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

Stephen A. Luebker, Scott A Koepsell

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Article number4324987
JournalInternational Journal of Proteomics
Volume2016
DOIs
StatePublished - Jan 1 2016

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Firearms
Paraffin
Proteomics
Formaldehyde
Urea
Tissue
Proteins
Workflow
Temperature
Buffers
Electrospray ionization
Thiourea
Peptides
Electrospray Ionization Mass Spectrometry
Liquid chromatography
Tandem Mass Spectrometry
Chemical analysis
Liquid Chromatography
Mass spectrometry
Digestion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics. / Luebker, Stephen A.; Koepsell, Scott A.

In: International Journal of Proteomics, Vol. 2016, 4324987, 01.01.2016.

Research output: Contribution to journalArticle

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