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Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between
two chromophores in which excitation is transferred from a donor molecule (fl uorophore)
to an acceptor molecule (quencher) without photon emission. Removal of the acceptor molecule
from donor?s proximity results in quantifi able fl uorescence (Figure 1). FRET is an important
technique and can be utilized in monitoring peptide substrate cleavage by proteases in diff erent
We report a simple, sensitive, specifi c FRET based platform for detection of proteases
profi les in diff erent biological samples by screening against a combinatorial library of internally
quenched peptide probes (IQFP?s). Th e library was utilized to determine the proteolytic profi le
of two clinically relevant biological fl uids, serum and bronchoalveolar lavage fl uid. Both fl uids
displayed a distinct and quantifi able proteolytic signature.
Th e library was further utilized to distinguish between the protease profi les of diff erent
Aspergillus species and also to identify peptide probes that are indicative of invasive aspergillosis
(IA) in a guinea pig model.
Substrate specifi city of biologically relevant recombinant enzymes such as prolyl
oligopeptidase (POP) and fi broblast activation protein (FAP) both of which are implicated in
number of human diseases was also determined using the IQFP library screen.
Th e approach provides a comprehensive fi nger print of the proteolytic activity of complex
biological fl uids as well as individual proteases. Th e technology is currently being applied to
identify proteases as biomarkers in a variety of disease states for subsequent development of in
Kalyani Jambunathan is a researcher at SRI International?s Center for Advanced Drug Research (CADRE), at
present working on NIAID funded project to develop FRET based assay to detect fungal derived proteolytic
activity during invasive Aspergillosis. She graduated with a BSc in chemistry from Madras Christian College
(MCC, India), MSc in chemistry from Indian Institute of Technology-Madras (IIT-M) and PhD in chemistry
under the guidance of Dr. H. Mario Geysen in Department of Chemistry at University of Virginia. Her areas of
specialization include but not limited to peptide science, molecular biology, assay design and development.
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