title: Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting.
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 cross-link 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, full-length, 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.
authors.affiliation: Clinical and Biomedical Proteomics Group, Cancer Research UK Clinical Centre, Leeds, UK.
authors.forename: Niroshini J
authors.initials: NJ
authors.lastname: Nirmalan
authors.forename: Patricia
authors.initials: P
authors.lastname: Harnden
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Selby
authors.forename: Rosamonde E
authors.initials: RE
authors.lastname: Banks
mesh_terms: D000199:Actins / Q000032:analysis; D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D002292:Carcinoma, Renal Cell / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007680:Kidney Neoplasms / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification; D016612:Paraffin Embedding; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics; D015203:Reproducibility of Results; D016707:Tissue Fixation
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D008565:Membrane Proteins; D011506:Proteins