Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four-dimensional imaging.
Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four-dimensional imaging.
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DOI：
10.1038/nprot.2018.040
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发表时间：
2018-06
期刊：
影响因子：
14.8
通讯作者：
Ellenberg J
中科院分区：
文献类型：
--
作者：
Politi AZ;Cai Y;Walther N;Hossain MJ;Koch B;Wachsmuth M;Ellenberg J
用荧光蛋白（FP）在蛋白质的内源位点标记蛋白质的能力使得能够在生理水平上定量地理解蛋白质动力学。基因组编辑技术现在已经使这种强大的方法常规适用于哺乳动物细胞和许多其他模型系统，开辟了系统和定量绘制细胞蛋白质组四维图的可能性。3D延时共聚焦显微镜（4D成像）是研究空间和时间蛋白质动力学的重要工具，但它缺乏进行系统分析所需的绝对和可比测量所需的定量能力。另一方面，荧光相关光谱（FCS）提供定量蛋白质组学和生物物理参数，如蛋白质浓度，流体动力学半径和寡聚化，但缺乏在4D空间和时间成像中的高通量应用的能力。在这里，我们提出了一种自动化的实验和计算工作流程，该工作流程集成了这两种方法并以高通量提供定量4D成像数据。处理该数据以产生将图像体素的荧光强度与绝对蛋白质丰度相关联的校准曲线。校准曲线允许将任意荧光强度转换为4D（3D加时间）成像堆栈的所有体素的蛋白质量。通过我们的工作流程，用户可以获取和分析数百个FCS校准的图像系列，以四维映射他们感兴趣的蛋白质。与其他协议相比，目前的协议不需要额外的校准标准，并提供了一个自动采集管道的FCS和成像数据。该方案可以在不到1天的时间内完成。
The ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables the quantitative understanding of protein dynamics at the physiological level. Genome editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool to investigate spatial and temporal protein dynamics, however it lacks the required quantitative power to make absolute and comparable measurements required for systems analysis. Fluorescence correlation spectroscopy (FCS) on the other hand provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius and oligomerization but lacks the ability for high-throughput application in 4D spatial and temporal imaging. Here, we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high throughput. This data is processed to yield a calibration curve relating the fluorescence intensities of image voxels to absolute protein abundance. The calibration curve allows the conversion of the arbitrary fluorescence intensities to protein amounts for all voxels of 4D (3D plus time) imaging stacks. With our workflow the user can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared to other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in less than 1 day.
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影响因子：
14.8
作者：
Koch B;Nijmeijer B;Kueblbeck M;Cai Y;Walther N;Ellenberg J
通讯作者：
Ellenberg J
影响因子：
48
作者：
Conrad, Christian;Wuensche, Annelie;Tan, Tze Heng;Bulkescher, Jutta;Sieckmann, Frank;Verissimo, Fatima;Edelstein, Arthur;Walter, Thomas;Liebel, Urban;Pepperkok, Rainer;Ellenberg, Jan
通讯作者：
Ellenberg, Jan
影响因子：
16.6
作者：
Lukinavičius G;Blaukopf C;Pershagen E;Schena A;Reymond L;Derivery E;Gonzalez-Gaitan M;D'Este E;Hell SW;Wolfram Gerlich D;Johnsson K
通讯作者：
Johnsson K
影响因子：
64.8
作者：
Cuylen S;Blaukopf C;Politi AZ;Müller-Reichert T;Neumann B;Poser I;Ellenberg J;Hyman AA;Gerlich DW
通讯作者：
Gerlich DW
影响因子：
46.9
作者：
Capoulade, Jeremie;Wachsmuth, Malte;Knop, Michael
通讯作者：
Knop, Michael