摘要
定量蛋白质组学研究是蛋白质组学研究的新方向,也是揭示生命活动变化规律的有效工具。细胞培养条件下稳定同位素标记定量蛋白质组学技术(stable isotope labeling by amino acids in cell culture, SILAC)是当前定量蛋白质组学研究的金标准,定量准度好、精度高,常用于组学水平上对两种不同处理或状态的比较分析,进而研究特定因素对细胞或动物蛋白质组的影响,可作为信号通路研究方法。
大肠杆菌是一种与人类生活和生产有着密切关系的革兰氏阴性棒状杆菌,是生命科学研究中常用的模式生物,且因培养简单、方便、价廉、快速、研究透彻、没有毒性、遗传操作方法成熟等诸多优点,已经成为基因工程研究和工业发酵生产的常用工具。SILAC方法经过十年的发展,已经广泛应用于酵母、培养的哺乳动物细胞,甚至果蝇和整体小鼠,但作为原核模式生物的大肠杆菌却没有有效的经过严格评价的标记蛋白质组的SILAC培养基。
M9是大肠杆菌常用的无机盐培养基;SC是一种包含无机盐及各种氨基酸的成分可知的酵母完全培养基,通过调节氨基酸组成可用于筛选基因缺陷型菌株。本课题在两种培养基的基础上,通过调节氨基酸的组成,发展了一种可用于SILAC标记的具有自主知识产权的合成培养基,不仅可以满足大肠杆菌的快速、旺盛生长,适合大肠杆菌的代谢研究,而且可实现野生型大肠杆菌培养过程中重稳定同位素标记氨基酸的蛋白质组完全标记。
我们将大肠杆菌常用菌株BL21(DE3)分别在轻标和重标的培养基中进行培养,等量菌体混合后进行SILAC实验分析,即零差异试验(purenull实验)分析,由此得到定量数据的信噪比(S/N)卡值以及平台的定量精准度。进而我们又在该培养基中通过IPTG进行了外源蛋白的诱导表达,得到诱导前及诱导后1小时的轻重标菌体。菌体等OD混合后,通过正反标SILAC定量实验分析了IPTG处理前后蛋白质组的变化。结果显示,外源蛋白主要以可溶性形式存在;诱导1小时后菌体绝大部分内源蛋白表达下调,包括核糖体蛋白,鞭毛蛋白,tRNA合成,TCA循环蛋白等;在表达水平升高显著的11种蛋白中,半乳糖苷酶LacZ、T7RNA聚合酶和外缘基因本身等三种蛋白的变化幅度最大,证明菌体集中大量能量用于目的蛋白的合成;FTSI、PBPA和LPXC等三种蛋白与细胞壁的合成有关,暗示在诱导后菌体表面积略有升高,细胞形态发生变化,并通过电子显微镜证实了这种变化。该SILAC培养基的开发和高精度定量蛋白质组学的发展可为大肠杆菌定量发酵调控研究和合成生物学探索提供了有效的手段。
The rapid development of mass spectrometry-based proteomic technologies, and the increased accessibility of powerful data analysis tools, have provided proteomic researchers the ability to look at changes beyond only qualitative measurements and into the quantitative intricacies of biological systems. SILAC, or Stable Isotope labeling by Amino acids in Cell culture, is the one of such technologies that has been applied effectively and provided highly reproducible and accurate quantitative measurements. SILAC's initial applications were limited to yeast and cultured cells but more recently has been applied to fruit flies and mice. The technology has not been readily applied to such organism as Escherichia coli because of the lackness of the availability of SILAC medium. Here we present a mass spectrometry-based quantitative proteomics method to first introduce SILAC technology into E. coli via development of a novel growth medium and secondly to examine global proteomic profile changes during IPTG inducing.
E. coli is a rod-shaped gram-negative bacteria that has become one of the most useful and characterized models because of its ease in culture and manipulation. Its genome has been completely sequenced and its proteome has also been well annotated. While E. coli has been widely used both in industry and laboratory research, it has rarely used in SILAC based methods because of a lack of proper labeling medium. Complete labeling is essential to the success of SILAC and the proper medium is an important prerequisite. We have developed a medium with a base composition of M9minimal growth medium and yeast synthetic complete (SC) medium. By adjusting the composition of various amino acids and concentrations of carbon sources, we were able to create a medium that allowed fast growth and complete labeling within just10hours.
To test the accuracy of our quantitative platform we performed a pure null experiment involving both light and heavy labeling of BL21(DE3) competent cells in our SILAC medium. The two cellular populations were mixed at a ratio of1:1according to OD600and quantitative proteomics was performed. The quantitative results showed us the accuracy of our platform is about7%which was the highest so far based on our published literature. In addition, the pure null experiment also allowed us to figure out the necessary signal cutoff and filter thresholds for our following experiments.
Induction by IPTG is commonly used to differentially express proteins in E. coli. During induction, the cells are driven to mass transcribe objective gene of interest. We induced an exogenous protein by IPTG for1hour in BL21(DE3) recombinant cells, and collected the cells before and after induction which were differentially cultured in light and heavy medium respectively. After LC-MS/MS analysis we filtered the data using the cutoffs and thresholds from our pure null experiment and summarized the results according to differential expression. SILAC results showed that most proteins were down-regulated. These proteins were but not limited to tRNA synthesis, tricarboxylic acid cycle (TCA), ribosomal function, and flagella function. There were11proteins that showed over-expression profiles,3of which are expected as they were T7RNA polymerase, LacZ, and the target protein. The remaining over-expressed proteins included3related to cell wall synthesis suggesting that cellular morphology is changed. This was validated by the observation with electro-microscope. This experiment shows that under such conditions as induction the cellular state is changed and the media we developed can be as a good tool for quantitative proteomics research.
引文
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