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.

Recent advances and opportunities in proteomic analyses of tumour heterogeneity

“…Formalin fixation significantly modifies proteins and nucleic acids, producing protein-protein (lysines and backbone amides), protein-DNA and DNA-DNA cross-links, which collectively have influenced much of the scientific community's focus on analyses of fresh tissues to avoid these prospectively confounding factors [22,23]. However, our group and others have shown that comparative analyses of archival FFPE versus fresh-frozen tissues with quantitative bottom-up proteomic techniques reveal negligible differences in peptide identification [24][25][26][27][28] or quantification [29,30], suggesting that modifications induced by formalin fixation have a marginal impact on peptide yields relative to frozen tissue. Also, techniques have been devised to facilitate investigation of tissue proteomes extracted from routine FFPE tissue sections in a standardized and multiplexed fashion [26].…”

Evaluation of Fast and Sensitive Proteome Profiling of FF and FFPE Kidney Patient Tissues

“…Our results on kidney FFPE tissues showed underrepresentation of lysine (K) residues, probably due to a reaction of the lysine sidechains with formaldehyde in FFPE tissues, which is in agreement with previously published work [ 31 , 37 ]. Significance of the experimental optimization of the protocols which use urea in protein extraction is also demonstrated in previous reports whereas maximum yield was observed after samples were incubated at elevated temperature (95 °C), and urea was added after decreasing the temperature of the sample solution to 60 °C [ 38 ]. However, in the case of the lysate of SK-MEL cells, rat spleen lysates, and pancreatic tumor lysates, the best performance was observed when digestion was performed with urea at room temperature [ 35 ].…”

Molecular Dynamics Simulation-assisted Ionic Liquid Screening for Deep Coverage Proteome Analysis

“…The cancer and para-carcinoma tissues of human liver samples were by guest on November 2, 2020 treated using i-FASP method. As a comparison, the same amounts of samples were back-to-back treated by using FASP method with 8 M urea as the extractant (46,47). Overall, an average of 7,176 proteins (corresponded to 56,492 peptides) were identified from liver cancer tissue by i-FASP method, 6% and 16% increase in comparison with the protein and peptide number identified from FASP method (shown in Table S11 and Supplementary Table 6).…”