Table of Contents
International Journal of Proteomics
Volume 2016 (2016), Article ID 4324987, 6 pages
http://dx.doi.org/10.1155/2016/4324987
Research Article

Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics

Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA

Received 20 June 2016; Accepted 9 August 2016

Academic Editor: Djuro Josic

Copyright © 2016 Stephen A. Luebker and Scott A. Koepsell. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.