title: Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates.
abstract: High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of 'tissue surrogates' as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80-100 degrees C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.
authors.affiliation: Department of Biophysics, Armed Forces Institute of Pathology, Rockville, MD 20850, USA.
authors.forename: Carol B
authors.initials: CB
authors.lastname: Fowler
authors.forename: Robert E
authors.initials: RE
authors.lastname: Cunningham
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Waybright
authors.forename: Josip
authors.initials: J
authors.lastname: Blonder
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: O'Leary
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Mason
mesh_terms: D002021:Buffers; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D013058:Mass Spectrometry; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification*; D016612:Paraffin Embedding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013696:Temperature; D013997:Time Factors; D014015:Tissue Banks
chemical_list: D002021:Buffers; D005404:Fixatives; D011506:Proteins; D005557:Formaldehyde; D009113:Muramidase