Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis

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 mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.

Figure 1

1D SDS-PAGE of fresh-frozen and FFPE mouse liver extracts: lane 1, fresh-frozen tissue (protocol-1FI); lane M, molecular weight marker; lane 2, FFPE tissue extracted with heat at 40,000 psi (protocol-1P); lane 3, FFPE tissue extracted with heat alone (protocol-1A).

Figure 2

Gene ontology analysis of proteins identified by LC-MS/MS. Proteins identified using fresh-frozen mouse liver (protocol-1FI) or FFPE liver extracted with heat and elevated pressure (protocol-1P) were categorized by subcellular localization (A) or biological process (B), using GoMiner gene ontology software.

Figure 3

Venn diagrams showing the number of unique and common proteins identified using LC MS/MS analysis: panel A, fresh-frozen tissue extracted on ice (protocol-1FI) or with heat (protocol-1FH); panel B, fresh-frozen tissue extracted with heat (protocol-1FH) and FFPE mouse liver extracted with elevated pressure (protocol-1P); panel C, fresh-frozen tissue extracted with heat (protocol-2FH) and FFPE mouse liver extracted with elevated pressure (protocol-2P).

Figure 4

Total number of unique proteins identified using LC-MS/MS by two or more unique, fully tryptic peptides in FFPE mouse tissue extracted with heat and elevated pressure (40,000 psi): A, 30-day-old FFPE mouse liver (protocol-1P); B, 1-year-old FFPE mouse liver (protocol-2P).