Proteomic analyses of pancreatic cyst fluids

Abstract

Objectives: There are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. Proteomic analyses were performed to explore pancreatic cyst fluids to yield effective diagnostic biomarkers.

Methods: We have prospectively recruited 20 research participants and prepared their pancreatic cyst fluids specifically for proteomic analyses. Proteomic approaches applied were as follows: (1) matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry peptidomics with LC/MS/MS (HPLC-tandem mass spectrometry) protein identification; (2) 2-dimensional gel electrophoresis; (3) GeLC/MS/MS (tryptic digestion of proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by LC/MS/MS).

Results: Sequencing of more than 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable; protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, making 2-dimensional gel proteomics unmanageable; GeLC/MS/MS revealed a panel of potential biomarker proteins that correlated with carcinoembryonic antigen (CEA).

Conclusions: Two homologs of amylase, solubilized molecules of 4 mucins, 4 solubilized CEA-related cell adhesion molecules (CEACAMs), and 4 S100 homologs may be candidate biomarkers to facilitate future pancreatic cyst diagnosis and risk-stratification. This approach required less than 40 microL of cyst fluid per sample, offering the possibility to analyze cysts smaller than 1 cm in diameter.

Figure 1. Alignment of some peptide clusters observed in cyst 13

A: bile salt activated lipase, Apolipoprotein alpha.

Figure 2. 2D-gel electrophoresis proteomics of pancreatic cyst fluids

Each panel of 2D gel from left to right is pH 3 to pH 10. A: Cyst 14 cystadenoma, B: Cyst 5 adenocarcinoma, C: Cyst 13 rich in pancreatic enzymes, D: Cyst 9 rich in pancreatic enzymes, E: Cyst 6 rich in plasma, F: Cyst 15 rich in plasma.

Figure 3. Representatives of 137 plasma proteins distributed among the pancreatic cyst samples

The numbers shown are emPAI roughly proportional to protein abundance. Dark (>1), medium (0.1 to 1), and light (<0.1) shading denotes relative protein abundance. No proteins were detected for the empty boxes. CEA = ng/mL. MCA=mucinous cystadenoma

Figure 4. Pancreatic enzyme proteins in the pancreatic cyst samples

Legends are the same as for Figure 3.

Figure 5. Proteomic potential biomarkers for mucins, CEACAMs, and S100s found in the pancreatic cyst samples

Legends are the same as for Figure 3.

Figure 6. SDS PAGE Western blotting validation of proteomic biomarkers in pancreatic cyst fluids

A: CEACAM6, B: S100A9.