Proteomic analysis of formalin-fixed paraffin-embedded pancreatic tissue using liquid chromatography tandem mass spectrometry

Abstract

Objectives: FFPE tissue is a standard method of specimen preservation for hospital pathology departments. Formalin-fixed paraffin-embedded tissue banks are a resource of histologically characterized specimens for retrospective biomarker investigation. We aim to establish liquid chromatography coupled with tandem mass spectrometry analysis of FFPE pancreatic tissue as a suitable strategy for the study of the pancreas proteome.

Methods: We investigated the proteomic profile of FFPE pancreatic tissue specimens, using liquid chromatography coupled with tandem mass spectrometry, from 9 archived specimens that were histologically classified as normal (n = 3), chronic pancreatitis (n = 3), and pancreatic cancer (n = 3).

Results: We identified 525 nonredundant proteins from 9 specimens. Implementing our filtering criteria, 78, 15, and 21 proteins were identified exclusively in normal, chronic pancreatitis, and pancreatic cancer specimens, respectively. Several proteins were identified exclusively in specimens with no pancreatic disease: spink 1, retinol dehydrogenase, and common pancreatic enzymes. Similarly, proteins were identified exclusively in chronic pancreatitis specimens: collagen α1 (XIV), filamin A, collagen α3 (VI), and SNC73. Proteins identified exclusively in pancreatic cancer included annexin 4A and fibronectin.

Conclusions: We report that differentially expressed proteins can be identified among FFPE tissue specimens originating from individuals with different pancreatic histologic findings. The mass spectrometry-based method used herein has the potential to enhance biomarker discovery and chronic pancreatitis research.

Figure 1. Experimental workflow

1) FFPE tissue specimens were obtained, 2) a 1.5 cm × 1 cm × 5 μm slice of sample was scraped into a tube, 3) paraffin was removed with heptane, 4) disulfide bonds were reduced with dithiotreitol (DTT) and alkylated with iodoacetamide, 5) sample was digested overnight with trypsin, 6) peptides were isolated, 7) LC-MS/MS analysis was performed, and 8) bioinformatics analysis was performed.

Figure 2. Overlap of proteins among the three cohorts

Venn diagrams showing unique and overlapping proteins among the three cohorts. Also indicated in the figure are the tables in which these sets of proteins are listed. Abbreviations: NP, normal pancreas; CP, chronic pancreatitis; PC, pancreatic cancer.

Figure 3. Gene ontology annotation

A) Subcellular localization. B) Molecular function. We include in this analysis all non-redundant proteins that were identified in each cohort. Abbreviations: NP, normal pancreas; CP, chronic pancreatitis; PC, pancreatic cancer.