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.