Purification and proteomics analysis of pancreatic zymogen granule membranes

Pancreatic zymogen granules (ZGs) are specialized for digestive enzyme storage and regulated secretion in 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 (ZGM) proteins from ZGs purified by Percoll gradient centrifugation. By combining multiple separation strategies including two-dimensional gel electrophoresis and two-dimensional HPLC with tandem mass spectrometry, we identified 101 proteins from purified ZGMs including a large number of proteins previously unknown on ZGMs. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomics strategy was used to measure the enrichment of intrinsic membrane proteins through the purification steps by labeling crude, KBr-, and Na(2)CO(3)-washed ZGMs with multiplexed isobaric tags (iTRAQtrade mark), 114, 116, and 117, respectively. The proteins with 117:114 ratios greater than one correlated well with intrinsic membrane proteins that contain either known or predicted transmembrane domains.