Proteomic Analysis of Formalin Fixed Tissue
Article first published online: 14 APR 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
PROTEOMICS - Clinical Applications
Special Issue: Proteomic Analysis of Formalin Fixed Tissue
Volume 7, Issue 3-4, pages 215–216, April 2013
How to Cite
(2013), Proteomic Analysis of Formalin Fixed Tissue. Prot. Clin. Appl., 7: 215–216. doi: 10.1002/prca.201370024
- Issue published online: 14 APR 2013
- Article first published online: 14 APR 2013
Formalin fixed paraffin embedded (FFPE) clinical archives constitute a major, hitherto unexplored tissue repository for retrospective biomarker discovery. Conventional proteomic technologies have failed to characterise this cohort of proteins that have been subjected to prolonged, irreversible fixation by formaldehyde cross-linking. Recent years have seen considerable advances in the development of novel methodologies to overcome this hurdle.
High temperature antigen retrieval concepts borrowed from immunohistochemistry have been successfully applied to formalin fixed tissues to achieve significant improvements in protein retrieval and analysis. In this issue Fu et al. report on improvements in protein extraction using a high temperature and high pressure combination. Improvements in commercial detergent quality, more significantly in relation to compatibility with mass spectrometry have enabled direct analytic options, minimising detergent clean-up steps and the inevitable protein loss associated with this process.
Incorporation of micro-dissection techniques have significantly improved the homogeneity of the tissues analysed, impacting positively on the validity of the inferences. Wisniewski et al. report significant improvements in the depth of mining using a combined laser capture microdissection and LC-MS/MS approach on a quadrupole orbitrap mass spectrometer. A novel, direct tissue analysis workflow combining on-tissue protein digestion and liquid micro-extraction reported here by Wisztorski et al. yielded a total of 983 and 792 unique protein groups in benign and malignant regions of tubo-ovarian cancer samples.
The pre-analytic variables associated with sample collection, processing and storage have been recognized as potential sources of error that could significantly confound inferences drawn from this cohort of samples. Definition of these variables and their influence on the FFPE proteome needs be stringently characterised to ensure the validity of this approach. In this issue, Thompson et al. review the potential impact of the effects of pre-analytic variables on experimental findings. Addis et al. report the application of 2D-DIGE techniques to archival samples. They conclude that while method could prove a useful tool to interrogate the archival proteome, the loss of pattern complexity in comparison to the fresh frozen control tissue samples is likely to be due to pre-analytic variables.
While novel extraction and analytic protocols have permitted a deeper mining of the FFPE proteome, there is as yet insufficient evidence regarding reproducibility of these methods. Sha-Rong et al. review FFPE protein extraction strategies and highlight the need for protocol standardisation to ensure quantitative and qualitative reproducibililty. A significant drawback that needs to be addressed in this respect would be the development of robust quantitative proteomic approaches. Craven et al. report on the efficacy of label-free quantitative approaches to deduce the effects of storage time on FFPE renal carcinoma samples. A label-free LC-MS/MS approach is also reported by Gamez-Pozo et al. to be effective in the quantitative identification of over 1000 protein groups in a cohort of triple negative breast cancer tissue samples.
The development of robust quantitative strategies will enable researchers to comparatively analyse methodological advances and define accurately the effects of pre-analytic variables on the FFPE proteome. Yet another aspect largely ignored in current studies centres around the ethical implications surrounding the use archival tissue resources. With rapid advances in protein extraction and identification methodologies, the time is now ripe to take a wider view of the analytic process, if we are to maximise outputs in this field of research. Furthermore, such an overview would be critical to ensure the development of standardised guidelines for prospective FFPE tissue banks and enable the wider use of the important resource in the future.
Jennifer E. Van Eyk