Identification of pancreas-specific proteins in endoscopically (endoscopic pancreatic function test) collected pancreatic fluid with liquid chromatography--tandem mass spectrometry

Abstract

Objectives: We aimed to establish the endoscopic pancreatic function test (ePFT) as a method that can safely obtain pancreatic fluid for mass spectrometric analysis from patients during upper endoscopy and to reproducibly identify pancreas-specific proteins.

Methods: We performed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry-based proteomic analysis (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry [GeLC-MS/MS]) on ePFT-collected pancreatic fluid from 3 individuals, without evidence of chronic pancreatitis, who were undergoing an upper endoscopy for dyspepsia and chronic abdominal pain.

Results: Pancreatic fluid was safely collected from all subjects. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of ePFT-collected pancreatic fluid revealed no significant variation (F statistic, 1.33, P = 0.29) in protein concentration during the 1-hour collection period and a visually reproducible protein banding pattern among the 3 subjects. The GeLC-MS/MS analysis of ePFT-collected fluid identified pancreas-specific proteins previously described from endoscopic retrograde cholangiopancreatography and surgical collection methods. Gene ontology further revealed that most of the proteins identified have a molecular function of proteases.

Conclusions: The ePFT is capable of collecting large amounts of pancreatic fluid for proteomic analysis enabling the identification of pancreas-specific proteins. This endoscopic collection method coupled with GeLC-MS/MS is a powerful technique, which can be used in future investigations to elucidate pathways involved in the development and progression of pancreatic disease.

Figure 1

Experimental workflow. A) SDS-PAGE gel image of proteins extracted from pancreatic fluid. B) Workflow of the GeLC-MS/MS experiment for the proteomic profiling of pancreatic fluid. C) Chromatogram of fraction 11 of the proteomic profiling of pancreatic fluid; the survey mass spectrum at 36.56 min; the product ion spectrum of the peptide at m/z 669.34 (marked with an arrowhead in the survey spectrum); database searches identified this MS/MS spectrum as being from VSAYIDWINEK from chymotrypsin C.

Figure 2

A-C) SDS-PAGE gels of time courses of secretin-stimulated ePFT-collected pancreatic fluid proteins of individuals with chronic abdominal pain (A: CAP1, B: CAP2, C: CAP3). D) Temporal variation of pancreatic protein concentration secretion following secretin stimulation for the three samples (CAP1, CAP2 and CAP3). Error bars are standard deviations of technical replicates.