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

Jessica H. van Wonderen, Jason C. Crack, Marcus J. Edwards, Thomas A. Clarke, Gerhard Saalbach, Carlos Martins, Julea N. Butt (Lead Author)

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Abstract

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.

Original languageEnglish
Article number184221
JournalBBA - Biomembranes
Volume1866
Issue number1
Early online date13 Sep 2023
DOIs
Publication statusPublished - 1 Jan 2024

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

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