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IntroductionCirculating anti-podocyte antibodies have been proposed as potential factors contributing to increased permeability in primary podocytopathies, such as Minimal Change Disease (MCD) and Focal Segmental Glomerulosclerosis (FSGS). The aim of the study was to to assess the levels of antibodies targeting synaptopodin and annexin 1 in the blood serum of patients diagnosed with nephrotic syndrome, with the aim of evaluating their potential utility in diagnosing primary podocytopathies and predicting therapeutic response.MethodsThe study included a total of 72 patients diagnosed with nephrotic syndrome, alongside 21 healthy subjects for comparison. Among the patients, 38 were diagnosed with FSGS, 12 with MCD, and 22 with MN. The levels of anti-synaptopodin and anti-annexin-1 antibodies were quantified using Enzyme-Linked Immunosorbent Assay.ResultsThe levels of antibodies to annexin 1 and anti-synaptopodin in the blood were found to be higher in patients diagnosed with MCD and FSGS compared to those with MN and healthy individuals. The elevated levels of antibodies to annexin 1 and synaptopodin showed area under the curve values of 0.826 (95% CI 0.732–0.923) and 0.827 (95% CI 0.741–0.879), respectively. However, a model incorporating both antibodies demonstrated higher sensitivity (80.9%) and specificity (81.3%) with an AUC of 0.859 (95% CI 0.760-0.957). Notably, serum levels of annexin 1 and anti-synaptopodin antibodies did not predict the response to prednisolone and/or CNI therapy.DiscussionLevels of antibodies targeting synaptopodin and annexin 1 were notably elevated in patients diagnosed with MCD and FSGS compared to those with MN and healthy controls. A panel comprising both antibodies demonstrated moderate to high sensitivity and specificity for diagnosis MCD or FSGS.

Background/Objectives: The development of blood tests for the early detection of individual predisposition to socially significant diseases remains a pressing issue. Methods: In this pilot study, multiple reaction monitoring mass spectrometry (MRM-MS) with a BAK-270 assay was applied for protein concentrations analysis in blood plasma from 21 healthy volunteers of the European cohort. Results: The levels of 138 plasma proteins were reliably and precisely quantified in no less than 50% of samples. The quantified proteins included 66 FDA-approved markers of cardiovascular diseases (CVD), and other potential biomarkers of pathologies such as cancer, diabetes mellitus, and Alzheimer’s disease. The analysis of individual variations of the plasma proteins revealed significant differences between the male (11) and female (10) groups. In total, fifteen proteins had a significantly different concentration in plasma; this included four proteins that exhibited changes greater than ±1.5-fold, three proteins (RBP4, APCS, and TTR) with higher levels in males, and one (SHBG) elevated in females. The obtained results demonstrated considerable agreement with the data collected from 20 samples of a North American cohort, which were analyzed with the similar MRM assay. The most significant differences between the cohorts of the two continents were observed in the level of 42 plasma proteins (including 24 FDA markers), of which 17 proteins showed a ≥1.5-fold change, and included proteins increased in North Americans (APOB, CRTAC1, C1QB, C1QC, C9, CRP, HP, IGHG1, IGKV4-1, SERPING1, RBP4, and AZGP1), as well as those elevated in Europeans (APOF, CD5L, HBG2, SELPLG, and TNA). Conclusions: The results suggest a different contribution of specific (patho)physiological pathways (e.g., immune system and blood coagulation) to the development of socially significant diseases in Europeans and North Americans, and they should be taken into account when refining diagnostic panels.

Background. Focal segmental glomerulosclerosis (FSGS) is a primary podocytopathy characterized by primary podocyte detection and high proteinuria. The search for biomarkers and factors associated with the progression of this disease is an important task nowdays. Aim. To assess the proteomic profile of urine in patients with FSGS and to isolate urinary biomarkers of podocytopathies. Materials and methods. The study included 41 patients diagnosed with chronic glomerulonephritis, 27 men and 14 women. According to the morphological study, 28 patients were diagnosed with FSGS, 9 with steroid-sensitive nephrotic syndrome and 14 with steroid-resistant nephrotic syndrome. The comparison group included 13 patients with membranous nephropathy. The study of the urinary proteome was carried out by targeted liquid chromatography-mass spectrometry using multiple reaction monitoring with synthetic stable isotope labelled peptide standards. Results. The main differences in the protein profile of urine were found in the subgroups of steroid-sensitive (SS) and steroid-resistant (SR) FSGS. In the FSGS SR group, at the onset of the disease, there was a high concentration of proteins reflecting damage to the glomerular filter (apo-lipoprotein A-IV, orosomucoid, cadherin, hemopexin, vitronectin), as well as proteins associated with tubulo-interstitial inflammation and accumulation of extracellular matrix (retinol- and vitamin D-binding proteins, kininogen-1, lumican and neurophilin-2). Compared with the membranous nephropathy group, FSGS patients had significantly higher urinary concentrations of carnosinase, orosomucoid, cadherin-13, tenascin X, osteopontin, and zinc-alpha-2-glycoprotein. Conclusion. Thus, in patients with SR FSGS, the proteomic profile of urine includes more proteins at elevated concentrations, which reflects severe damage to various parts of the nephron compared with patients with SS FSGS and membranous nephropathy.

The objects of the study are extractants and optimal extraction conditions that contribute to the full release of the substances being determined: the concentration and volume of the extractant, the extraction time and temperature regime. This work includes the technology of sample preparation for determining the fatty acid composition of milk with a fat mass fraction of more than 3 %, which is centrifuged for 10 minutes at 10000 rpm, 20 µl of oil is taken from the centrifuged laboratory sample into a test tube from the upper part, then dissolved in 2 cm3 of organic solvent (hexane), then mixed manually for 1-2 minutes, 100 ml of sodium methylate solution of 2 molar concentration is added to the resulting solution with a pipette and the tube is closed with a stopper, then intensively mixed manually for 2 minutes, insist for 5 minutesand filter through a paper filter the top layer containing methyl esters, the resulting solution will be ready for examination by gas chromatography. The proposed new approach to technology development reduces the sample preparation time (~19 min), reduces the amount of solvent consumed by more than 10 times, minimizes the number of actions when working with samples, and requires a minimum amount of equipment.

The aim of this study was to examine the hepatoprotective activity of multicomponent mixtures of natural origin in the BALB/C mouse model, with subacute liver failure (SALF) induced by the administration of toxin carbon tetrachloride (CCl4). The hepatoprotective activity of activated hydrolytic lignin (BP-Cx-1), humic acid peloids (HA), and isoflavones from kudzu Pueraria lobata roots (IFL) was evaluated using mass spectrometry (MS)-based omics technologies. Our MS-based approach revealed new insights into the molecular mechanisms of the hepatoprotective activity of multicomponent mixtures of natural origin. Significant differences were observed in the proteome and metabolome profiles of the urine and liver of BALB/c mice with SALF between a control group with CCl4 administration, intact controls, and groups receiving potential hepatoprotectors of natural origin (BP-Cx-1, HA, IFL). Proteomic and metabolomics analyses demonstrated that among the hepatoprotectors, IFL possessed the highest hepatoprotective potential, which correlated well with the relative effectiveness of the drugs recorded during in vitro studies. These results correlate with the relative effectiveness of the drugs recorded in previous in vitro and in vivo studies. The leading IFL activity may be attributed to a higher content of active polyphenolic components compared to heterogeneous HA and BP-Cx-1. Enrichment with active components by fractionation is a direction that can be explored for developing hepatoprotective agents based on natural complex polyphenols. [ABSTRACT FROM AUTHOR]

This review covers the results of the application of mass spectrometric (MS) techniques to study the diversity of beta‐amyloid (Aβ) peptides in human samples. Since Aβ is an important hallmark of Alzheimer's disease (AD), which is a socially significant neurodegenerative disorder of the elderly worldwide, analysis of its endogenous variations is of particular importance for elucidating the pathogenesis of AD, predicting increased risks of the disease onset, and developing effective therapy. MS approaches have no alternative for the study of complex samples, including a wide variety of Aβ proteoforms, differing in length and modifications. Approaches based on matrix‐assisted laser desorption/ionization time‐of‐flight and liquid chromatography with electrospray ionization tandem MS are most common in Aβ studies. However, Aβ forms with isomerized and/or racemized Asp and Ser residues require the use of special methods for separation and extra sensitive and selective methods for detection. Overall, this review summarizes current knowledge of Aβ species found in human brain, cerebrospinal fluid, and blood plasma; focuses on application of different MS approaches for Aβ studies; and considers the potential of MS techniques for further studies of Aβ‐peptides. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has exposed a number of key challenges that need to be urgently addressed. Mass spectrometric studies of blood plasma proteomics provide a deep understanding of the relationship between the severe course of infection and activation of specific pathophysiological pathways. Analysis of plasma proteins in whole blood may also be relevant for the pandemic as it requires minimal sample preparation. The frozen whole blood samples were used to analyze 203 plasma proteins using multiple reaction monitoring (MRM) mass spectrometry and stable isotope-labeled peptide standards (SIS). A total of 131 samples (FRCC, Russia) from patients with mild (n=41), moderate (n=39) and severe (n=19) COVID-19 infection and healthy controls (n=32) were analyzed. Levels of 94 proteins were quantified and compared. Significant differences between all of the groups were revealed for 44 proteins. Changes in the levels of 61 reproducible COVID-19 markers (SERPINA3, SERPING1, ORM1, HRG, LBP, APOA1, AHSG, AFM, ITIH2, etc.) were consistent with studies performed with serum/plasma samples. The best-performing classifier built with 10 proteins achieved the best combination of ROC-AUC (0.97–0.98) and accuracy (0.90–0.93) metrics and distinguished patients from controls, as well as patients by severity. Here, for the first time, frozen whole blood samples were used for proteomic analysis and assessment of the status of patients with COVID-19. The results obtained with frozen whole blood samples are consistent with those from plasma and serum. [ABSTRACT FROM AUTHOR]

Introduction: Identifying early risks of developing Alzheimer's disease (AD) is a major challenge as the number of patients with AD steadily increases and requires innovative solutions. Current molecular diagnostic modalities, such as cerebrospinal fluid (CSF) testing and positron emission tomography (PET) imaging, exhibit limitations in their applicability for large-scale screening. In recent years, there has been a marked shift toward the development of blood plasma-based diagnostic tests, which offer a more accessible and clinically viable alternative for widespread use. Furthermore, advances in large-scale proteomics technologies have boosted an interest in identifying novel biomarkers and developing panels of AD-associated proteins. Areas covered: This review mainly examines the results of recent searches for proteomic markers of AD in blood plasma (from 2022–2024 PubMed), focuses on some aspects for special attention in further studies, and discusses the prospects for their further application. Expert opinion: Recent advances in AD plasma/serum proteomic studies are largely driven using novel Olink/PEA and SomaScan/aptamer technologies, which complement the 'gold standard' of MS-based quantitative proteomics (MRM/SRM), and particularly expand the capabilities for studying low-abundant proteins. [ABSTRACT FROM AUTHOR]

BackgroundMinimal change disease and focal segmental glomerulosclerosis are primary podocytopathies that are clinically presented in adults presenting with severe nephrotic syndrome. The pathogenesis of these diseases is not clear and many questions remain to be answered. A new concept about the role of changes in the antigenic determinant of podocytes and the production of anti-podocyte antibodies that cause podocyte damage is being developed. The aim of the study is to evaluate the levels of anti-CD40 and anti-ubiquitin carboxyl-terminal hydrolase L1 (anti-UCH-L1) antibodies in patients with podocytopathies in comparison with other glomerulopathies.MethodsOne hundred and six patients with glomerulopathy and 11 healthy subjects took part in the study. A histological study revealed primary FSGS in 35 patients (genetic cases of FSGS and secondary FSGS in the absence of NS were excluded), 15 had MCD, 21 - MN, 13 - MPGN, 22 patients - IgA nephropathy. The effect of steroid therapy was evaluated in patients with podocytopathies (FSGS and MCD). The serum levels of anti-UCH-L1 and anti-CD40 antibodies were measured by ELISA before steroid treatment.ResultsThe levels of anti-UCH-L1 antibodies were significantly higher in MCD patients and anti-CD40 antibodies were higher in MCD and FSGS than in the control group and other groups of glomerulopathies. In addition, the level of anti-UCH-L1 antibodies was higher in patients with steroid-sensitive FSGS and MCD, and anti-CD40 antibodies were lower than in patients with steroid-resistant FSGS. An increase in anti-UCH-L1 antibody levels above 6.44 ng/mL may be a prognostic factor of steroid-sensitivity. The ROC curve (AUC = 0.875 [95% CI 0.718–0.999]) for response to therapy showed a sensitivity of 75% and specificity of 87.5%.ConclusionAn increase in the level of anti-UCH-L1 antibodies is specific for steroid-sensitive FSGS and MCD, while an increase in anti-CD40 antibodies – for steroid-resistant FSGS, compared with other glomerulopathies. It suggests that these antibodies could be a potential factor for differential diagnosis and treatment prognosis.

Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS).Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) — 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) — 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro).Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (а-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc.Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.

Background/Objectives: The development of blood tests for the early detection of individual predisposition to socially significant diseases remains a pressing issue. Methods: In this pilot study, multiple reaction monitoring mass spectrometry (MRM-MS) with a BAK-270 assay was applied for protein concentrations analysis in blood plasma from 21 healthy volunteers of the European cohort. Results: The levels of 138 plasma proteins were reliably and precisely quantified in no less than 50% of samples. The quantified proteins included 66 FDA-approved markers of cardiovascular diseases (CVD), and other potential biomarkers of pathologies such as cancer, diabetes mellitus, and Alzheimer's disease. The analysis of individual variations of the plasma proteins revealed significant differences between the male (11) and female (10) groups. In total, fifteen proteins had a significantly different concentration in plasma; this included four proteins that exhibited changes greater than ±1.5-fold, three proteins (RBP4, APCS, and TTR) with higher levels in males, and one (SHBG) elevated in females. The obtained results demonstrated considerable agreement with the data collected from 20 samples of a North American cohort, which were analyzed with the similar MRM assay. The most significant differences between the cohorts of the two continents were observed in the level of 42 plasma proteins (including 24 FDA markers), of which 17 proteins showed a ≥1.5-fold change, and included proteins increased in North Americans (APOB, CRTAC1, C1QB, C1QC, C9, CRP, HP, IGHG1, IGKV4-1, SERPING1, RBP4, and AZGP1), as well as those elevated in Europeans (APOF, CD5L, HBG2, SELPLG, and TNA). Conclusions: The results suggest a different contribution of specific (patho)physiological pathways (e.g., immune system and blood coagulation) to the development of socially significant diseases in Europeans and North Americans, and they should be taken into account when refining diagnostic panels. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a complex and multifaceted obstetric syndrome characterized by several distinct molecular subtypes. It complicates up to 5% of pregnancies and significantly contributes to maternal and newborn morbidity, thereby diminishing the long-term quality of life for affected women. Due to the widespread dissatisfaction with the effectiveness of existing approaches for assessing PE risk, there is a pressing need for ongoing research to identify newer, more accurate predictors. This study aimed to investigate early changes in the maternal serum proteome and associated signaling pathways. The levels of 125 maternal serum proteins at 11–13 weeks of gestation were quantified using liquid chromatography–multiple reaction monitoring mass spectrometry (LC-MRM MS) with the BAK-125 kit. Ten serum proteins emerged as potential early markers for PE: Apolipoprotein M (APOM), Complement C1q subcomponent subunit B (C1QB), Lysozyme (LYZ), Prothrombin (F2), Albumin (ALB), Zinc-alpha-2-glycoprotein (AZGP1), Tenascin-X (TNXB), Alpha-1-antitrypsin (SERPINA1), Attractin (ATRN), and Apolipoprotein A-IV (APOA4). Notably, nine of these proteins have previously been associated with PE in prior research, underscoring the consistency and reliability of our findings. These proteins play key roles in critical molecular processes, including complement and coagulation cascades, platelet activation, and insulin-like growth factor pathways. To improve the early prediction of PE, a highly effective Support Vector Machine (SVM) model was developed, analyzing 19 maternal serum proteins from the first trimester. This model achieved an area under the curve (AUC) of 0.91, with 87% sensitivity and 95% specificity, and a hazard ratio (HR) of 13.5 (4.6–40.8) with p < 0.001. These findings demonstrate that serum protein-based SVM models possess significantly higher predictive power compared to the routine first-trimester screening test, highlighting their superior utility in the early detection and risk stratification of PE. [ABSTRACT FROM AUTHOR]

Abstract Despite broad application of different analytical techniques for studies on organic matter of chondrite meteorites, information about composition and structure of individual compounds is still very limited due to extreme molecular diversity of extraterrestrial organic matter. Here we present the first application of isotopic exchange assisted Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for analysis of alkali extractable fraction of insoluble organic matter (IOM) of the Murchison and Allende meteorites. This allowed us to determine the individual S-containing ions with different types of sulfur atoms in IOM. Thiols, thiophenes, sulfoxides, sulfonyls and sulfonates were identified in both samples but with different proportions, which contribution corroborated with the hydrothermal and thermal history of the meteorites. The results were supported by XPS and thermogravimetric analysis coupled to FTICR MS. The latter was applied for the first time for analysis of chondritic IOM. To emphasize the peculiar extraterrestrial origin of IOM we have compared it with coal kerogen, which is characterized by the comparable complexity of molecular composition but its aromatic nature and low oxygen content can be ascribed almost exclusively to degradation of biomacromolecules.

Aim. Conducting a pilot study to assess the effect of thermal heliox on the state of the respiratory tract by studying of the exhaled breath condensate protein composition before the thermal heliox procedure, immediately after and after three hours of relaxation Materials and methods. A comparative study of the exhaled breath condensates (EBC) protein composition of five non-smoking healthy donors was carried out. The EBC was taken before the respiratory procedure, immediately after a 20-minute inhalation by mixture of He/O2 gases (70/30) heated to 70C and 3 hours later. The protein composition was determined by chromatography-mass spectrometric analysis after selective tryptic hydrolysis. The results were processed using the Mascot program and the UniProt database. Results. After the heliox procedure, the volume of the collected condensate (11.5 ml) decreases by an average of 32% and is practically restored after three hours of relaxation. Most proteins were consistent for all samples, regardless of the thermal heliox procedure. These are keratins, several proteins of the immune system (immunoglobulins, compliment proteins), tubulin. In samples after thermal heliox, the appearance of small amounts of additional proteins is observed. These are proteins of muscle metabolism (actin and calmodulin), fibrinogen, traces of hemoglobin, apolipoprotein, type B creatine kinase. After three hours of relaxation, tubulin disappears in the EBC. Conclusion. Most exhaled proteins are the same before, after the procedure, and for three hours of relaxation. The results obtained demonstrate the relative safety of the use of high temperature heliox as a therapeutic agent.

Metastasis is a serious and often life-threatening condition, representing the leading cause of death among women with breast cancer (BC). Although the current clinical classification of BC is well-established, the addition of minimally invasive laboratory tests based on peripheral blood biomarkers that reflect pathological changes in the body is of utmost importance. In the current study, the serum proteome and lipidome profiles for 50 BC patients with (25) and without (25) metastasis were studied. Targeted proteomic analysis for concertation measurements of 125 proteins in the serum was performed via liquid chromatography–multiple reaction monitoring mass spectrometry (LC–MRM MS) using the BAK 125 kit (MRM Proteomics Inc., Victoria, BC, Canada). Untargeted label-free lipidomic analysis was performed using liquid chromatography coupled to tandem mass-spectrometry (LC–MS/MS), in both positive and negative ion modes. Finally, 87 serum proteins and 295 lipids were quantified and showed a moderate correlation with tumor grade, histological and biological subtypes, and the number of lymph node metastases. Two highly accurate classifiers that enabled distinguishing between metastatic and non-metastatic BC were developed based on proteomic (accuracy 90%) and lipidomic (accuracy 80%) features. The best classifier (91% sensitivity, 89% specificity, AUC = 0.92) for BC metastasis diagnostics was based on logistic regression and the serum levels of 11 proteins: alpha-2-macroglobulin, coagulation factor XII, adiponectin, leucine-rich alpha-2-glycoprotein, alpha-2-HS-glycoprotein, Ig mu chain C region, apolipoprotein C-IV, carbonic anhydrase 1, apolipoprotein A-II, apolipoprotein C-II and alpha-1-acid glycoprotein 1.

Bleomycin, which is widely used as an antitumor agent, possesses serious adverse effects such as pulmonary toxicity. Local nanoaerosol deposition for lung cancer treatment is a promising alternative to drug delivery to lung lesions. The aim of this work is to test the hypothesis that bleomycin nanoaerosol can be effectively used to treat multiple lung metastases. To obtain bleomycin nanoaerosol, an aerosol generator based on electrospray of a solution of a nonvolatile substance with gas-phase neutralization of charged aerosol particles was used. Lung metastases in murine Lewis lung carcinoma and B16 melanoma animal models were counted. The effect of inhaled bleomycin nanoparticles on the number and volume of metastases, as well as pulmonary side effects, was investigated. Using a mouse exposure chamber, the dose-dependent effect of inhaled bleomycin on tumor volume was evaluated in comparison with intraperitoneal administration. Bleomycin nanoaerosol reduced the volume of metastases and produced a higher antitumor effect at much lower doses. It has been established that long-term exposure to nanoaerosol with a low dose of bleomycin is capable of suppressing cancer cell growth. The treatment was well tolerated. In the lungs, minor changes were found in the form of focal-diffuse infiltration of the lung parenchyma.

Glomerulopathies with nephrotic syndrome that are resistant to therapy often progress to end-stage chronic kidney disease (CKD) and require timely and accurate diagnosis. Targeted quantitative urine proteome analysis by mass spectrometry (MS) with multiple-reaction monitoring (MRM) is a promising tool for early CKD diagnostics that could replace the invasive biopsy procedure. However, there are few studies regarding the development of highly multiplexed MRM assays for urine proteome analysis, and the two MRM assays for urine proteomics described so far demonstrate very low consistency. Thus, the further development of targeted urine proteome assays for CKD is actual task. Herein, a BAK270 MRM assay previously validated for blood plasma protein analysis was adapted for urine-targeted proteomics. Because proteinuria associated with renal impairment is usually associated with an increased diversity of plasma proteins being present in urine, the use of this panel was appropriate. Another advantage of the BAK270 MRM assay is that it includes 35 potential CKD markers described previously. Targeted LC-MRM MS analysis was performed for 69 urine samples from 46 CKD patients and 23 healthy controls, revealing 138 proteins that were found in ≥2/3 of the samples from at least one of the groups. The results obtained confirm 31 previously proposed CKD markers. Combination of MRM analysis with machine learning for data processing was performed. As a result, a highly accurate classifier was developed (AUC = 0.99) that enables distinguishing between mild and severe glomerulopathies based on the assessment of only three urine proteins (GPX3, PLMN, and A1AT or SHBG).