Proteome of Pancreatic Zymogen Granules

胰酶原颗粒的蛋白质组

• 批准号: 7067535
• 负责人: MICHAEL J. RINDLER
• 金额: $ 16.5万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7067535
• 关键词: SDS polyacrylamide gel electrophoresis; acinar cell; biological signal transduction; centrifugation; digestion; exocytosis; granule; immunocytochemistry; laboratory rat; mass spectrometry; membrane proteins; pancreas enzyme; peptide library; protein purification; protein sequence; protein structure function; proteomics; western blottings; zymogens

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.

描述（由申请人提供）：该提案涉及通过利用蛋白质测序技术和基因组序列鉴定的最新改进，鉴定储存在外分泌胰腺分泌颗粒中的新蛋白质。分泌颗粒是储存许多重要蛋白质的地方，包括消化酶，在刺激时释放。分泌颗粒蛋白不仅调节消化，还调节新陈代谢、生长、神经传递和免疫反应。颗粒膜蛋白在胞吐过程中起作用，并作为消化酶的前体，但其在胞吐调节中的作用在分子水平上尚不清楚。工作假设是胰腺颗粒含有尚未确定的酶和其他具有代谢意义的蛋白质。虽然在确定主要成分蛋白方面取得了进展，但在确定不太重要的成分或膜蛋白方面却很少付出努力。部分原因是很难从小鼠或人类身上获得足够的材料来与他们的基因组数据库进行比较。美国国立卫生研究院国家人类基因组研究所最近宣布完成大鼠基因组，为使用大鼠组织作为原始材料提供了可能性。高灵敏度质谱分析，能够一次确定超过50个肽的序列，将用于建立酶原颗粒蛋白质的蛋白质组学谱。具体目的是：1)从大鼠胰腺中获得高纯度的分泌颗粒及其膜；2)经SDS/PAGE和胰蛋白酶消化后，用质谱法测定颗粒蛋白的序列；3)通过将获得的序列与数据库中的序列进行比较，鉴定出感兴趣的新蛋白质；4)通过颗粒部分免疫印迹和免疫细胞化学证实新鉴定的蛋白在胰腺外分泌和颗粒中的存在。此外，这些蛋白质在其他外分泌器官中的存在将使用类似的技术进行分析。目标是发现以前未被认为储存在胰腺中的蛋白质，以及参与消化，信号转导和胞吐的新蛋白质。预计这些大鼠蛋白将在人类中具有相应的意义，对影响胰腺功能的异常代谢状况（如胰腺炎、囊性纤维化和癌症）具有重要意义。