Specific mutations in H5N1 mainly impact the magnitude and velocity of the host response in mice

Tchitchek, Nicolas; Eisfeld, Amie J; Tisoncik-Go, Jennifer; Josset, Laurence; Gralinski, Lisa E; Bécavin, Christophe; Tilton, Susan C; Webb-Robertson, Bobbie-Jo; Ferris, Martin T; Totura, Allison L; Li, Chengjun; Neumann, Gabriele; Metz, Thomas O; Smith, Richard D; Waters, Katrina M; Baric, Ralph; Kawaoka, Yoshihiro; Katze, Michael G

All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Ammonium bicarbonate and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA), and sequencinggrade modified trypsin was purchased from Promega (Madison, WI). Bicinchoninic acid (BCA) assay reagents and standards were obtained from Pierce (Rockford, IL); and purified, deionized water, >18 MΩ, (Nanopure Infinity ultrapure water system, Barnstead, Dubuque, IA) was used to make all aqueous buffers. Dissected lung tissues were directly frozen at −80°C. To prepare protein extracts, thawed tissues were suspended in 1 ml of 8 M urea in 50 mM NH 4 HCO 3 and homogenized using a TissueLyser or Magnalyser. Protein concentrations of the cleared homogenates were determined by BCA protein assay and diluted to uniform volume in 50 mM ammonium bicarbonate, pH 7.8. Proteins were reduced with 10 mM dithiothreitol, followed by alkylation of free sulfhydryl groups with 40 mM iodoacetamide at 37°C in the dark; each reaction was performed for 1 h at 37°C with constant shaking at 550 rpm. Denatured and reduced samples were diluted 10-fold with 50 mM ammonium bicarbonate, pH 7.8, and CaCl 2 was added to a final concentration of 1 mM prior to enzymatic digestion. Sequencing-grade modified trypsin was activated by adding 20 μL of 50 mM ammonium bicarbonate, pH 7.8, to 20 μg lyophilized trypsin and incubating for 10 min at 37°C. Activated trypsin was then added to the samples at 1:50 (w/w) trypsin-to-protein ratio, and samples were digested at 37°C for 3 h with constant shaking at 800 rpm; reactions were quenched by rapid freezing in liquid nitrogen. Digested samples were desalted using solid phase extraction columns (Discovery C18, Supelco, Bellefonte, PA), according to the manufacturer's instructions. Samples were concentrated to 100 μLin vacuo (Speed-Vac SC 250 Express, Thermo Savant, Holbrook, NY), and a BCA protein assay was performed to verify final peptide concentrations. Samples were stored at −80°C until strong cation exchange fractionation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) or quantitative LC-MS analyses.