Isolation and Preparation of Spore Proteins and Subsequent Characterisation by Electrophoresis and Mass Spectrometry

Nicola C. Thorne*, Haroun N. Shah, Saheer Gharbia

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

The critical stage of sample preparation in bacterial spore proteomics, particularly in the case of Clostridium difficile, is the step of purifying spores from vegetative cells, germinating spores and cellular debris, which seems to be more of a challenge for C. difficile as previous methods used for Bacillus spores do not appear to be rigorous enough. Enzymatic lysis of vegetative cells or lysis with cold water washing, heat, etc., releases large concentrations of cellular debris that requires subsequent thorough removal so that the starting material for protein extraction and analysis contains only free spores. Thereafter many varied methods are available for the solubilisation of proteins of the spore coat without the need to disrupt the spores themselves, or mechanical methods have been employed previously to crack open the spores to analyse the full spore proteome or in certain cases to extract the small acid soluble proteins (SASPs) located in the spore's core. Proteins in the different spore layers are likely to have different solubility characteristics and so the best approach is to use multiple methods aimed at solubilising these different protein fractions to get the best coverage of the sample in mass spectral analysis.

Original languageEnglish
Title of host publicationMass Spectrometry for Microbial Proteomics
PublisherJohn Wiley and Sons
Pages143-156
Number of pages14
ISBN (Print)9780470681992
DOIs
Publication statusPublished - 15 Jun 2010

Keywords

  • B. subtilis, highly amenable to genetic manipulation
  • Bacterial spores of Clostridia
  • Bacterial spores, vehicles for widespread contamination in environments
  • C. difficile, major cause of hospital acquired infections
  • Pressure cycling technology-and protein extraction
  • Sonication, commonly used method-pulsed, high frequency sound waves shearing bacterial cells and spores
  • Spore protein extraction and solubilisation
  • Spore protein isolation, preparation and electrophoresis and mass spectrometry characterisation
  • Spores formed via 'sporulation'-Sporulation cascade of B. subtilis

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