Sensitive single-molecule protein quantification and protein complex detection in a microarray format.
Sensitive single-molecule protein quantification and protein complex detection in a microarray format.
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DOI：
10.1002/pmic.201100361
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发表时间：
2011-12
期刊：
影响因子：
3.4
通讯作者：
Mitra RD
中科院分区：
文献类型：
--
作者：
Tessler LA;Mitra RD
单分子蛋白质分析提供了敏感的蛋白质定量与数字读出，是有希望的研究生物系统和临床检测生物标志物。然而，目前的单分子平台依赖于一次对一种蛋白质的定量。传统的抗体微阵列可以同时检测多种蛋白质，但它们依赖于荧光分子的总体平均，灵敏度较低，定量较少。在这里，我们展示了一种微阵列格式的单分子蛋白质检测，该检测由超低背景表面和单分子成像实现。通过使用混合数模量化，数字读出提供高灵敏度，低飞摩尔检测极限和4个数量级的动态范围。从粗细胞裂解液中，我们平行测量了p53和MDM2的水平，证明了用于蛋白质组学分析的数字抗体微阵列的概念。我们还应用单分子芯片检测细胞裂解液中的p53-MDM2蛋白复合物。我们的研究为单分子蛋白质方法的发展和应用提供了前景，因为它代表了单一和高度多重研究之间的技术桥梁。
Single-molecule protein analysis provides sensitive protein quantitation with a digital read-out and is promising for studying biological systems and detecting biomarkers clinically. However, current single-molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less-sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here we demonstrate a single-molecule protein assay in a microarray format enabled by an ultra-low background surface and single-molecule imaging. The digital read-out provides a highly sensitive, low femtomolar limit of detection and 4 orders of magnitude of dynamic range through the use of hybrid digital-analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single-molecule microarray to detect the p53-MDM2 protein complex in cell lysate. Our study is promising for development and application of single-molecule protein methods because it represents a technological bridge between single-plex and highly multiplex studies.