Liquid-chromatography mass spectrometry describes post-translational modification of Shewanella outer membrane proteins

van Wonderen, Jessica H., Crack, Jason C., Edwards, Marcus J., Clarke, Thomas A. ORCID:, Saalbach, Gerhard, Martins, Carlos and Butt, Julea N. ORCID: (2024) Liquid-chromatography mass spectrometry describes post-translational modification of Shewanella outer membrane proteins. BBA - Biomembranes, 1866 (1). ISSN 0005-2736

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Electrogenic bacteria deliver excess respiratory electrons to externally located metal oxide particles and electrodes. The biochemical basis for this process is arguably best understood for species of Shewanella where the integral membrane complex termed MtrCAB is key to electron transfer across the bacterial outer membranes. A crystal structure was recently resolved for MtrCAB from S. baltica OS185. However, X-ray diffraction did not resolve the N-terminal residues so that the lipidation status of proteins in the mature complex was poorly described. Here we report liquid chromatography mass spectrometry revealing the intact mass values for all three proteins in the MtrCAB complexes purified from Shewanella oneidensis MR-1 and S. baltica OS185. The masses of MtrA and MtrB are consistent with both proteins being processed by Signal Peptidase I and covalent attachment of ten c-type hemes to MtrA. The mass of MtrC is most reasonably interpreted as arising from protein processed by Signal Peptidase II to produce a diacylated lipoprotein containing ten c-type hemes. Our two-step protocol for liquid-chromatography mass spectrometry used a reverse phase column to achieve on-column detergent removal prior to gradient protein resolution and elution. We envisage the method will be capable of simultaneously resolving the intact mass values for multiple proteins in other membrane protein complexes.

Item Type: Article
Additional Information: Funding information: This research was funded by the U.K. Biotechnology and Biological Sciences Research Council (BBSRC), grant numbers BB/S002499/1 and BB/P01819X/1. Data Availability Statement: Mass spectrometry proteomics results are presented in the ProteomeXchange Consortium via the Proteomics IDEntifications (PRIDE) partner repository with dataset identifier PXD044260 and 10.6019/PXD044260. Datasets used to make figures are deposited at Figshare (DOI: 10.6084/m9.figshare.21725531). For the purposes of open access, the author has applied a Creative Commons Attribution (CC BY) license to any author accepted manuscript version arising.
Uncontrolled Keywords: integral membrane protein,mass spectrometry,detergent,lipidation,integral membrane protein;intact mass analysis,electromicrobiology,detergent,electromicrobiology,integral membrane protein,lipidation,intact mass analysis,mass spectrometry,biophysics,biochemistry,cell biology,2* ,/dk/atira/pure/subjectarea/asjc/1300/1304
Faculty \ School: Faculty of Science > School of Biological Sciences
Faculty of Science > School of Chemistry
UEA Research Groups: Faculty of Science > Research Groups > Centre for Photonics and Quantum Science
Faculty of Science > Research Groups > Molecular Microbiology
Faculty of Science > Research Groups > Energy Materials Laboratory
Faculty of Science > Research Groups > Chemistry of Light and Energy
Faculty of Science > Research Groups > Chemistry of Life Processes
Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry
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Depositing User: LivePure Connector
Date Deposited: 01 Sep 2023 09:30
Last Modified: 28 Sep 2023 08:30
DOI: 10.1016/j.bbamem.2023.184221


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