Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting

Nirmalan, NJ; Harnden, P; Selby, PJ; Banks, RE

Abstract

The development of efficient formaldehyde cross-link reversal strategies will make the vast
diagnostic tissue archives of pathology departments amenable to prospective and retrospective
translational research, particularly in biomarker-driven proteomic investigations.
Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of crosslink
reversal, potentially enabling archival tissue usage for proteomic applications outside
its current remit of immunohistochemistry (IHC). While most successes achieved so far have
been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches,
attempts at extracting full-length, non-degraded, immunoreactive proteins from archival
tissue have proved challenging. We have developed a novel heat-induced antigen retrieval
strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from
formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization
and comparison of extraction efficacies with frozen tissues and current leader methodology
is presented. Quantitative validation of methodology was carried out in a cohort of matched
tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting
for a selected panel of seven proteins known to be differentially expressed in renal
cancer. Our data show that the protocol enables efficient extraction of non-degraded, fulllength,
immunoreactive protein, with tumour versus normal differential expression profiles
for a majority of the panel of proteins tested being comparable to matched frozen tissue
controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in
extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation
of quantitative data from this subset of proteins. The method provides a viable,
cost-effective quantitative option for the validation of potential biomarker panels through a
range of clinical samples from existing diagnostic archives, provided that validation of the
method is first carried out for the specific proteins under study.

Citation

Nirmalan, N., Harnden, P., Selby, P., & Banks, R. (2009). Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting. The Journal of pathology, 217(4), 497-506. https://doi.org/10.1002/path.2504

• Journal Article Type: Article
• Publication Date: Jan 1, 2009
• Deposit Date: Dec 20, 2011
• Journal: The Journal of Pathology
• Print ISSN: 1096-9896
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 217
• Issue: 4
• Pages: 497-506
• DOI: https://doi.org/10.1002/path.2504
• Publisher URL: http://dx.doi.org/10.1002/path.2504