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Updated: Jun 11, 2026
A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Published on: March 30, 2020
Citric acid antigen retrieval (CAAR) for tryptic peptide imaging directly on archived formalin-fixed
Johan O R Gustafsson1, Martin K Oehler, Shaun R McColl
1Adelaide Proteomics Centre, School of Molecular and Biomedical Science, The University of Adelaide, SA 5005, Adelaide, Australia.
Insights
We developed a new method to analyze proteins in decades-old formalin-fixed paraffin-embedded (FFPE) tissues using imaging mass spectrometry (IMS). This allows detailed proteomic analysis of archived patient samples for disease research.
Area of Science:
- Biochemistry
- Proteomics
- Pathology
Background:
- Imaging mass spectrometry (IMS) is vital for mapping molecular distributions in tissues, particularly for neoplastic diseases.
- Current IMS applications are largely restricted to fresh frozen tissue specimens.
- Analyzing formalin-fixed paraffin-embedded (FFPE) tissues is highly desirable due to extensive archival patient data.
Purpose of the Study:
- To develop a protocol for analyzing the proteome of archived FFPE tissues using IMS.
- To enable the study of long-term stored tissue samples linked with clinical information.
- To overcome the limitations of FFPE tissue cross-linking for proteomic analysis.
Main Methods:
- Developed a high-temperature citric acid buffer antigen retrieval protocol to reverse FFPE protein cross-linking.
- Utilized automated deposition of trypsin and matrix for generating peptide ion distribution images.
- Employed in situ peptide fragmentation and downstream HPLC-MS strategies for protein identification.
Main Results:
- Generated meaningful peptide ion distribution images from FFPE tissues.
- Identified high-abundance proteins like Actin and Collagen via in situ fragmentation.
- Identified up to 106 proteins using downstream HPLC-MS, with 67 correlating to IMS data from archived FFPE ovarian tissue.
Conclusions:
- The developed citric acid antigen retrieval (CAAR) method enables proteomic analysis of archived FFPE tissues.
- This method complements existing antigen retrieval protocols.
- It represents a significant advancement for comprehensive proteomic analysis of historical FFPE tissue archives.
Abstract:
Imaging mass spectrometry (IMS) is a powerful technology for mapping distributions of biological molecules like proteins and peptides within tissue sections. It is therefore potentially extremely useful for the analysis of pathological conditions such as neoplastic diseases. The use of IMS is typically limited to fresh frozen tissue specimens. However, there is a high interest in the possibility of being able to analyze the tissue proteome of formalin-fixed paraffin-embedded (FFPE) specimens that have been stored together with the clinicopathological information of patients in huge archives over many decades. We have therefore developed an antigen-retrieval protocol using a high temperature citric acid buffer to allow partial reversal of FFPE protein cross-linking. Coupled with automated deposition of trypsin and matrix, our method allows the generation of meaningful peptide ion distribution images. In situ peptide fragmentation provided identification of high abundance proteins such as Actin and Collagen. Furthermore, downstream application of three different HPLC-MS strategies allowed identification of a maximum of 106 proteins, 67 of which were mass correlated to ions from IMS analysis of archived FFPE ovarian tissue. The CAAR method presented here complements previously described antigen-retrieval protocols and is an important step in being able to fully analyze the proteome of archived FFPE tissue.
