Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.

Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level. The peptidase/protease probes were prepared on the basis of a 7-amino-4-methylcoumarin fluorophore. The introduction of a phosphonic acid to the core scaffold made the probe suitable for use in a microdevice-based assay, while phosphonic acid served as the handle for the affinity separation of the probe using Phos-tag. Using this semi-automated scheme, 48 fluorogenic probes for the single-molecule peptidase/protease activity analysis were prepared. Activity-based screening using blood samples revealed altered single-molecule activity profiles of CD13 and DPP4 in blood samples of patients with early-stage pancreatic tumors. The study shows the power of single-molecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.

Thioester-Based Coupled Fluorogenic Assays in Microdevice for the Detection of Single-Molecule Enzyme Activities of Esterases with Specified Substrate Recognition.

Single-molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single-molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water-soluble thiol-reacting probes based on phosphonate-substituted boron dipyrromethene (BODIPY). The system enables the detection of cholinesterase activities in blood samples at single-molecule level, and it is shown that the dissecting alterations of single-molecule esterase activities can serve as an informative platform for activity-based diagnosis.