Proteome of Pancreatic Zymogen Granules

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DESCRIPTION (provided by applicant): This proposal concerns the identification of novel proteins stored in secretory granules from exocrine pancreas by taking advantage of recent improvements in protein sequencing technology and in genomic sequence identification. Secretory granules are the sites of storage of many important proteins, including digestive enzymes, for release upon a stimulus. Secretory granule proteins not only regulate digestion, but metabolism, growth, neurotransmission, and the immune response. Granule membrane proteins function in exocytosis and serve as precursors of digestive enzymes, but their role in regulating exocytosis is not well understood at the molecular level. The working hypothesis is that pancreatic granules contain as yet unidentified enzymes and other proteins of metabolic significance. While progress has been made in determining the major content proteins, little effort has been devoted to identifying less prominent components or membrane proteins. In part, the reason for this has been the difficulty in obtaining sufficient material from mouse or human sources for comparison to their genomic databases. The recent announcement by the NIH National Human Genome Research Institute of the completion of the rat genome affords the possibility of using rat tissue as source material. Highly sensitive mass spectrometric analysis, capable of determining the sequences of more than 50 peptides at a time, will be used to establish a proteomic profile of the proteins of zymogen granules. The specific aims are: 1) To obtain highly purified secretory granules and their membranes from rat pancreas; 2) To determine sequences of granule proteins using mass spectrometry after subjecting the preparations to SDS/PAGE and tryptic digestion; 3) To identify novel proteins of interest by comparing the sequences obtained to those in the databases; 4) To confirm the presence of the newly identified proteins in exocrine pancreas and in granules by immunoblotting of granule fractions and by immunocytochemistry. In addition, the presence of these proteins in other exocrine organs will be analyzed using similar techniques. The goal is to discover proteins not previously recognized to be stored in the pancreas as well as novel proteins involved in digestion, signal transduction and exocytosis. It is anticipated that these rat proteins will have human counterparts of significance to abnormal metabolic conditions affecting the function of the pancreas such as pancreatitis, cystic fibrosis, and cancer.