Quantitative Proteomics Analysis of Pancreatic Zymogen Granule Membrane Proteins

Pancreatic zymogen granules (ZG) are specialized for digestive enzyme storage and regulated secretion in the exocrine pancreas and are a classical model for studying secretory granule function. To understand the function of this organelle, we have conducted a proteomic study to identify the ZG membrane proteins from ZGs purified by Percoll gradient centrifugation. By combining multiple separation strategies including two-dimensional gel electrophoresis and two-dimensional liquid chromatography with tandem mass spectrometry (TMS), we identified 101 proteins from purified ZG membranes including a large number of proteins previously unknown on ZG membranes. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomics strategy was developed to measure the enrichment of intrinsic membrane proteins through the purification steps by labeling crude, KBr-, and Na₂ CO₃ -washed ZG membranes with multiplexed isobaric tags (iTRAQ™), 114, 116 and 117, respectively. The proteins with 117:114 ratios greater than one correlated well with known or predicted intrinsic membrane proteins.