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.
Highlights
The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research
These 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
High-MW and basic proteins are known to be the most difficult to extract, separate and identify within an FFPE tissue proteome [5,6]. This leads to the hypothesis, clearly and consistently verified in this study, that sample preparation procedures based on a protein extraction step may imply a depletion of high-MW proteins compared to methods founded on a whole tissue trypsinization
Summary
The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. With respect to shotgun LC-MS/MS, direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) are the most commonly used strategies; more recently, an efficient alternative for processing FFPE protein extracts through the filter-aided sample preparation (FASP) workflow has been introduced [3,8,11]. These sample preparation workflows are all preceded by tissue deparaffinization and rehydration and usually comprise a high temperature treatment, but exhibit several differences in the other steps. In spite of the growing number of FFPE tissue proteomics papers, there is currently no consensus on the optimal protocol for shotgun proteomic analysis of FFPE tissue samples, and no studies critically comparing the performance of DT, ISD and/or FASP with FFPE specimens have been reported so far [3,8,11]
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