{Reference Type}: Journal Article
{Title}: Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.
{Author}: Sakamoto S;Hiraide H;Minoda M;Iwakura N;Suzuki M;Ando J;Takahashi C;Takahashi I;Murai K;Kagami Y;Mizuno T;Koike T;Nara S;Morizane C;Hijioka S;Kashiro A;Honda K;Watanabe R;Urano Y;Komatsu T;
{Journal}: Cell Rep Methods
{Volume}: 4
{Issue}: 1
{Year}: 2024 Jan 22
暂无{DOI}: 10.1016/j.crmeth.2023.100688
{Abstract}: 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.