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首页 » 干细胞&iPS » MCP:鉴定出小鼠胚胎成纤维细胞和人胚胎干细胞的分泌蛋白质组

MCP:鉴定出小鼠胚胎成纤维细胞和人胚胎干细胞的分泌蛋白质组

来源:生物谷 2012-09-27 00:08

2012年9月26日 讯 /生物谷BIOON/ --来自美国北卡罗来纳州立大学的研究人员开发出一种新技术来鉴定细胞分泌的蛋白。这种新方法应当有助于研究人员在细胞生物学上收集精确的数据。

在这项新研究中,研究人员开发出的这种新方法是基于一个事实:每个细胞利用它的分泌途径来包裹它的蛋白。每个细胞合成蛋白,然后通过这种途径运输它:将它包裹在袋状的膜中,最终将它运输到胞外。

在这种新技术中,研究人员采集细胞样品,分离出分泌途径中含有蛋白的细胞器。他们然后利用质谱分析这些细胞器中的物质以便观察细胞正在分泌哪些蛋白。利用这种方法,他们能够鉴定出小鼠胚胎成纤维细胞和人胚胎干细胞分泌的蛋白。

这种新方法能够评估与在细胞培养基中发现的蛋白相关联的问题。但是它也允许研究人员追踪当细胞对一种刺激(比如接触一种化学物)作出反应而导致它分泌的蛋白所产生的变化。(生物谷:Bioon.com)

Targeted proteomics of the secretory pathway reveals the secretome of mouse embryonic fibroblasts and human embryonic stem cells

Prasenjit Sarkar, Shan M. Randall, David C. Muddiman and Balaji M. Rao

Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of hESC self-renewal and differentiation. Current MS-based approaches for identifying secreted proteins rely predominantly on MS analysis of cell culture supernatants. Here we show that targeted proteomics of secretory pathway organelles is a powerful alternate approach to interrogate the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse embryonic fibroblasts (MEFs) and hESCs that are enriched in secretory pathway organelles, while ensuring retention of the secretory cargo. MS analysis of these fractions from hESCs cultured in MEF conditioned medium (MEF-CM) or MEFs exposed to hESC medium revealed 99 and 129 proteins putatively secreted by hESCs and MEFs, respectively. Of these, 53 and 62 proteins have been previously identified in cell culture supernatants of MEFs and hESCs respectively, thus establishing the validity of our approach. Furthermore, 76 and 37 putatively secreted proteins identified in this study, in MEFs and hESCs respectively, have not been reported in previous MS analyses. Identification of low abundance secreted proteins by MS analysis of cell culture supernatants typically necessitates the use of altered culture conditions such as serum-free medium. However, an altered medium formulation might directly influence the cellular secretome. Indeed, we observed significant differences between the abundances of several secreted proteins in subcellular fractions isolated from hESCs cultured in MEF-CM and those exposed to unconditioned hESC medium for 24 hours. By contrast, targeted proteomics of secretory pathway organelles does not require the use of customized media. We expect that our approach will be particularly valuable in two contexts highly relevant to hESC biology – to obtain a temporal snapshot of proteins secreted in response to a differentiation trigger, and to identify proteins secreted by cells that are isolated from a heterogeneous population.

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