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Updated: Jul 2, 2026
Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples
Published on: June 29, 2022
Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS
Maurizio Ronci1, Elena Bonanno, Alfredo Colantoni
1Centro Studi Sull'Invecchiamento, Chieti, Italy.
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-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.
Insights
Archival formalin-fixed paraffin-embedded (FFPE) tissues can yield valuable proteomic data. A new method using heat-induced antigen retrieval and on-target trypsin hydrolysis improves peptide recovery for mass spectrometry analysis.
Area of Science:
- Biochemistry
- Proteomics
- Histology
Background:
- Archival formalin-fixed paraffin-embedded (FFPE) tissues are rich in clinical and biological information.
- Protein profiling using MALDI-TOF imaging MS (MALDI-IMS) is influenced by tissue preparation.
- Previous studies primarily used cryo-preserved tissues, limiting FFPE utility.
Purpose of the Study:
- To develop and optimize protein extraction protocols for FFPE tissues.
- To enable proteomic investigation of archival FFPE specimens.
- To improve peptide recovery for mass spectrometry (MS) analysis.
Main Methods:
- Developed an in vitro approach using
- tissue surrogate
- samples to test protein unlocking procedures.
- Compared protocols using MALDI-TOF MS and nLC-MS(E) on surrogate and human breast cancer FFPE samples.
- Applied optimized protocols for FFPE tissue section preparation for MALDI-IMS.
Main Results:
- A two-step procedure combining heat-induced antigen retrieval (HIAR) with EDTA and on-target trypsin hydrolysis was effective.
- Valuable peptide mass spectra profiles were obtained from FFPE preparations.
- Multivariate statistical evaluation correlated molecular distributions with tissue morphology.
Conclusions:
- The developed protocol enables valuable peptide recovery from FFPE tissues.
- This method enhances the utility of archival FFPE samples for proteomic analysis.
- Optimized FFPE preparation is crucial for successful MALDI-IMS.
