Kavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands.
Department of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands.
Proc Natl Acad Sci U S A. 2018 Mar 27;115(13):3338-3343. doi: 10.1073/pnas.1707207115. Epub 2018 Mar 12.
Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.
蛋白质组学分析为细胞系统的分子途径和生物体的状态提供了重要信息。质谱分析目前是蛋白质组学分析的首选方法。然而，对大量样本的需求使得小规模蛋白质组学研究具有挑战性。在这里，我们展示了基于单分子 FRET 的蛋白质指纹分析的概念验证。我们利用 AAA+蛋白酶 ClpXP 来扫描肽段。通过使用供体荧光标记的 ClpP，我们依次从肽段的受体标记氨基酸读取 FRET 信号。这种重新设计的 ClpXP 表现出单向加工、高连续性，并有可能检测到低丰度的蛋白质。我们的技术是使用少量样本对蛋白质底物进行测序的一种很有前途的方法。
