Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations
Nirmalan, NJ, Hughes, C, Peng, J, McKenna, T, Langridge, J, Cairns, DA, Harnden, P, Selby, PJ and Banks, RE 2011, 'Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations' , Journal of Proteome Research, 10 (2) , pp. 896-906.
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Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography-mass spectrometry- based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250-300 proteins per 500 ng of tissue with 1D LC-MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/ normal differential expression patterns (205 proteins, r ) 0.682; p < 10-15). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.
|Themes:||Subjects outside of the University Themes|
|Schools:||Colleges and Schools > College of Science & Technology > School of Environment and Life Sciences > Ecosystems and Environment Research Centre|
|Journal or Publication Title:||Journal of Proteome Research|
|Publisher:||American Chemical Society|
|Depositing User:||Users 29196 not found.|
|Date Deposited:||20 Dec 2011 15:49|
|Last Modified:||28 Jul 2014 11:25|
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