Protein extraction of formalin-fixed, paraffin-embedded tissue enables robust proteomic profiles by mass spectrometry.
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.
Scicchitano
Marshall S
MS
Department of Safety Assessment, 709 Swedeland Road, Mail Stop UE0364, King of Prussia, PA 19406, USA. Marshall.2.Scicchitano@gsk.com
Dalmas
Deidre A
DA
Boyce
Rogely W
RW
Thomas
Heath C
HC
Frazier
Kendall S
KS
D000818:Animals; D000069059:Atorvastatin; D015415:Biomarkers / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D005404:Fixatives; D005557:Formaldehyde; D006538:Heptanoic Acids / Q000494:pharmacology; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D016612:Paraffin Embedding; D020543:Proteome / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D005404:Fixatives; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D020543:Proteome; D011758:Pyrroles; D005557:Formaldehyde; D000069059:Atorvastatin