Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

Abstract

BackgroundProteomic 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.Principal FindingsIn 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.ConclusionsThese 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.

Improving the Proteomic Analysis of Archival Tissue by Using Pressure- Assisted Protein Extraction: A Mechanistic Approach

“…RNase A and lysozyme, which have a high number of formaldehyde-reactive residues, were identified with sequence coverage comparable to the native protein mixture (59 and 69% sequence coverage, respectively) at pH 4 at 40,000 psi. Myoglobin, which was included as a low-abundance component, was identified by 2 or more fully tryptic peptides in the pressure-extracted multi-protein FFPE tissue surrogates [12]. Thus, we demonstrated that heat augmented with high pressure improved the total recovery of protein and eliminated the extraction bias seen at atmospheric pressure.…”

“…Further, some studies report high rates of false-positive protein identification and are limited to the analysis of tryptic digests of FFPE tissues by LC/MS. To develop improved extraction protocols for FFPE tissue, our laboratory has taken a mechanistic approach, by first studying the reactions of formaldehyde with proteins and ways to reverse these reactions [9], 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 [10-12], and finally FFPE mouse liver tissue [13]. …”

“…By SDS-PAGE, the high-pressure extracted tissue surrogate samples (Figure 3, lanes 2 and 3) consisted of a number of well-resolved bands with the same mobility as the unfixed component proteins (Figure 3, lane 1). In contrast, only lower molecular species were extracted at low (atmospheric) pressure (Figure 3, lane 4) [12]. …”

“…For example, the LC/MS trace of the tryptic digests of the surrogates extracted at atmospheric pressure with heat at pH 4 or 8 showed a number of broad, late eluting peaks, suggesting that the material was either poorly digested, or remained cross-linked (Figure 4, panel C, pH 8). There were only a total of 5 correctly identified spectra, representing 2 unique peptides (false ID rate of 42%), for the surrogate extracted at pH 4 and ambient pressure, and no correctly identified tryptic peptides for the surrogate extracted at pH 8 and ambient pressure (Tables 1 and 2) [12]. In contrast, the surrogates extracted with heat and elevated pressure (Figure 4, panel B) compared favorably with the corresponding native, unfixed protein mixture (Figure 4, panel A).…”

Advancing formaldehyde cross-linking towards quantitative proteomic applications

“…MALDI-IMS is an imaging technique in which MS analysis is performed directly on tissue sections and not only enables identification of proteins but also provides information about their location within the tissue. In essence, MALDI-IMS enables visualisation of proteins and [68] works in an unbiased way, thereby complementing classical immunohistological approaches, which are restricted to specific proteins. Because of the nature of this method, all steps leading to MS analysis are performed on the slide itself, which means antigen retrieval must be performed gently, directly followed by protease digestion.…”