Protein unlocking procedures of formalin‐fixed paraffin‐embedded tissues: Application to MALDI‐TOF Imaging MS investigations

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Abstract
Archival formalin‐fixed paraffin‐embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI‐TOF imaging MS (MALDI‐IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo‐preserved tissues. We have developed an in vitro approach using “tissue surrogate” samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI‐TOF MS and nLC‐MSE analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI‐IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.