Toward Cadaveric Proteomics: A Theoretical Framework for Mass Spectrometry Analysis of Embalmed Human Tissues.

Abstract

Mass spectrometry-based proteomics has been applied with considerable success to the analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, and this success raises the question of whether the same methods can be applied to the embalmed human cadaveric specimens that are used in anatomical education and research. In this review, a theoretical framework is presented for the application of proteomic methods to embalmed cadaveric tissues, in which the feasibility of the approach is evaluated on the basis of the methods that have been established for FFPE tissue, and the critical gaps in the evidence are identified. Four questions are addressed: which of the methods that have been established for FFPE tissue can be transferred to embalmed tissue; how the post-mortem interval and the chemistry of the embalming fluid limit that transfer; which acquisition and quantification strategies are suited to proteins that carry a heavy burden of chemical modification; and which applications are already established in preserved human tissue and which of them still require validation in embalmed specimens. A comprehensive review of the literature was carried out on the proteomic methods for FFPE tissue, on the kinetics of post-mortem protein degradation, on the chemistry of embalming, and on the strategies for the reversal of crosslinks. Studies of post-mortem protein degradation place the critical threshold for embalming at 24 h if protein integrity is to be maintained; preservation is best when embalming takes place within 6-12 h. Embalming with formaldehyde produces extensive crosslinks between the proteins that are similar to those of the FFPE processing, and this suggests that the strategies for the reversal of the crosslinks that have been established for FFPE tissue may be compatible with embalmed tissue. The alternative methods of embalming, which include the ethanol-based and the salt-based approaches, may offer a better preservation of the proteins. However, the extended periods of preservation and the multi-component chemical environment of cadaveric embalming present challenges that exceed those of the clinical FFPE specimens. No published study has yet carried out a full proteomic analysis of an embalmed human cadaver. The rates of protein identification and the effectiveness of the processing remain theoretical projections that require experimental validation. Although the theoretical considerations suggest that the approach is feasible, there are significant gaps in the evidence between the established methods for FFPE tissue and the requirements of cadaveric specimens. The framework identifies the priority areas for experimental validation, which are the optimization of the reversal of the crosslinks, the assessment of the recovery of proteins with the different methods of embalming, and the development of specialized protocols for processing. The successful development of cadaveric proteomics could improve anatomical education through the characterization of the tissues at the molecular level, could support forensic applications, and could enable biomedical research on human specimens that are well characterized. However, extensive experimental validation will be required before what is possible in theory can be turned into a method that is used in practice.