TY - DATA T1 - Analysis of Low Abundance Membrane-Associated Proteins from Rat Pancreatic Zymogen Granules PY - 2010/10/01 AU - Heike Borta AU - Miguel Aroso AU - Cornelia Rinn AU - Maria Gomez-Lazaro AU - Rui Vitorino AU - Dagmar Zeuschner AU - Markus Grabenbauer AU - Francisco Amado AU - Michael Schrader UR - https://acs.figshare.com/articles/figure/Analysis_of_Low_Abundance_Membrane_Associated_Proteins_from_Rat_Pancreatic_Zymogen_Granules/2725993 DO - 10.1021/pr100052q.s002 L4 - https://ndownloader.figshare.com/files/4401994 KW - tandem mass spectrometry KW - suborganellar proteomics approach KW - RMCP KW - immunofluorescence studies KW - apical secretion KW - 46 spots KW - membrane proteins KW - ZG subfractions KW - exocrine pancreas KW - storage organelles KW - rat mast cell chymase KW - pancreatic AR 42J cells KW - ZG formation KW - validation experiments KW - Rat Pancreatic Zymogen GranulesZymogen granules KW - ZG localization KW - rat pancreas KW - zymogen granules N2 - Zymogen granules (ZG) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging, and regulated apical secretion of digestive enzymes. As there is a critical need for further understanding of the key processes in regulated secretion to develop new therapeutic options in medicine, we applied a suborganellar proteomics approach to identify peripheral membrane-associated ZG proteins. We focused on the analysis of a “basic” group (pH range 6.2−11) with about 46 spots among which 44 were identified by tandem mass spectrometry. These spots corresponded to 16 unique proteins, including rat mast cell chymase (RMCP-1) and peptidyl-prolyl cis−trans isomerase B (PpiB; cyclophilin B), an ER-resident protein. To confirm that these proteins were specific to zymogen granules and not contaminants of the preparation, we conducted a series of validation experiments. Immunoblotting of ZG subfractions revealed that chymase and PpiB behaved like bona fide peripheral membrane proteins. Their expression in rat pancreas was regulated by feeding behavior. Ultrastructural and immunofluorescence studies confirmed their ZG localization. Furthermore, a chymase−YFP fusion protein was properly targeted to ZG in pancreatic AR42J cells. Interestingly, for both proteins, proteoglycan-binding properties have been reported. The importance of our findings for sorting and packaging during ZG formation is discussed. ER -