Comparative proteomics of clathrin-coated vesicles.
Comparative proteomics of clathrin-coated vesicles.
复制标题
网格蛋白涂层囊泡的比较蛋白质组学。
DOI：
10.1083/jcb.200607164
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发表时间：
2006-11-20
影响因子：
7.8
通讯作者：
Robinson, Margaret S
中科院分区：
文献类型：
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作者：
Borner, Georg H H;Harbour, Michael;Hester, Svenja;Lilley, Kathryn S;Robinson, Margaret S
网格蛋白包被的囊泡（CCV）促进跨高尔基体网络，内体和质膜之间的货物运输。本研究首次对CCV进行了比较蛋白质组学研究。从HeLa细胞中分离CCV富集级分，从网格蛋白耗尽的细胞中分离“模拟CCV”级分。我们使用二维差异凝胶电泳和同量异位素标签的相对和绝对定量（iTRAQ）结合质谱分析和比较两个馏分的组合。总共鉴定了63种真正的CCV蛋白，其中包括28种先前未建立的与CCV相关的蛋白。这些包括许多后高尔基体陷阱; AP-3，retromer和BLOC-1复合物的亚基;溶酶体酶; CHC 22;和五种功能未知的新蛋白质。本文中概述的策略应广泛适用于区分真正的细胞器成分的污染物。
Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a “mock CCV” fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.