Proteomic analysis (GeLC-MS/MS) of ePFT-collected pancreatic fluid in chronic pancreatitis

Abstract

Chronic pancreatitis is characterized by inflammation, fibrosis, pain, and loss of exocrine function of the pancreas. We aimed to identify differentially expressed proteins in the ePFT-collected pancreatic fluid from individuals with chronic pancreatitis (CP; n = 9) and controls with chronic abdominal pain not associated with the pancreas (NP; n = 9). Using GeLC-MS/MS techniques, we identified a total of 1391 different proteins in 18 pancreatic fluid samples. Of these proteins, 257 and 413 were identified exclusively in the control and chronic pancreatitis cohorts, respectively, and 721 were identified in both cohorts. Spectral counting and statistical analysis thereof revealed an additional 38 and 77 proteins that were up- or down-regulated, respectively, in the pancreatic fluid from individuals with chronic pancreatitis. As expected, gene ontology analysis illustrated that the largest percentage of differentially regulated proteins was secreted/extracellular in origin. In addition, proteins that were down-regulated with statistical significance in the chronic pancreatitis cohort were determined to have biological function of proteases, corresponding to the canonical pancreatic insufficiency associated with chronic pancreatitis. Proteins enriched in the pancreatic fluid of chronic pancreatitis patients had roles in fibrosis, inflammation, and pain, whereas digestive enzymes were significantly less abundant. Our workflow provided a mass spectrometry-based approach for the further study of the pancreatic fluid proteome, which may lead to the discovery potential biomarkers of chronic pancreatitis.

Figure 1

Experimental workflow. The general workflow for the overall analysis is as follows: 1) ePFT sample collection, 2) protein extraction with tricholoracetic acid (TCA), 3) SDS-PAGE protein fractionation, 4) in-gel tryptic digestion, 5) LC-MS/MS peptide mass determination, and 6) bioinformatic data processing.

Figure 2

SDS-PAGE protein fractionation. Each gel lane represents approximately 100 μg of ePFT-collected pancreatic fluid that has been TCA precipitated from a particular patient (9 individual patients per gel). A) NP (non-pancreatitis) controls samples. B) CP (chronic pancreatitis) samples.

Figure 3

The number of unique A) proteins and B) peptides identified in each pancreatic fluid sample analyzed. Grey bars represent NP (non-pancreatitis) controls samples and white bars represent CP (chronic pancreatitis) samples.

Figure 4

Venn diagrams comparing proteins identified in the NP (non-pancreatitis) and CP (chronic pancreatitis) cohorts. A) Qualitative assessment of proteins in each cohort, depicting those exclusive to either cohort. B) Quantitative assessment of proteins in common between the two cohorts. Bayes factor ≥ 10 indicates a statistically significant difference of the protein between the two cohorts.

Figure 5

Gene ontology (GO) analysis of subcellular localization and molecular function that were statistically up- or down- regulated in chronic pancreatitis. GO characterization using the categories listed for proteins with statistically significant differences was performed manually with the UniProt database or using the GoFact online tool^, . A) Subcellular localization and B) Molecular function of proteins down-regulated in CP (white bars), absent in CP (light grey). exclusive to CP (medium grey), up-regulated in CP (darker grey bars), and identified in both NP and CP (black bars). CP, chronic pancreatitis; NP, non-pancreatitis controls.