title: Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics.
abstract: Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue.
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA.
authors.forename: Stephen A
authors.identifier: 0000-0001-6140-7654
authors.initials: SA
authors.lastname: Luebker
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Koepsell