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Mycobacterial proteome extraction: comparison of disruption methods
Mark D Lanigan1, Jill A Vaughan, Brian J Shiell
1Protein Biochemistry and Proteomics Group, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, Victoria, Australia. mark.lanigan@csiro.au
Abstract:
Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.
Insights
Bead beating is an effective method for disrupting hardy mycobacterial cells for proteomic analysis. This technique offers a rapid and safe alternative to sonication for Mycobacterium avium subsp. paratuberculosis research.
Area of Science:
- Microbiology
- Proteomics
- Veterinary Science
Background:
- Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants.
- Mycobacterial cells are hardy, requiring harsh lysis conditions for proteomic analysis.
Purpose of the Study:
- To compare bead beating and sonication for Mycobacterium cell lysis.
- To evaluate proteome extraction efficiency for Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis.
Main Methods:
- Cell lysis using bead beating and sonication.
- Proteomic analysis via two-dimensional gel electrophoresis (2D-PAGE).
- Comparison of protein spot counts from silver-stained gels.
Main Results:
- Both bead beating and sonication yielded similar representations of mycobacterial proteomes.
- Differences in total protein yield were observed between species and ages.
- Bead beating provided comparable proteomic data to sonication.
Conclusions:
- Bead beating is a rapid and effective method for mycobacterial proteome extraction.
- It offers a safer alternative to open-tube sonication for Mycobacterium species.
- This method facilitates proteomic studies of Johne's disease-causing bacteria.
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