Organellar proteomics - Analysis of pancreatic zymogen granule membranes
Organellar proteomics - Analysis of pancreatic zymogen granule membranes
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DOI：
10.1074/mcp.m500172-mcp200
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发表时间：
2006-02-01
影响因子：
7
通讯作者：
Andrews, PC
中科院分区：
文献类型：
--
作者：
Chen, XQ;Walker, AK;Andrews, PC
酶原颗粒（ZG）是胰腺腺泡细胞中储存消化酶和调节分泌的特化细胞器，是研究分泌颗粒功能的模型。在初步努力全面了解这个细胞器的功能，我们进行了蛋白质组学研究，以确定从高度纯化的ZG膜蛋白。通过双向凝胶电泳和双向液相色谱-串联质谱技术，从纯化的ZG膜中鉴定出101个蛋白，包括28个已知的ZG蛋白和73个未知蛋白，包括SNAP 29、Rab 27 B、Rab 11 A、Rab 6、Rap 1和肌球蛋白Vc。此外，几个假设的蛋白质被确定为代表潜在的新蛋白质。通过免疫细胞化学进一步证实了这些蛋白质中的9个的ZG定位。为了区分内在膜蛋白与可溶性和外周膜蛋白，使用定量蛋白质组学策略来测量通过纯化过程的内在膜蛋白的富集。iTRAQ（TM）比率与已知的或跨膜隐马尔可夫模型预测的可溶性或膜蛋白相关性良好。通过结合亚细胞分离与高分辨率分离和蛋白质的全面鉴定，我们已经开始阐明酶原颗粒的功能，通过蛋白质组学和随后的功能分析其膜组件。
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.