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

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-MS(E) 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.