**5. Conclusions**

Thus, the proteomic profile of urine from patients with FSGS and MCD is characterized by a large number of excreted proteins, but no significant differences between these two forms were revealed. However, the FSGS patients showed high variability inside the group and clustered into two subgroups, which could be reliably distinguished basing on the proteomic profile. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the

two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. In patients with severe FSGS 2, the urine proteome panel reflects damage to podocytes (Vitronectin, Hemopexin, Gelsolin, Apolipiprotein A), complement activation (Complement component C4b, C9, Complement factor B and I), and accumulation of the extracellular matrix and tubular damage (Cystatin C, Vitamin D-binding protein, Retinol-binding protein 4, Alpha-2-HS-glycoprotein, Plasma protease C1 inhibitor, Lumican, Clusterin).

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms232012607/s1.

**Author Contributions:** Conceptualization, N.V.C. and A.S.K.; methodology, D.N.K.; software, A.G.B.; formal analysis, A.G.B. and D.N.K.; investigation, A.V., A.E.B., M.I.I. and N.V.C.; data curation, M.L. and S.M.; writing—original draft preparation, N.V.C. and A.S.K.; writing—review and editing, E.N.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Science Foundation, grant #21-74-20173.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by Ethical Committee of the Medical Research and Educational Center of Lomonosov University, Approval Code: Protocol 12/21, Approval Date: 13 December 2021.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.
