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Proteomic Protocols for the Study of Filamentous Fungi

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Laboratory Protocols in Fungal Biology

Part of the book series: Fungal Biology ((FUNGBIO))

Abstract

In the last few years, proteomics has experienced rapid improvement in technologies and applications. Gel-based strategies have become the method of choice for both identification and quantification of proteins in most studies. The workflow of a standard gel-based proteomic experiment includes experimental design, sampling, protein extraction, protein separation, mass spectrometry analysis, protein identification, data statistical analysis, validation of the identification, quantification, and data analysis. The appropriate protocol to be used depends on and must be optimized for the biological system (i.e., fungal species, plant species, organ, tissue, cells). Preliminary steps are relevant. The choice of a good extraction protocol in a proteomic experiment is crucial because only if you can extract and solubilize a protein you have a chance of detecting and identifying it. This is more important in the case of filamentous fungi, which, owing to their particular cellular characteristics, can be considered recalcitrant biological material, making it difficult to obtain quality protein samples to proteomic analysis.

Fungi have an exceptionally robust cell wall, consisting largely of chitin, which makes up the majority of the cell mass. Because of its rigidity, cell lysis is an important element in fungal proteomics. For protein extraction, various buffer- and precipitation-based protocols are available. In most of these protocols, trichloroacetic acid (TCA) and/or acetone are used for protein precipitation, or a phenol extraction is made, where proteins are solubilized in the phenolic phase and then are precipitated with methanol and ammonium sulfate. These methods also eliminate some contaminants abundant in fungal material (such as polysaccharides, lipids, nucleic acids, or phenolic compounds) that affect the protein isoelectrofocusing and electrophoresis processes.

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Notes

  1. 1.

     Broad Institute Database, http://broadinstitute.org/­science/project/fungal-genomeinitiative

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Acknowledgments

This work was supported by the Spanish Ministry of Science and Innovation (BotBank Project, EUI2008-03686), the Regional Government of Andalusia (Junta de Andalucía) and the University of Córdoba (AGR-0164: Agricultural and Plant Biochemistry and Proteomics Research Group).

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  1. Department of Biochemistry and Molecular Biology, University of Córdoba, Agro-forestry and Plant Biochemistry and Proteomics Research Group, Ed. Severo Ochoa, planta baja. Campus de Rabanales, Córdoba, 14071, Spain

    Raquel González Fernández & Jesús V. Jorrín Novo

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  1. Raquel González Fernández
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Correspondence to Raquel González Fernández .

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  1. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Vijai Kumar Gupta

  2. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Maria G. Tuohy

  3. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Manimaran Ayyachamy

  4. , Manufacturing Sciences and Technology, Pfizer Ireland Pharmaceuticals, The Pfizer Biotech Campus at Grange Castle, Clondalkin, N/A, Dublin, Ireland

    Kevin M. Turner

  5. , School of Natural Sciences, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Anthonia O’Donovan

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Fernández, R.G., Novo, J.V.J. (2013). Proteomic Protocols for the Study of Filamentous Fungi. In: Gupta, V., Tuohy, M., Ayyachamy, M., Turner, K., O’Donovan, A. (eds) Laboratory Protocols in Fungal Biology. Fungal Biology. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-2356-0_24

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