Biotinylated Protein Purification and Digestion

After organ preparation, biotinylated proteins were purified as previously described (13 (link)). Briefly, biotinylated proteins were purified from homogenized liver (1 mg protein) using a Bravo AssayMap platform and AssayMap streptavidin cartridges (Agilent). Briefly, cartridges were prewashed with 50 mM ammonium bicarbonate (pH 8), and then samples were loaded. Nonbiotinylated proteins were removed by extensively washing the cartridges with 8 M urea in 50 mM ammonium bicarbonate buffer (pH 8). Cartridges were washed with rapid digestion buffer (Promega; rapid digestion buffer kit), and bound proteins were subjected to on-column digestion using mass spectrometry-grade trypsin/Lys-C rapid digestion enzyme (Promega, Madison, WI) at 70°C for 2 h. Released peptides were desalted in the Bravo platform using AssayMap C₁₈ cartridges, and the organic solvent was removed by vacuum centrifugation (SpeedVac). Samples were stored at −20°C prior to liquid chromatography tandem mass spectrometry (LC–MS/MS) SWATH (sequential window acquisition of all theoretical fragment ion spectra) analysis.