Proteomic analysis of insulin secretory granules in INS-1cells by protein correlation profiling.
Proteomic analysis of insulin secretory granules in INS-1cells by protein correlation profiling.
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通过蛋白质相关图谱对 INS-1 细胞中胰岛素分泌颗粒进行蛋白质组学分析
DOI：
10.1007/s41048-018-0061-3
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发表时间：
2018-01-01
影响因子：
--
通讯作者：
Hou, Junjie
中科院分区：
文献类型：
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作者：
Li, Min;Du, Wen;Hou, Junjie
摘要：胰岛素分泌颗粒（Insulin secretory granules, ISGs）是胰腺β细胞中一组独特的细胞器，负责胰岛素的储存和分泌，以维持血糖稳态。ISG生物发生、成熟、运输和胞外分泌的分子机制仍然很大程度上是未知的，因为参与这些不同步骤的蛋白质尚未完全确定。密度梯度离心亚细胞分馏法已成功地用于分析许多细胞器的蛋白质组。然而，使用这种方法来阐明ISG蛋白质组受到共分离污染物的限制，因为ISG是非常动态的，并且与其他细胞器（如高尔基体、溶酶体和核内体）有大量的交换或接触。在这项研究中，我们开发了一种基于蛋白质相关分析（PCP）的蛋白质组学鉴定ISG蛋白的新策略，其中包括使用OptiPrep密度梯度离心纯化ISG，无标记定量蛋白质组学，以及通过关联候选ISG标记物和已知ISG标记物之间的分离谱来鉴定ISG蛋白。使用这种方法，我们能够鉴定81个ISG蛋白。其中，九跨膜蛋白TM9SF3被认为是PCP网络中突出的高置信度ISG候选蛋白。进一步的生化和免疫荧光分析表明，TM9SF3定位于ISG，提示它是一个潜在的新的ISG标志物。图形化的简介:
ABSTRACT: Insulin secretory granules (ISGs), a group of distinguishing organelles in pancreatic beta cells, are responsible for the storage and secretion of insulin to maintain blood glucose homeostasis. The molecular mechanisms of ISG biogenesis, maturation, transportation, and exocytosis are still largely unknown because the proteins involved in these distinct steps have not been fully identified. Subcellular fractionation by density gradient centrifugation has been successfully employed to analyze the proteomes of numerous organelles. However, use of this method to elucidate the ISG proteome is limited by co-fractionated contaminants because ISGs are very dynamic and have abundant exchanges or contacts with other organelles, such as the Golgi apparatus, lysosomes, and endosomes. In this study, we developed a new strategy for identifying ISG proteins by protein correlation profiling (PCP)-based proteomics, which included ISG purification by OptiPrep density gradient centrifugation, label-free quantitative proteome, and identification of ISG proteins by correlating fractionation profiles between candidates and known ISG markers. Using this approach, we were able to identify 81 ISG proteins. Among them, TM9SF3, a nine-transmembrane protein, was considered a high confidence ISG candidate protein highlighted in the PCP network. Further biochemical and immunofluorescence assays indicated that TM9SF3 localized in ISGs, suggesting that it is a potential new ISG marker.GRAPHICAL ABSTRACT: