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.
Highlights
We recently applied a urea in-solution digestion (UISD) method to prepare protein from formalin-fixed paraffin-embedded (FFPE) tissue for liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS), providing a more efficient workflow relative to Qproteome FFPE Tissue Kit [1]
Trypsin digestion efficiency was reduced by carbamylation, and UISD had more missed cleavages and a lower lysine to arginine (K : R) peptide terminal amino acid ratio relative to the commercially available Qproteome FFPE Tissue Kit (Qkit) [1]
The benefits of UISD as a preparation method outweigh the drawback of reduced cleavage efficiency, and in this study we optimize its application to FFPE tissue
Summary
We recently applied a urea in-solution digestion (UISD) method to prepare protein from FFPE tissue for liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS), providing a more efficient workflow relative to Qproteome FFPE Tissue Kit [1]. Trypsin digestion efficiency was reduced by carbamylation, and UISD had more missed cleavages and a lower lysine to arginine (K : R) peptide terminal amino acid ratio relative to the commercially available Qproteome FFPE Tissue Kit (Qkit) [1]. Ammonium bicarbonate, included in the UISD extraction buffer, inhibits carbamylation when using urea as a protein denaturant, but its efficacy is greatly reduced above 37∘C [4, 5]. Despite having this drawback, UISD preparation detected similar numbers of peptides and proteins obtained using Qkit and some not detected by Qkit with a bias toward arginine terminal peptides. The benefits of UISD as a preparation method outweigh the drawback of reduced cleavage efficiency, and in this study we optimize its application to FFPE tissue
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