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

Abstract

Background: The 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 design: DT, 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.

Results: DT 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.

Conclusions: 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. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.

Keywords: Archival tissues; FASP; FFPE; LC-MS/MS; Protein extraction.

Figure 1

Qualitative and quantitative reproducibility of DT (blue), FASP (red) and ISD (green) methods. A) Top: Venn diagrams depicting distribution of identified proteins among replicates. Percentages of common proteins are indicated in yellow. Bottom: correlation of protein abundance between all replicates combinations for every method. Pearson correlation coefficients are also reported. B) Same as Panel A but at the peptide level.

Figure 2

Qualitative and quantitative method comparison. Top: Unsupervised hierarchical cluster analysis based on protein (A) and peptide (B) label-free quantitative data, respectively. Middle: Venn diagrams illustrating distribution of all identified proteins (C) and peptides (D). Percentage of common proteins and peptides are indicated in yellow. Bottom: Dot plots describing correlation of protein (E) and peptide (F) abundance between DT and FASP (purple), DT and ISD (blue-green), FASP and ISD (bronze). Pearson correlation coefficients are also reported.

Figure 3

Quantitative protein distribution according to subcellular localization. Mean and SD value of NSAF percentage for three independent experimental replicates are shown. NSAF values were expressed as percentage of the annotated proteins. Asterisks indicate statistical significance according to Student’s t-test (p value < 0.05); the blue ones indicate statistically significant difference versus DT, the red ones versus FASP, the green ones versus ISD and the black ones versus all other methods, respectively.

Figure 4

Quantitative protein distribution according to physicochemical features. Quantitative protein distribution according to MW (A), pI (B), number of transmembrane domains (TMD, C) and hydrophobicity (GRAVY score, D). Mean and SD value of NSAF percentage for three independent experimental replicates are shown. NSAF values were expressed as percentage of all proteins. Asterisks indicate statistical significance according to Student’s t-test (p value < 0.05); the blue ones indicate statistically significant difference versus DT, the red ones versus FASP, the green ones versus ISD and the black ones versus all other methods, respectively.

Figure 5

Impact of non-tryptic and formaldehyde-modified peptides. A) Left: Venn diagrams showing distribution of peptides identified with ‘trypsin’ and ‘no enzyme’ searches in DT (blue), FASP (red) and ISD (green) samples. Right: Venn diagram showing distribution of non-tryptic peptides among all methods. B) Left: Venn diagrams showing distribution of peptides identified with standard search (‘no mod’) and search comprising formaldehyde-induced modifications (‘mod’) in DT (blue), FASP (red) and ISD (green) samples. Right: Venn diagram showing distribution of formaldehyde-modified peptides among all methods.