Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

Abstract

BackgroundThe growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.Experimental designDT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.ResultsDT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.ConclusionsThese results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.

Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost

“…Both methods surprisingly identified a significant number of unique proteins with each method replicate despite analyzing serial FFPE sections of a homogenous tumor. The variance in protein identifications between method replicates is high (only 74% common for Qkit and 64% for UISD), but the levels are similar to those found in a recent study comparing three methods: 58.0% for direct tissue trypsinization, 65.2% for filter aided sample preparation, and 69.5% for in solution digestion . Incomplete tissue solubilization leads to bias which prevents current work with FFPE tissue from being completely quantitative .…”

Analysis of Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue Via Proteomic Techniques and Misconceptions of Antigen Retrieval

“…In spite of this, SDS is, ironically, the best candidate for a high-quality extraction buffer. This was first acknowledged by Wisniewski et al in 2011 (33) and 2013 (34), and then again by Tanca et al 2014 (32). These two teams employed filter-assisted sample preparation (FASP), which uses membrane filters to facilitate SDS/urea exchange of FFPE samples to allow further downstream processing via tryptic digestion and LC-MS/MS analysis.…”

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini

“…From these, a total of 486 N termini were shared between both preservation methods (among all replicates of cryopreserved and FFPE tissues, respectively). Previous studies indicate that proteins extracted from FFPE tissues are susceptible to a ϩ12 Da addition at N termini, lysine, tryptophan, tyrosine, serine, and threonine residues, as well as a ϩ30 Da addition at cysteine, histidine, lysine, and arginine residues (20,33,34). In both cryopreserved and FFPE samples, the fraction of peptides displaying these modifications remained below the 5% false discovery rate (data not shown).…”