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.
Ronci
Maurizio
M
Centro Studi Sull'Invecchiamento, Chieti, Italy.
Bonanno
Elena
E
Colantoni
Alfredo
A
Pieroni
Luisa
L
Di Ilio
Carmine
C
Spagnoli
Luigi Giusto
LG
Federici
Giorgio
G
Urbani
Andrea
A
D001943:Breast Neoplasms / Q000737:chemistry* / Q000473:pathology; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis*; D016612:Paraffin Embedding / Q000379:methods; D012710:Serum Albumin, Bovine; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
D009363:Neoplasm Proteins; D005557:Formaldehyde; D012710:Serum Albumin, Bovine