Development of a single-molecule protease activity analysis platform to elucidate disease-related alterations of circulating proteoform signatures

Protein function represents an important output of biological regulation, yet its direct measurement in circulation has been hindered by limited sensitivity and the inability to discriminate among different proteoforms. We developed scaffolds for single-molecule enzyme activity analysis probes that can resolve functional proteoforms of proteases/peptidases in blood samples. The single-molecule assay uncovered multiple proteoforms of dipeptidyl peptidase 4 (DPP4)/fibroblast activating protein α (FAPα) complexes and aminopeptidase N (CD13) with altered activity patterns in blood samples of pancreatic ductal adenocarcinoma (PDAC) patients. These findings serve as a proof of concept for functional proteoform discovery in complex biofluids and lead to a better understanding of how the remodeled tumor microenvironment imprints functional enzyme signatures in circulation.