Proteomic Analysis of Formalin-Fixed Paraffin Embedded (FFPE) Samples: Pitfalls and Potentials

Vladimir   A.   Valera; Beatriz   A.   Walter; W.   Marston   Linehan; David   D.   Roberts; Maria   J.   Merino

Abstract

Proteomic analysis of clinical samples has been traditionally performed using fresh or frozen tissues. However, millions of embedded samples are stored worldwide in pathology repositories with linked long-term outcome data. Such samples are believed to be unsuitable for proteome analysis because of the extensive protein modification attained during tissue processing. Formaldehyde, in the form of buffered formalin, is the most commonly used fixative during processing before embedding in paraffin. These formalin-fixed paraffin embedded (FFPE) tissue samples are widely used in pathology labs for qualitative protein analysis using immunohistochemistry (IHC). For this purpose, FFPE tissues are treated in different ways in order to reverse chemical modifications by formaldehyde that limit protein antigen detection by specific antibodies. Accordingly, intensive research efforts are now attempting to adapt the sample pretreatments used in immunohistochemistry to facilitate high throughput proteome analysis of FFPE tissues. Two such approaches have been successful: a liquid-based proteomic approach and an in situ approach, mainly based on separation and identification technologies such as liquid chromatography and mass spectrometry. These efforts have proven that similar protein profiles and numbers of polypeptides can be attained as using frozen sections. In this article, a review is provided of methods and current trends for the extraction of peptides and proteins from FFPE tissues for use proteomic analysis. Limitations in these methods and future directions to overcome these are also outlined. Ongoing improvements in protein extraction technology and analysis techniques will enable identification of novel disease biomarkers from FFPE tissues.

Keywords: Proteomics, formalin-fixed paraffin embedded tissues, antigen retrieval, immunohistochemistry, protein extraction, imaging mass spectrometry, liquid chromatography, mass spectrometry