Determination of absolute protein numbers in single synapses by a GFP-based calibration technique
Determination of absolute protein numbers in single synapses by a GFP-based calibration technique
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DOI：
10.1038/nmeth783
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发表时间：
2005-09-01
期刊：
影响因子：
48
通讯作者：
Okabe, S
中科院分区：
文献类型：
--
作者：
Sugiyama, Y;Kawabata, I;Okabe, S
为了建立神经元分子组织的定量模型，了解单个突触上蛋白质分子的数量是至关重要的。在这里，我们开发了一种方法，通过校准带有单个EGFP分子的微球的荧光强度来估计实际兴奋性突触上单个蛋白质的绝对数量。在培养的海马神经元中，我们观察到在神经元分化和随后的稳定过程中，每个单个突触的突触后蛋白数量单调增加。成熟时，我们计算出单个兴奋性突触后部位包含100-450个单独的突触后蛋白，如PSD-95、GKAP、Shank和Hmer。突触后蛋白含量的这一狭窄范围表明，突触后分子组织的化学计量学相对简单。基于EGFP的校准技术为估计大分子复合体中的蛋白质数量提供了一种前所未有的通用方法。
To build a quantitative model of molecular organization of neurons, it is essential to have information about the number of protein molecules at individual synapses. Here we developed a method to estimate absolute numbers of individual proteins at actual excitatory synapses by calibrating the fluorescence intensity of microspheres with single EGFP molecules. In cultured hippocampal neurons, we observed a monotonous increase of postsynaptic protein numbers per single synapse during neuronal differentiation and subsequent stabilization. At maturity we calculated that a single excitatory postsynaptic site contains 100-450 of individual postsynaptic proteins, such as PSD-95, GKAP, Shank and Homer. This narrow range of postsynaptic protein content suggests relatively simple stoichiometry of postsynaptic molecular organization. The EGFP-based calibration technique provides an unprecedented general method for estimating the amounts of proteins in macromolecular complexes.