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Updated: May 18, 2026
A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions
Published on: April 18, 2025
Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas
Kyoko Kojima1, Gregory J Bowersock, Chinatsu Kojima
1Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Abstract:
A number of reports have recently emerged with focus on extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D-nanoLC-MS(MS)(2) following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease.
Insights
This study validates a robust proteomics method for formalin-fixed paraffin-embedded (FFPE) tissues, showing high comparability to frozen controls. This approach enables reliable proteomic analysis of FFPE samples for disease research.
Area of Science:
- Proteomics
- Biochemistry
- Cancer Research
Background:
- Formalin-fixed paraffin-embedded (FFPE) tissues are valuable for retrospective studies, but protein extraction for mass spectrometry (MS) analysis faces challenges in reproducibility and robustness.
- Limited studies have focused on validating FFPE proteomics methods against gold-standard frozen controls.
Purpose of the Study:
- To identify and validate a practical and highly robust approach for proteomics analysis of FFPE tissues.
- To assess the comparability of FFPE proteomics data with that from matched flash-frozen tissues.
Main Methods:
- Proteomic analysis using 1D-nanoLC-MS(MS)(2) on mouse FFPE and matched frozen pancreatic tissues (n=8).
- Utilized commercially available kits for sample workup.
- Applied the validated FFPE approach to archived human FFPE pancreatic cancer specimens (n=11) and uninvolved tissues (n=8).
Main Results:
- The FFPE proteomics approach demonstrated high comparability to frozen controls, with similar numbers of unique peptides identified (958 for FFPE vs. 1070 for frozen).
- Approximately 80% protein identification overlap was observed between FFPE and frozen samples.
- Analysis of human FFPE samples identified 47 significantly increased potential pancreatic ductal adenocarcinoma markers, including 28 previously reported, with strong pathway associations to cancer, such as estrogen receptor α.
Conclusions:
- A straightforward and highly robust method for FFPE tissue proteomics analysis has been validated.
- This approach is suitable for translational studies, enabling the use of valuable archived FFPE samples for disease biomarker discovery.
