Journal: Cell reports methods

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

doi: 10.1016/j.crmeth.2023.100688

Figure Lengend Snippet: Figure 1. Development of PMAC as a core scaffold for the microdevice-based assay (A) Concept of single-molecule enzyme activity analysis using microfabricated chambered device. (B) The design of the microfabricated chambered device for single-molecule enzyme activity analysis. (C) Structure of PMAC-based fluorogenic probes of amidases, peptidases, and proteases. (D) Absorbance spectra of EP-PMAC (5 mM) before (black line) and after (red line) a reaction with recombinant DPP4 (300 ng/mL) in Tris-HCl buffer (pH 7.4, 100 mM) containing CHAPS (0.1%) at 25C. (E) Fluorescence spectra of EP-PMAC in the same condition as in (d). Fluorescence spectra were measured every 10 min. lex. = 350 nm. (F) Structures of GP-AMC and EP-PMAC and their kinetic parameters in the reaction with DPP4 in 384-well plate-based assay. (G) Fluorescence images of microdevice loaded with GP-PMAC or EP-PMAC (50 mM) and recombinant DPP4 (10 ng/mL) in HEPES buffer (pH 7.4, 100 mM) containing CaCl2 (1 mM), MgCl2 (1 mM), DTT (1 mM), and Triton X-100 (3 mM). Images were taken after incubation at 37C for 2 h.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-DPP4 (CD26) mouse monoclonal antibody Origene Cat. #TA500733 Lot #F002; RRID: AB_11140529 Anti-CD13 rabbit monoclonal antibody Abcam Cat. #ab108310 Lot #GR3306688-9; RRID:AB_10866195 Anti-mouse IgG HRP-linked antibody Cytiva Cat. #NA931V Lot #17479274; RRID: AB_772210 Anti-rabbit IgG HRP-linked F(ab’)2 fragment Cytiva Cat. #NA9340V Lot #17402176; RRID: AB_772191 Chemicals, peptides, and recombinant proteins DPP4, human recombinant R&D Systems Cat. #9168-SE Lot #DFQM0420081 Aminopeptidase N, human recombinant R&D Systems Cat. #3815-ZN Lot #PQP0221041 Bovine serum albumin Wako Cat. #016-15091 Lot #LEQ2235 FC-70 Sigma-Aldrich Cat. #F9880 Fomblin PFPE Solvay Cat. #YL VAC 25/6 Bestatin Tokyo Chemical Industries Cat. #U0111 Saxagliptin Tokyo Chemical Industries Cat. #A3435 NHS-activated Sepharose 4 Fast Flow Cytiva Cat. #17-0906-01 Critical commercial assays IEF gels (pH 3–7) Invitrogen Cat. #EC6645BOX Bis-Tris Native PAGE gels (4–16%) Invitrogen Cat. #BN1004BOX Native Mark unstained protein standard Invitrogen Cat. #LC0725 Sainome diced electrophoresis gel assay plate Sainome Corp. Cat. #SAI-386-PEB HDL purification kit (ultracentrifugation free) Cell Biolabs Inc. Cat. #STA-607 Bio-Rad protein assay dye reagent concentrate Bio-Rad Cat. #5000006JA

Techniques: Activity Assay, Recombinant, Fluorescence, Incubation

Journal: Cell reports methods

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

doi: 10.1016/j.crmeth.2023.100688

Figure Lengend Snippet: Figure 6. Identification of high- and low-activity species of single-molecule DPP4 and its alteration in blood samples of patients with pancreatic tumors (A) Fluorescence images of microdevice loaded with EP-PMAC (50 mM) and plasma samples (1:5,000 dilution; top) or recombinant DPP4 (1 ng/mL; bottom). The histograms indicate the activity distribution of single-molecule enzymes. White arrows indicate the low-activity DPP4 species detected in plasma. (B) Histograms of single-molecular activities of EP-PMAC (50 mM) with blood samples (1:5,000 dilution) of patients with early-stage pancreatic tumor (stages I and II, 30 individuals) and healthy subjects (30 individuals). Samples with aberrant ratio of high- and low-activity species of DPP4 were indicated by red square. (C) Left: dotted plot of the proportion of high-activity DPP4 species in plasma of early-stage (stages I and II) pancreatic tumor patients or of healthy subjects in (B). p value was calculated using Student’s t test. Right: dot plot of the total DPP4 activity in plasma samples monitored using conventional 384-well plate-based fluorometric assay. EP-PMAC (10 mM) was incubated with plasma samples (1:500 dilution) in HEPES buffer (pH 7.4, 100 mM) containing CaCl2 (1 mM), MgCl2 (1 mM), DTT (1 mM), and CHAPS (0.1%) at 25C for 30 min. p value was calculated using Student’s t test. (D) Western blot analysis of DPP4 of plasma samples of patients with pancreatic tumors that showed altered DPP4 populations or that of healthy subjects.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-DPP4 (CD26) mouse monoclonal antibody Origene Cat. #TA500733 Lot #F002; RRID: AB_11140529 Anti-CD13 rabbit monoclonal antibody Abcam Cat. #ab108310 Lot #GR3306688-9; RRID:AB_10866195 Anti-mouse IgG HRP-linked antibody Cytiva Cat. #NA931V Lot #17479274; RRID: AB_772210 Anti-rabbit IgG HRP-linked F(ab’)2 fragment Cytiva Cat. #NA9340V Lot #17402176; RRID: AB_772191 Chemicals, peptides, and recombinant proteins DPP4, human recombinant R&D Systems Cat. #9168-SE Lot #DFQM0420081 Aminopeptidase N, human recombinant R&D Systems Cat. #3815-ZN Lot #PQP0221041 Bovine serum albumin Wako Cat. #016-15091 Lot #LEQ2235 FC-70 Sigma-Aldrich Cat. #F9880 Fomblin PFPE Solvay Cat. #YL VAC 25/6 Bestatin Tokyo Chemical Industries Cat. #U0111 Saxagliptin Tokyo Chemical Industries Cat. #A3435 NHS-activated Sepharose 4 Fast Flow Cytiva Cat. #17-0906-01 Critical commercial assays IEF gels (pH 3–7) Invitrogen Cat. #EC6645BOX Bis-Tris Native PAGE gels (4–16%) Invitrogen Cat. #BN1004BOX Native Mark unstained protein standard Invitrogen Cat. #LC0725 Sainome diced electrophoresis gel assay plate Sainome Corp. Cat. #SAI-386-PEB HDL purification kit (ultracentrifugation free) Cell Biolabs Inc. Cat. #STA-607 Bio-Rad protein assay dye reagent concentrate Bio-Rad Cat. #5000006JA

Techniques: Activity Assay, Fluorescence, Clinical Proteomics, Recombinant, Incubation, Western Blot