Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

Abstract

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)-embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT-embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis.

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

“…Proteins from sample paraffin tissue blocks were extracted using the protocols established previously (17)(18). The protein concentration was measured by D c protein assay (Bio-Rad).…”

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

“…The extracted RNA from formalin-fixed paraffin-embedded tissue is used for quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) (Castiglione et al 2007), DNA array technologies (Lehmann and Kreipe 2001), and mRNA copy number analysis (von Smolinski et al 2005). Protein expression in formalin-fixed paraffin-embedded tissue has been analyzed by immunohistochemistry, western blot analysis and mass spectrometry (Crockett et al 2005;Ostasiewicz et al 2010;Scicchitano et al 2009). Gene and protein expression profiles of formalin-fixed paraffin-embedded tissue can provide insights into molecular mechanisms of diseases, but there are difficulties because of the marked degradation and cross-linking of biomolecules by fixation.…”

Comprehensive proteome analysis of fresh frozen and optimal cutting temperature (OCT) embedded primary non-small cell lung carcinoma by LC–MS/MS

“…However, the polymers in OCT medium (i.e., PVA and PEG) interfere with MS analysis by suppressing ion formation [7], and thus need to be removed from samples. A few proteomic studies have been conducted in OCTembedded tissues [8][9][10][11][12]. In some studies, OCT was cleared in subsequent experimental steps (e.g., any separation approach that involved running polyacrylamide gels) [8,9,12].…”

“…In some studies, OCT was cleared in subsequent experimental steps (e.g., any separation approach that involved running polyacrylamide gels) [8,9,12]. While in other studies, the number of proteins identified was compromised due to incomplete elimination of OCT medium [10,11]. One challenge associated with fresh frozen tissues is that sample size/amount may be limiting and finite.…”