Unifying Fluorescence Microscopy and Mass Spectrometry for Studying Protein Complexes in Cells
Unifying Fluorescence Microscopy and Mass Spectrometry for Studying Protein Complexes in Cells
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DOI：
10.1074/mcp.m800397-mcp200
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发表时间：
2009-06-01
影响因子：
7
通讯作者：
Krutchinsky, Andrew N.
中科院分区：
文献类型：
--
作者：
Deng, Changhui;Xiong, Xinghua;Krutchinsky, Andrew N.
我们开发并应用了一种将荧光显微镜和质谱相结合的方法来研究酵母细胞中蛋白质和蛋白质复合物的时空特性。为了结合这些技术，首先我们生产了各种DNA结构，可用于模块化荧光和亲和标签的蛋白质基因组标记。模块化标签由融合到各种小亲和力表位的单体荧光蛋白的多个版本之一组成。在这一步之后，我们通过用各种荧光探针和亲和探针标记酿酒酵母中参与内吞作用的大蛋白复合物eisosomes的核心成分两种酵母蛋白Pil1和Lsp1来测试构建物。在所产生的模块化标签中，我们发现了几种最适合确定亚细胞定位和纯化标记蛋白质和蛋白质复合物的组合，以便通过质谱进行详细分析。最后，我们将设计的方法应用于寻找新的异构体蛋白组分，并对通过可逆磷酸化和去磷酸化调节异构体组装和拆卸的分子机制有了新的认识。我们的研究结果表明，这种将荧光显微镜和质谱相结合的方法为研究活细胞中蛋白质复合物的组成和动态特性的分子机制提供了独特的视角。中国生物医学工程学报（英文版），2009。
We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. After this step we tested the constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosis in Saccharomyces cerevisiae, with a variety of fluorescent and affinity probes. Among the modular tags produced we found several combinations that were optimal for determining subcellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and dephosphorylation. Our results indicate that this approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells. Molecular & Cellular Proteomics 8: 1413-1423, 2009.