*2.1. Optimization of the Urine Preparation Protocol for Proteomic Analysis*

All clinical urine samples were characterized by proteinuria of varying severity (Table 1). In order to select the optimal method of urine sample preparation for LC-MS/MS analysis 3 previously published methods for concentrating, purifying and hydrolyzing proteins were tested: (1) precipitation of proteins with ice-cold acetone [15]; (2) concentration and hydrolysis of proteins on filters (filter-aided sample preparation (FASP), Microcon (Millipore) filters were used) [16]; (3) ultrafiltration to purify proteins from low molecular weight components of urine [17]. The main criteria for the optimization of urine sample preparation were the robustness and ease of reproducibility of all steps; and the second the effectiveness of the protocol in the view of the number of detected proteins. It was decided not to use the third method due to its excessive laboriousness and poor reproducibility of the ultrafiltration stage for urine samples with proteinuria.


**Table 1.** Calculation of the FSGS severity index.

Comparison of the two remaining methods of sample preparation showed that more different proteins was detected using the acetone precipitation method (Table 2, Figure 1). The first protocol allowed to detect the highest number of proteins in the test samples with proteinuria (5, 7, 10 mg/mL of total protein) and was used for further studies with minor modifications.

**Table 2.** Efficiency of urine proteins extraction by two methods (Acetone precipitation by ice-cold acetone protein precipitation; FASP—protein concentration and hydrolysis on filters). The number of identified proteins is indicated in Table.

