Eggeling C, Jäger S, Winkler D, Kask Peet
Max-Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Goettingen, Germany.
Curr Pharm Biotechnol. 2005 Oct;6(5):351-71. doi: 10.2174/138920105774370571.
We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.
我们比较了多种用于研究Förster共振能量转移（FRET）分析的荧光涨落光谱（FFS）方法的准确性。例如，使用基于荧光强度、荧光相关光谱（FCS）和荧光强度分布分析（FIDA）的方法监测并分析蛋白酶胰蛋白酶对双标记的FRET活性肽底物的切割。将所呈现的荧光数据与同一分析中获得的高压液相色谱（HPLC）数据进行比较。HPLC分析揭示了FRET方法的一些普遍缺点，如标记不完全和需要等分试样。然而，同时使用两个监测两种标记荧光信号的光子探测器可显著改善分析。特别是，两种全局分析工具二维荧光强度分布分析（2D-FIDA）和双色全局荧光相关光谱（2CG-FCS）突出了FFS和FRET相结合的潜力。虽然传统的FIDA和FCS自相关或交叉相关分析给用户留下了双色和FRET应用中固有的缺点，但这些影响通过分子水平的全局分析得以克服。此外，当将任何荧光相关方法与FRET结合时，分析未归一化而非归一化的相关数据是有利的，因为未归一化数据的分析引入了更高的准确性，并且对实验缺点不太敏感。
