Table 3.

Detailed TMPRSS2 mass spectrometry detection biochemical assay.

Step #	Process	Notes
1	200 nL of peptide substrate dispensed into 384-well plates.	Peptide (Cbz-SKPSKRSFIED, 250x, dissolved in DMSO) was dispensed using an ECHO 655 acoustic dispenser (LabCyte) into a Greiner 384-well plate (Cat #781201).
2	Compounds and controls (200 nL) were pre-spotted into 384-well plates.	Inhibitor (250x) or vehicle control (DMSO) was dispensed using an ECHO 655 acoustic dispenser (LabCyte). Compounds in dose-response were dispensed to columns 5–24 and controls dispensed into columns 1–4.
3	TMPRSS2 diluted in assay buffer dispensed into 384-well plates.	TMPRSS2 (reconstituted in 50% glycerol at 8.75 μM, 22x) in assay buffer (50 mM Tris pH 8, 150 mM NaCl) was dispensed using a BioRAPTR (Beckman Coulter). Total reaction volume of 50 μL.
4	Incubated at RT for 1 hr	Final assay conditions are 10 μM peptide and 0.4 μM TMPRSS2 in assay buffer (50 mM Tris-HCl pH 8, 150 mM NaCl)
5	Addition of quench solution (50 μL)	Quench solution (90:10 ACN:H2O + 0.1% formic acid + 100 nM of IS (Cbz-SK-{13C5 15N Pro}-SKR. Total volume of 100 μL.
6	Dilution of reaction (10-fold)	Dilution of reaction 10-fold using 20 mM ammonium formate + 0.1% formic acid.
7	Measure mass detection on SciEx 6500	The LC buffers were A: 0.1% difluoroacetic acid in water and B: acetonitrile, and a gradient elution was used that went from 5% Buffer B to 90% Buffer B in 0.5 min with a flow rate of 0.70 mL/min. The product cleavage fragment (Cbz-SKPSKR): 418.8/702.4 m/z. The IS cleavage fragment: 421.64/708.24