title: Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.
abstract: BACKGROUND
  Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.
  
  
  RESULTS
  In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.
  
  
  CONCLUSIONS
  These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.
authors.affiliation: Department of Biophysics, Armed Forces Institute of Pathology, Rockville, Maryland, United States of America. carol.fowler1@us.army.mil
authors.forename: Carol B
authors.initials: CB
authors.lastname: Fowler
authors.forename: Ingrid E
authors.initials: IE
authors.lastname: Chesnick
authors.forename: Cedric D
authors.initials: CD
authors.lastname: Moore
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: O'Leary
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Mason
mesh_terms: D000818:Animals; D002645:Chickens; D003432:Cross-Linking Reagents / Q000737:chemistry; D004529:Egg White / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D009113:Muramidase / Q000737:chemistry; D010232:Paraffin / Q000737:chemistry*; D016612:Paraffin Embedding / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D005404:Fixatives; D011506:Proteins; D005557:Formaldehyde; D010232:Paraffin; D009113:Muramidase