Purified EBOV (strain Mayinga 1976, Zaire) GP protein was solubilized in ultrapure water to a final concentration of 1 mg/ml. The protein was then incubated with 5 μM Y18 or 1% DMSO for an additional 30 min and exposed to UV light (365 nm) for 10 min on ice. The probe-labeled protein was analyzed by peptide mapping. After an overnight trypsin digest at 37°C, the reaction was quenched by the addition of trifluoroacetic acid (TFA). The tryptic peptides were resuspended and separated by C18 reversed-phase HPLC (RP-HPLC) (Easy-nLC II; Thermo Fisher Scientific, CA) with in-line UV detection. The fractions were collected, dried, and then resuspended in TFA. Each fraction was further separated on an analytical C18 column. The peptides were identified by MALDI (MS and MS/MS) analysis. The photoaffinity labeling of HIV gp41 with Y20 and of influenza virus HA with Y21 and subsequent MS measurements were performed using the same procedure.

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