Chapter 7. Isolation and Preparation of Spore Proteins and Subsequent Characterisation by Electrophoresis and Mass Spectrometry

  1. Haroun N. Shah and
  2. Saheer E. Gharbia
  1. Nicola C. Thorne,
  2. Haroun N. Shah and
  3. Saheer E. Gharbia

Published Online: 15 JUN 2010

DOI: 10.1002/9780470665497.ch7

Mass Spectrometry for Microbial Proteomics

Mass Spectrometry for Microbial Proteomics

How to Cite

Thorne, N. C., Shah, H. N. and Gharbia, S. E. (2010) Isolation and Preparation of Spore Proteins and Subsequent Characterisation by Electrophoresis and Mass Spectrometry, in Mass Spectrometry for Microbial Proteomics (eds H. N. Shah and S. E. Gharbia), John Wiley & Sons, Ltd, Chichester, UK. doi: 10.1002/9780470665497.ch7

Editor Information

  1. Department for Bioanalysis and Horizon Technologies, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK

Author Information

  1. Department for Bioanalysis and Horizon Technologies, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK

Publication History

  1. Published Online: 15 JUN 2010
  2. Published Print: 23 JUL 2010

ISBN Information

Print ISBN: 9780470681992

Online ISBN: 9780470665497

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Keywords:

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

Summary

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.