ACE2 substrate peptide cleavage activity in plasma samples in the presence and absence of SARS-CoV-2 spike protein synthetic peptides

To evaluate the specificity of ACE2 substrate cleavage activity by the plasma, we designed tiled SARS-CoV-2 spike protein RBD peptides which covered RBD amino acid sequence from residues Arg319 to Phe541 including receptor-binding motif from Ser438 to Gln506 based on previous comprehensive structure analysis on viral-host interaction (37) and had them synthesized by SB-PEPTIDE (SmartBioscience SAS, France; Table 2). Plasma samples and positive control, purified recombinant human ACE2 protein provided in the ACE2 assay kit (Abcam, Cat. #ab273297), were incubated with or without fivefold serially diluted peptide pools at room temperature for 15 min before adding the substrate. Samples incubated with ddH₂O and dimethyl sulfoxide (DMSO) which were equivalent to the concentrations used in the peptide pool reconstitution served as negative controls. Then 50 µL of ACE2 substrate (pre-diluted according to the manufacturer’s protocol) was added into the wells, and the samples were assayed. RFUs were measured, and data were analyzed as described below.

SARS-CoV-2 spike RBD peptides for ACE2-like activity competition inhibition assay