Mass spectrometry of intact protein samples was performed with an Agilent 6210 Time of Flight Mass Spectrometer with a 4GHz upgrade and an Agilent 1200 Rapid Resolution HPLC. The samples were analyzed on an Agilent Zorbax 300 Extend C18 Rapid Resolution column at 70°C, using reverse phase chromatography with a gradient from 20 to 90% acetonitrile containing 0.1% formic acid. Data were analyzed using Agilent Masshunter B.06 Qualitative Analysis with the Bioconfirm upgrade.
1 nmol of NS5A protein in 1 mM TCEP, 20 mM iodoacetamide, 8M urea was incubated with 1 μg of Lys-C (Roche) and incubated for 4 hr at 37ºC. The digests were diluted 4 fold and incubated with 5 μg of trypsin for 12 hr at 37ºC. The digests were acidified with 0.1% formic acid and desalted with C-18 spin columns (Thermo Scientific). The eluted peptides were dried and resuspended in 10 μL of acetonitrile and 0.01% formic acid. The mixture of peptides was analyzed using accurate-mass nano-LC-MS/MS with an Agilent 6530 Quadrupole Time of Flight mass spectrometer. Chromatographic separation of peptides was performed with an Agilent 1260 Capillary/Nano-HPLC system coupled to a chip cube with a Phosphochip (Agilent Technologies) using a 0 to 90% acetonitrile gradient containing 0.1% formic acid. Data were analyzed by searching a customized protein database containing the sequence of NS5A using SpectrumMill (Agilent).
1 nmol of NS5A protein in 1 mM TCEP, 20 mM iodoacetamide, 8M urea was incubated with 1 μg of Lys-C (Roche) and incubated for 4 hr at 37ºC. The digests were diluted 4 fold and incubated with 5 μg of trypsin for 12 hr at 37ºC. The digests were acidified with 0.1% formic acid and desalted with C-18 spin columns (Thermo Scientific). The eluted peptides were dried and resuspended in 10 μL of acetonitrile and 0.01% formic acid. The mixture of peptides was analyzed using accurate-mass nano-LC-MS/MS with an Agilent 6530 Quadrupole Time of Flight mass spectrometer. Chromatographic separation of peptides was performed with an Agilent 1260 Capillary/Nano-HPLC system coupled to a chip cube with a Phosphochip (Agilent Technologies) using a 0 to 90% acetonitrile gradient containing 0.1% formic acid. Data were analyzed by searching a customized protein database containing the sequence of NS5A using SpectrumMill (Agilent).