2012年9月26日 訊 /生物谷BIOON/ --來自美國北卡羅來納州立大學(xué)的研究人員開發(fā)出一種新技術(shù)來鑒定細胞分泌的蛋白,。這種新方法應(yīng)當(dāng)有助于研究人員在細胞生物學(xué)上收集精確的數(shù)據(jù),。
在這項新研究中，研究人員開發(fā)出的這種新方法是基于一個事實：每個細胞利用它的分泌途徑來包裹它的蛋白,。每個細胞合成蛋白,，然后通過這種途徑運輸它：將它包裹在袋狀的膜中，最終將它運輸?shù)桨狻?/p>
在這種新技術(shù)中,，研究人員采集細胞樣品,，分離出分泌途徑中含有蛋白的細胞器。他們?nèi)缓罄觅|(zhì)譜分析這些細胞器中的物質(zhì)以便觀察細胞正在分泌哪些蛋白,。利用這種方法,，他們能夠鑒定出小鼠胚胎成纖維細胞和人胚胎干細胞分泌的蛋白,。
這種新方法能夠評估與在細胞培養(yǎng)基中發(fā)現(xiàn)的蛋白相關(guān)聯(lián)的問題,。但是它也允許研究人員追蹤當(dāng)細胞對一種刺激(比如接觸一種化學(xué)物)作出反應(yīng)而導(dǎo)致它分泌的蛋白所產(chǎn)生的變化。(生物谷：Bioon.com)
doi: 10.1074/mcp.M112.020503
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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.
