Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues face challenges due to inefficient protein extraction and limited understanding of formaldehyde-induced protein modifications.
• Previous work introduced 'tissue surrogates' as a model system to study FFPE processing and protein retrieval.

Purpose of the Study:

• To demonstrate the efficacy of high hydrostatic pressure for efficient protein recovery from FFPE tissue surrogates.
• To investigate the reversal of formaldehyde-induced protein modifications under high pressure conditions.
• To optimize pressure-based extraction for improved proteomic analysis of FFPE samples.

Main Methods:

• Utilized lysozyme tissue surrogates subjected to high hydrostatic pressure (45,000 psi) at elevated temperatures (80-100°C).
• Employed Tris buffers with sodium dodecyl sulfate and glycine at pH 4, with additives like trimethylamine N-oxide or copper (II) chloride.
• Analyzed recovered proteins using mass spectrometry to assess sequence coverage and formaldehyde adducts.

Main Results:

• High hydrostatic pressure treatment (45,000 psi, 80-100°C) effectively reversed formaldehyde-induced protein adducts and crosslinks in FFPE tissue surrogates.
• Optimized conditions yielded 70% sequence coverage of lysozyme, identified all formaldehyde-reactive amino acids, and confirmed trypsin cleavage site hydrolysis.
• Additives reduced acid-catalyzed aspartic acid cleavage while maintaining efficient crosslink reversal.

Conclusions:

• Elevated hydrostatic pressure is a promising method for enhancing protein recovery from FFPE tissues.
• This technique improves the quality and quantity of proteins available for proteomic analysis.
• The findings offer a new strategy for analyzing archival FFPE samples.