Sequencing grade modified trypsin lysc

Avertin (Tribromoethanol; Sigma-Aldrich, 250 mg/kg i.p.) was used to anesthetize the animals. Each sample (i.e., brains removed from mice) was dissolved in 100 μL of 5% SDS, reduced with 20 mM dithiothreitol in 50 mM NH₄HCO₃ for 10 min at 95 °C, and alkylated with 40 mM iodoacetamide in 50 mM NH₄HCO₃ for 30 min under the light blocking. S-TRAP™ (Protifi) was used for the fast and reproducible preparation of proteomics samples. Denatured, non-digested proteins were bound to the S-TRAP™. Each sample was incubated overnight at 37 °C with 12.5 μg sequencing grade modified trypsin/LysC (Promega) in 50 mM NH₄HCO₃ buffer (pH 7.8) on S-TRAP column. Eluted peptide sample was dried down and quantified. The samples were re-suspended in 0.1% formic acid and dried for LC-MS analysis.

Each dried urine sample was dissolved in 100 µL of 8 M urea, reduced with 20 mM dithiothreitol in 50 mM NH₄HCO₃ for 60 min at 25 °C, and alkylated with 40 mM iodoacetamide in 50 mM NH4HCO3 for 60 min in the dark. Urea concentration was diluted to less than 1.0 M. Each urine sample was incubated overnight at 37 °C with 12.5 µg sequencing grade modified trypsin/LysC (Promega, Madison, WI, USA) in 50 mM NH4HCO3 buffer (pH 7.8), followed by quenching with 10uL of 5% formic acid and lyophilization with a cold trap. The samples were re-suspended in 0.1% formic acid, desalted using C18 ZipTips (Millipore, Burlington, MA, USA), and dried for LC-MS analysis.