Rapid digestion buffer kit

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.

All sample preparation and mass spectrometry were conducted at the proteomics core at Sanford Burnham Prebys. Protein concentration was determined using a bicinchoninic acid protein assay (Thermo Scientific). Disulfide bridges were reduced with 5 mM tris (2-carboxyethyl)phosphine at 30°C for 60 min, and cysteines were subsequently alkylated with 15 mM iodoacetamide in the dark at room temperature for 30 min. Affinity purification was carried out in a Bravo AssayMap platform (Agilent) using AssayMap streptavidin cartridges (Agilent). Briefly, cartridges were first primed with 50 mM ammonium bicarbonate and then proteins were slowly loaded onto the streptavidin cartridge. Background contamination was removed with 8 M urea, 50 mM ammonium bicarbonate. Finally, cartridges were washed with Rapid digestion buffer (Promega, Rapid digestion buffer kit), and proteins were subjected to on-cartridge digestion with mass spec grade Trypsin/Lys-C Rapid digestion enzyme (Promega, Madison, WI, USA) at 70°C for 1 hr. Digested peptides were then desalted in the Bravo platform using AssayMap C18 cartridges and dried down in a SpeedVac concentrator.

Affinity purification and digestion of biotinylated proteins were carried out in an automated fashion in a Bravo AssayMap platform (Agilent) using AssayMap streptavidin cartridges (Agilent). Briefly, cartridges were first primed with 50 mM ammonium bicarbonate and then proteins were slowly loaded onto the streptavidin cartridge. Background contamination was removed with 8 M urea, 50 mM ammonium bicarbonate. Finally, cartridges were washed with Rapid digestion buffer (Promega, Madison, WI, USA, Rapid digestion buffer kit) and proteins were subjected to on-cartridge digestion with mass spec grade Trypsin/Lys-C Rapid digestion enzyme (Promega) at 70 °C for 1 h. Digested peptides were then desalted in the Bravo platform using AssayMap C18 cartridges and dried down in a SpeedVac concentrator.