Autoimmune pancreatic disease: Preparation of pancreatic juice for proteome analysis

Abstract

The identification of pancreatic proteins is generally hampered by the high content and activity of proteases produced by this organ. The aim of this work was the development of a protocol for the analysis of pancreatic juice by two-dimensional (2-D) gel electrophoresis allowing consistent and reproducible protein analysis encompassed by high-resolution protein 2-D maps and subtle protein spot recognition without substantial losses due to proteases. Immobilized pH gradient (IPG) strips were used for the first dimension, the second dimension was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the key step was the sample preparation technique. Improvements were achieved by using several protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, L-1-chloro-3-[4-tosyl-amido]-7-amino-2-heptanine (TLCK)-HCI, Complete) to prevent degradation of the proteins. The application of different pH-ranges was a valuable step for getting an overview of the expressed protein pattern. These investigations resulted in well-resolved 2-D maps with a high reproducibility.

Proteomic Profiling of Pancreatic Cancer for Biomarker Discovery

“…With recent progress in 2-DE and MS, several groups have started to employ a proteomics approach to study pancreatic juice. A detailed procedure for preparing pancreatic juice for 2-DE analysis was documented (92). In the study, the authors discovered that the addition of protease inhibitors resulted in well-resolved 2-D maps with a high reproducibility.…”

Protein and antibody microarray technology

“…However, it took till the mid-nineties, until the introduction of immobilised pH gradient isoelectric focussing increased the reproducibility as well as the resolution by an order of magnitude [5][6][7][8]. Based upon this improvement, 2D-gel electrophoresis today allows separation and detection from a wide variety of sources and permits the characterisation of samples by different expression profiles [9][10][11], Moreover, the resolution allows separation of proteins isoforms that differ by post-translational modifications, such as glycosilation [12], deamination [13] and phosphorylation [14].…”

Mass spectrometry‐based proteomics of endoscopically collected pancreatic fluid in chronic pancreatitis research

“…Various methods of protein fractionation 37–44 are commonly used for proteomic analysis. Prior to the application of MS for protein identification, distinct patterns of proteins in pancreatic fluid have been investigated using HPLC 45, 46 and 2‐DE 47, 48. Although such methodologies could determine that specific proteins differed in the fluid of normal and diseased pancreata, the biomolecular mechanisms of disease remain undetermined.…”