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Preserving the yeast proteome from sample degradation

Authors

  • Julia Grassl,

    1. UCD Conway Institute of Biomolecular and Biomedical Research, School of Biomolecular and Biomedical Science, University College Dublin, Ireland
    Current affiliation:
    1. ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley WA 6009, Perth, Australia
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  • Jules A. Westbrook,

    1. Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Ireland
    Current affiliation:
    1. Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, LS9 7TF, UK
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  • Aisling Robinson,

    1. Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Ireland
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  • Mats Borén,

    1. Denator AB, Biotech Center, Gothenburg, Sweden
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  • Michael J. Dunn,

    1. Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Ireland
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  • Rosemary K. Clyne

    Corresponding author
    1. UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland
    • UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland Fax: +353-1-716-6701
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Abstract

Sample degradation is a common problem in all types of proteomic analyses as it generates protein and peptide fragments that can interfere with analytical results. An important step in preventing such artefacts is to preserve the native, intact proteome as early as possible during sample preparation prior to proteomic analysis. Using the budding yeast Saccharomyces cerevisiae, we have evaluated the effects of trichloroacetic acid (TCA) and thermal treatments prior to protein extraction as a means to minimise proteolysis. TCA precipitation is commonly used to inactivate proteases; thermal stabilisation is used to heat samples to approximately 95°C to inactivate enzyme activity. The efficacy of these methods was also compared with that of protease inhibitors and lyophilisation. Sample integrity was assessed by 2-D PAGE and a selection of spots was identified by MS/MS. The analysis showed that TCA or thermal treatment significantly reduced the degree of degradation and that these pre-treatment protocols were more effective than treatment with either protease inhibitors or lyophilisation. This study establishes standardised sample preparation methods for the reproducible analysis of protein patterns by 2-D PAGE in yeast, and may also be applicable to other proteomic analyses such as gel-free-based quantitation methods.

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