Novel methods for proteomics analysis of formalin-fixed, paraffin-embedded tissues (FFPE), and their application for biomarker discovery

A wealth of information on proteins involved in many aspects of disease is encased within formalin-fixed paraffin-embedded (FFPE) tissue repositories stored in hospitals worldwide. However, FFPE protein extracts, as described to date, often exhibit a low pattern complexity, and a poor suitability for downstream gel-based proteomic techniques. Thus, an optimised method for extraction of full-length proteins from FFPE tissues was developed. The results obtained analysing FFPE muscle, liver, and thyroid extracts, by GeLC-MS/MS, western immunoblotting, protein arrays, and ELISA, are presented and discussed. Moreover, 2D-PAGE-MS and 2D-DIGE-MS analyses of proteins extracted from fixed skeletal muscle and liver tissues are reported. Finally, the application of 2D-DIGE-MS and GeLC-MS/MS for differential proteomic investigation of FFPE diseased samples was pursued. First, a proteomic comparison between sheep pathological liver samples was carried out, and several stress biomarkers were detected. Subsequently, a thorough biomarker discovery study was conducted, analysing human lung neuroendocrine cancer tissues. GeLC-MS/MS analysis of 3 typical carcinoid and 3 small cell lung carcinoma cases led to the identification of over 400 unique proteins per disease class. According to statistical analysis, a panel of over 30 differentially expressed putative biomarkers is presented. In addition, also 2D-DIGE-MS data clearly support biomarkers identification.