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.
Chen
Xuequn
X
Department of Biological Chemistry, The University of Michigan, Ann Arbor, MI, USA.
Andrews
Philip C
PC
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D010179:Pancreas / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D020543:Proteome; D022142:Secretory Vesicles / Q000737:chemistry / Q000648:ultrastructure*; D014357:Trypsin
D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; D014357:Trypsin