*4.3. Label-Free Untargeted LC-MS/MS Urine Proteomic Analysis*

The resulting tryptic peptide mixture was analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method based on a nano-HPLC Dionex Ultimate3000 system (Thermo Fisher Scientific, Madison, WI, USA) and a timsTOF Pro (Bruker Daltonics, Billerica, MA, USA) mass spectrometer. A packed emitter column (C18, 25 cm × 75 μm 1.6 μm) (Ion Optics, Parkville, Australia) was used to separate peptides at a flow rate of 400 nL/min by gradient elution from 4% to 90% of phase B during 40 min. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile.

Mass spectrometric analysis was performed using the parallel accumulation serial fragmentation (PASEF) acquisition method. An electrospray ionization (ESI) source was operated at 1500 V capillary voltage, 500 V end plate offset and 3.0 L/min of dry gas at temperature of 180 ◦C. The measurements were carried out in the m/z range from 100 to 1700 Th. The ion mobility was in the range from 0.60 to 1.60 V s/cm2. The total cycle time was 1.88 s and the number of PASEF MS/MS scans was set to 10.

*Targeted quantitative LC-MS/MS using multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS).*

Targeted quantitative LC-MS analysis was carried out using synthetic stable-isotope labeled internal standard (SIS) and natural (NAT) synthetic proteotypic peptides for measurements of the corresponding proteins in urine. The selected 22 SIS and NAT synthetic peptides had been previously validated for use in LC/MRM-MS experiments [55]. LC-MS parameters, such as the LC gradient and the MRM parameters (Q1 and MRM scans) were adapted and optimized based on the previous studies [56]. The SIS peptide mixture was spiked in each urine sample at a balanced concentration which was optimized in experiments with dilution series of urine samples with proteinuria. Standard curves were generated using NAT and SIS peptide standards with a pooled urine sample as matrix.

All samples were analyzed in duplicate by HPLC-MS system consisting of an ExionLC™ (UHPLC system (ThermoFisher Scientific, Waltham, MA, USA) coupled online to a SCIEX QTRAP 6500+ triple quadrupole mass spectrometer (SCIEX, Toronto, ON, Canada). The loaded sample volume was 10 μL per injection. HPLC separation was carried out using Zorbax Eclipse Plus C18 RRHD column (150 × 2.1 mm, 1.8 μm) (Agilent, Santa Clara, CA, USA) with gradient elution. Mobile phase A was 0.1% FA in water; mobile phase B was 0.1% FA in acetonitrile. LC separation was performed at a flow rate of 0.4 mL/min using a 53 min gradient from 2 to 45% of mobile phase B. Mass-spectrometric measurements were carried out using the MRM acquisition method. The electrospray ionization (ESI) source settings were as follows: ion spray voltage 4000 V, temperature 450 ◦C, ion source gas 40 L/min. The corresponding transition list for MRM experiments with retention times values and Q1/Q3 masses for each peptide were adapted from the previous studies [56].

Skyline Quantitative Analysis software (version 20.2.0.343, University of Washington) was used for quantitative analysis.
