Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas obtained from mouse and human

Abstract

A number of reports have recently emerged with focus on extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D-nanoLC-MS(MS)(2) following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease.

Figure 1

FFPE tissue workflow for proteomic analysis. Workflow for the proteomic analysis of the frozen and FFPE tissues.

Figure 2

LC-MS analysis of FFPE tissue proteome with high reproducibility. (A) Top, representative LC-MS ion chromatogram plotted as m/z (x-axis) versus retention time (RT, y-axis). Bottom, one randomly chosen area within the plot (arrow) was expanded across all LC-MS runs as an example. This process was used to illustrate the quality and reproducibility of the individual LC-MS runs (rep = replicate). (B) Scatter plot of ln individual normalized ion intensity (x-axis) versus natural log (ln) average normalized ion intensities (y-axis). The lines correlate with ±2- and ±4-fold changes. The Pearson’s correlation coefficient was calculated for each of the eight FFPE samples with an average and standard deviation of R² = 0.97 ± 0.01 and R² = 0.98 ± 0.01 for the frozen samples.

Figure 3

Comparison of FFPE tissue and matched frozen tissue. (A) Venn diagram of the total number of protein groups identified from eight animals per group with two or more peptides per protein and >95% confidence from two (10 mm × 10 mm × 10 µm) tissue sections. Greater than 80% of the protein IDs overlapped between two groups. (B) Venn diagram of all peptides identified with >95% confidence. (C) Top, pancreatic amylase amino acid sequence. Red letters: peptides identified in this study. Yellow highlights: peptides identified in both FFPE and frozen groups. Cyan highlights: peptides identified only in the frozen group. Bottom, table of peptide sequences identified in this study for pancreatic amylase with number of repeated identification of the specific peptide indicated.

Figure 4

Markers of pancreatic ductal adenocarcinoma (PDAC) via proteomic analysis of human FFPE tissues. (A) Venn diagram of identified protein groups with 95% confidence in control and PDAC. (B) Heat map of normalized spectral counts in control and PDAC, corresponding to those proteins that were shared by both groups that also met the criteria of SAM >|±0.8|, fold change>|±2|, and Wilcoxon rank sum test < 0.05.

Figure 5

Pathway analysis of human pancreatic cancer FFPE tissue proteome. (A) Potential relationship between COL6A3 and KRT19 by systems biology pathway analysis (red arrows). Pink to red double circle: increase in pancreatic ductal adenocarcinoma (PDAC). Light blue double circle: decrease in PDAC. Cyan highlight: upstream regulators. (B) Bar charts representing the normalized spectral counts with standard deviation for 14 statistically significant proteins comparing the control (blue) and PDAC (red) groups.