The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ TM ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.
Molecular & Cellular Proteomics 5:306 -312, 2006.Pancreatic acinar cells are the functional units of digestive enzyme synthesis, storage, and secretion and have been a classical model for studying regulated exocytosis (1, 2). In acinar cells digestive enzymes are stored in a specialized organelle, the zymogen granule (ZG).1 Stimulation of acinar cells by secretagogues triggers fusion of ZG membranes with the apical membrane and the subsequent release of digestive enzymes. Early studies using SDS-PAGE indicated a relatively simple protein structure for the ZG membrane. Several more recent studies using 2D GE led to the identification of two additional major ZG membrane components, GP3 (3) and membrane dipeptidase (4). In another study, 14 spots were identified as small GTPases by [ 35 S]GTP␣S overlay on a 2D gel of ZG membrane proteins, but the identities of the spots remained unknown (5). In addition, a number of low abundance proteins, including Rab3D and several SNARE proteins, have been identified on ZG membranes by immunoblotting and immunocytochemistry (2). Despite its importance, a comprehensive proteomic analysis of the membrane protein components of ZGs has not been achieved.By combining 2D GE and 2D LC with tandem mass spectrometry, we report the identification of 101 proteins on ZG membranes, many of which had not been localized previously on ZGs including multiple small GTP-binding proteins, SNARE proteins, and molecular motor proteins. In addition, to distinguish intrinsic membrane proteins from peripheral and soluble proteins, a quantitative proteomic strategy was used to measure the r...