Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues

Abstract

Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.

Quantitative Proteomic Analysis of Formalin-fixed and Paraffin-embedded Nasopharyngeal Carcinoma Using iTRAQ Labeling, Two-dimensional Liquid Chromatography, and Tandem Mass Spectrometry

“…In 2005, a report described the successful application of shotgun proteome analysis combined with trypsin-mediated 18 O labeling to FFPE tissues of cancerous prostate lesions and benign prostate hyperplasia, opening the door to comparative proteomic analyses of FFPE tissues (Hood et al 2005). In 2007, the absolute quantitation of abundance method coupled to LC-based multiple reaction monitoring was applied to achieve picogram-level quantitation of prostate-specific antigen (Hwang et al 2007). Later, in a proteomic analysis of squamous cell carcinoma of the head and neck (HNSCC), FFPE tissue sections of normal oral epithelium and well-, moderately, and poorly differentiated HNSCCs were microdissected and processed for nano-reversed-phase liquid chromatography followed by tandem MS; KRT4, KRT16, vimentin, and desmoplakin were chosen to be validated by IHC.…”

Vascular proteomics

“…However, to overcome this heterogeneity a new methodology, termed direct tissue proteomics [20], which allows protein identification directly from formalin fixed paraffin-embebed tissue samples, has been applied to study the proteome of human coronary arteries and atherosclerotic plaques. The analysis of 35 human coronary atherosclerotic samples allowed to identify a total of 806 proteins [21].…”

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

“…However, contrary to the classical workflow, tissue section chemical treatment involved a first step of scrapping each FFPE tissue spot with a razor blade from the glass slide. The tissues were then transferred into a tube and processed with RIPA buffer and finally submitted to boiling as an AR step (Hwang et al, 2007). Afterward, several teams proved that it was possible to perform the AR directly on tissue sections.…”

“…Chaotropic agents can also be used for proteins retrieval from FFPE tissues (Guo et al, 2007). RIPA buffers were used with different amounts of NaH 2 PO 4 , NaHPO 4 , NaCl, Triton, fluoride, sodium cholate, sodium azide, and ethylenediamine tetraacetic acid (EDTA) on colorectal carcinoma (Ikeda et al, 1998), lymphoma (Crockett et al, 2005), and prostate cancer specimens (Hwang et al, 2007). Organic solvents can also be employed at different concentrations as solubilizing agents such as 30% acetonitrile, which was applied on pancreatic (Pan et al, 2011) and colon cancer tissues (Kakimoto et al, 2012).…”