*4.2. Urine Sample Preparation for LC-MS/MS*

First morning urine samples were collected from all examined patients. The middle portion of freshly passed morning urine was collected in 10 mL test tubes, centrifuged at 3000 rpm for 15 min, the supernatant was frozen in 1 mL aliquots and stored at −20 ◦C.

Urine aliquots with a volume of 0.1 mL were quickly thawed and 0.5 mL of cold acetone was added to precipitate proteins overnight at −20 ◦C. Then the samples were centrifuged at 20,000× *g* for 10 min, the supernatant was removed, the precipitate was dissolved in 50 μL of 8 M urea/200 mM Tris-HCL, pH 8.5. The proteins were restored with 5 mM dithiotreitol for 30 min at 37 ◦C, alkylated with 20 mM iodoacetamide in the dark for 30 min. Before hydrolysis, 200 μL of deionized water was diluted, trypsin (Trypsin Gold, Promega, Madison, WI, USA) was added in an enzyme-protein ratio of 1:25, incubated overnight at 37 ◦C. The reaction was stopped by adding formic acid to the final concentration of 1%. Peptides were centrifuged at 18,000× *g*, the supernatant was left for desalting. Desalting was carried out by solid-phase extraction using plates (Oasis HLB 96-well Microelution Plate, Waters, Beverley, MA, USA). The eluate was lyophilized and dissolved in 0.1% formic acid to a concentration of 0.5mg/mL for further LC-MS/MS analysis.
