Initial Development and Validation of a Novel Extraction Method for Quantitative Mining of the Formalin-Fixed, Paraffin-Embedded Tissue Proteome for Biomarker Investigations

Abstract

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography−mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250−300 proteins per 500 ng of tissue with 1D LC−MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10−15). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

“…Indeed, the main concern about the use of these FFPE tissues is to apply a procedure that could give rise to the largest molecular information, compared to the one available using fr/fr tissues. Generally, fr/fr tissues give a higher number of proteins compared to FFPE tissues (Giusti and Lucacchini, 2013), but some studies reported a better proteins identifications recovery with FFPE tissues (Nirmalan et al, 2011;Palmer-Toy et al, 2005;Shi et al, 2006). These comparisons highlight the importance of the chemical treatments for efficient FFPE tissue preparation.…”

“…50% trifluoroethanol (TFE) for lung tissue processing was also tested (Tian et al, 2009). Commercial solutions exist for FFPE tissue processing such as the acid-deteriorating detergent RapiGest (Nirmalan et al, 2011), Liquid Tissue MS protein Prep kit (Nakatani et al, 2012;Prieto et al, 2005), Qproteome FFPE tissue kit (Becker et al, 2007), NDME-U (Chu et al, 2005), and FFPE Protein Extraction Solution (Agilent Technologies, Santa Clara, CA). Moreover, all these treatments methods can be applied with a digestion step done on Microcon (Millipore, Billerica, MA) or Vivacon (Vivaproducts, Littleton, USA) units.…”

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini

“…Several quantitative proteomic techniques have been utilized to investigate FFPE tissues, such as trypsin-mediated 18 O labeling (2), isobaric tag for relative and absolute quantitation (iTRAQ) (39 -41), label-free quantitation (42)(43)(44)(45)(46), and chemical dimethylation (47). The successful application of iTRAQ and chemical dimethylation are especially noteworthy, as these labeling techniques target primary amines, which is of importance to N-terminal degradomic analysis.…”

Important Clues of Current Phosphoproteomic Approaches for Clinical Research

“…Technological advances such as protein/ antibody chips, depletion of multiple high abundance proteins by affinity columns, and affinity enrichment of targeted protein analytes as well as multidimensional chromatographic fractionation, have all expanded the dynamic range of detection for low abundance proteins by several orders of magnitude in serum or plasma, making it possible to detect the more abundant disease-relevant proteins in these complex biological matrices [63][64][65][66][67][68][69][70][71]. However, plasma and cell-extract based discovery research studies aimed to identify low abundance proteins (e.g.…”