Multifunctional Concentric FRET-Quantum Dot Probes for Tracking and Imaging of Proteolytic Activity

Proteolysis has many important roles in physiological regulation. It is involved in numerous cell signaling processes and the pathogenesis of many diseases, including cancers. Methods of visualizing and assaying proteolytic activity are therefore in demand. Förster resonance energy transfer (FRET) probes offer several advantages in this respect. FRET supports end-point or real-time measurements, does not require washing or separation steps, and can be implemented in various assay or imaging formats. In this chapter, we describe methodology for preparing self-assembled concentric FRET (cFRET) probes for multiplexed tracking and imaging of proteolytic activity. The cFRET probe comprises a green-emitting semiconductor quantum dot (QD) conjugated with multiple copies of two different peptide substrates for two target proteases. The peptide substrates are labeled with different fluorescent dyes, Alexa Fluor 555 and Alexa Fluor 647, and FRET occurs between the QD and both dyes, as well as between the two dyes. This design enables a single QD probe to track the activity of two proteases simultaneously. Fundamental cFRET theory is presented, and procedures for using the cFRET probe for quantitative measurement of the activity of two model proteases are given, including calibration, fluorescence plate reader or microscope imaging assays, and data analysis. Sufficient detail is provided for other researchers to adapt this method to their specific requirements and proteolytic systems of interest.