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 singlemolecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.