*4.4. Enzymatic Digestion in-Solution*

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.
