Molecular architecture of mouse and human pancreatic zymogen granules: protein components and their copy numbers.
Molecular architecture of mouse and human pancreatic zymogen granules: protein components and their copy numbers.
复制标题
DOI：
10.1007/s41048-018-0055-1
复制
发表时间：
2018
影响因子：
--
通讯作者：
Chen X
中科院分区：
文献类型：
--
作者：
Lee JS;Caruso JA;Hubbs G;Schnepp P;Woods J;Fang J;Li C;Zhang K;Stemmer PM;Jena BP;Chen X
胰腺酶原颗粒（ZG）的分子模型是了解其功能的关键。我们已经广泛地表征了大鼠ZG蛋白的组成和膜拓扑结构。在这项研究中，我们报告了有针对性的蛋白质组学方法的发展，以量化代表性的小鼠和人类ZG蛋白质使用LC-SRM和重同位素标记的合成肽。小鼠Rab 3D和VAMP 8的绝对量被确定为1242 ± 218和2039 ± 151（平均值土SEM）拷贝/ZG。通过原子力显微镜测定ZGs的尺寸分布和平均直径750 ± 23 nm（平均值± SEM）。然后使用半定量Western印迹法以纯化的GST-Rab 3D蛋白作为内标物验证Rab 3D的绝对定量。为了将我们的蛋白质组学分析扩展到人胰腺，使用从胰岛移植中心获得的人腺泡纯化ZG。首次鉴定了180个人ZG蛋白，包括膜蛋白和内容物蛋白。此外，人Rab 3D和VAMP 8的每ZG拷贝数确定为1182 ± 45和485 ± 15（平均值± SEM）。对小鼠和人类胰腺ZG的全面蛋白质组学分析具有鉴定物种特异性ZG蛋白的潜力。胰腺ZGs上蛋白质拷贝数的测定代表了使用靶向蛋白质组学方法建立原型分泌囊泡的定量分子模型的重大进展。人ZG蛋白的鉴定为后续研究胰腺疾病中ZG组成和分泌的改变奠定了基础。本文的在线版本（10.1007/s41048-018-0055-1）包含补充材料，可供授权用户使用。
A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the composition and membrane topology of rat ZG proteins. In this study, we report the development of targeted proteomics approaches to quantify representative mouse and human ZG proteins using LC-SRM and heavy isotope-labeled synthetic peptides. The absolute quantities of mouse Rab3D and VAMP8 were determined as 1242 ± 218 and 2039 ± 151 (mean ± SEM) copies per ZG. The size distribution and the averaged diameter of ZGs 750 ± 23 nm (mean ± SEM) were determined by atomic force microscopy. The absolute quantification of Rab3D was then validated using semi-quantitative Western blotting with purified GST-Rab3D proteins as an internal standard. To extend our proteomics analysis to human pancreas, ZGs were purified using human acini obtained from pancreatic islet transplantation center. One hundred and eighty human ZG proteins were identified for the first time including both the membrane and the content proteins. Furthermore, the copy number per ZG of human Rab3D and VAMP8 were determined to be 1182 ± 45 and 485 ± 15 (mean ± SEM). The comprehensive proteomic analyses of mouse and human pancreatic ZGs have the potential to identify species-specific ZG proteins. The determination of protein copy numbers on pancreatic ZGs represents a significant advance towards building a quantitative molecular model of a prototypical secretory vesicle using targeted proteomics approaches. The identification of human ZG proteins lays a foundation for subsequent studies of altered ZG compositions and secretion in pancreatic diseases. The online version of this article (10.1007/s41048-018-0055-1) contains supplementary material, which is available to authorized users.