title: Improving the Proteomic Analysis of Archival Tissue by Using Pressure-Assisted Protein Extraction: A Mechanistic Approach.
abstract: Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE tissue. Our laboratory has taken a mechanistic approach to developing improved protein extraction protocols, by first studying the reactions of formaldehyde with proteins and ways to reverse these reactions, then applying this approach to a model system called a "tissue surrogate", which is a gel formed by treating high concentrations of cytoplasmic proteins with formaldehyde, and finally FFPE mouse liver tissue. Our studies indicate that elevated pressure improves the recovery of proteins from FFPE tissue surrogates by hydrating and promoting solubilization of highly aggregated proteins allowing for the subsequent reversal (by hydrolysis) of formaldehyde-induced protein adducts and cross-links. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency and up to a 30-fold increase in the number of non-redundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of non-redundant proteins identified in the FFPE tissue was nearly identical to that of the corresponding frozen tissue.
authors: Laboratory of Proteomics and Protein Science, Office of Research and Development, Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA.
authors: Carol B
authors: CB
authors: Fowler
authors: Veterans Health Administration, Washington, DC, USA.
authors: Timothy J
authors: TJ
authors: O'Leary
authors: Laboratory of Proteomics and Protein Science, Office of Research and Development, Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA.
authors: Jeffrey T
authors: JT
authors: Mason
keywords: Antigen Retrieval; FFPE; Formalin-fixed paraffin-embedded; High-pressure protein extraction; Mass spectrometry; Proteomics