Difference gel electrophoresis identifies differentially expressed proteins in endoscopically collected pancreatic fluid

Abstract

Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses.

Analysis of Endoscopic Pancreatic Function Test (ePFT)-Collected Pancreatic Fluid Proteins Precipitated Via Ultracentrifugation

“…In contrast, proportionally more peptidase (protease) activity-related proteins, as well as serine peptidase inhibitor (a protease inhibitor) activity-related proteins, were identified in the ultracentrifugation-precipitated sample. These classes of proteins are of particular interest in that several proteases and protease inhibitors have been identified as potential biomarkers of chronic pancreatitis (Table 3) [8, 9]. …”

“…In our prior studies using TCA protein precipitation, we identified several dozen potential protein biomarkers for chronic pancreatitis [8, 9]. Several of these proteins (including pancreatic enzymes (amylases, lipases, chymotrypsinogens, and carboxypeptidases), enzyme inhibitors (alpha-1-antitrypsin and alpha-1-antichymotrypsin), mucins, haptoglobin and neprilysin) were also identified in ultracentrifugation-precipitated pancreatic fluid (Table 3).…”

“…We have demonstrated previously that pancreatic fluid can be readily collected via the secretin-stimulated endoscopic pancreatic function test (ePFT) in volumes of 2 to 8 mL, and as such is a suitable specimen for mass spectrometry-based proteomic investigations [7, 8, 9, 10, 11]. We have screened a series of precipitation strategies to optimize protein extraction from pancreatic fluid [11], and we have demonstrated that pancreatic fluid can be maintained at 4°C for several hours with minimal proteolysis.…”