PM


  • HAL Id : pastel-00987029, version 1



Joseph Frederick Gault. Development of Top-Down Mass Spectrometry Approaches for the Analysis of Type IV Pili. Analytical chemistry. Ecole Polytechnique X, 2013. English. ⟨pastel-00987029⟩



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