Quantitative measurement of proteolytic rates with quantum dot-peptide substrate conjugates and Förster resonance energy transfer

An important challenge in biology is the development of probes for visualizing and quantitatively tracking enzyme activity. Proteases are an important class of enzyme with value as both diagnostic and therapeutic targets. In this chapter, we describe the preparation of quantum dot (QD)-peptide substrate conjugates as probes for measuring proteolytic activity. QDs have several highly advantageous optical properties that make these materials especially well suited for applications in bioanalysis and bioimaging. Further, peptide substrates for proteases can be controllably self-assembled to QDs and this capability, in combination with Förster resonance energy transfer (FRET), enables the design of quantitative in vitro assays capable of directly reporting on proteolytic activity. We present a detailed method for the preparation, calibration, and application of such QD probes, along with methods of analysis to generate progress curves for the proteolytic digestion of substrate. Representative data are illustrated for two different proteases and two different QD-fluorescent dye FRET pairs. The general methodology is likely to be applicable with other hydrolytic enzymes in addition to proteases. Overall, the method is straightforward to implement with commercially available materials and does not require specialized expertise.