Ultracentrifugation (UC) has long been considered the “gold standard” for extracellular vesicle (EV) isolation. However, due to its drawbacks such as high cost of an ultracentrifuge and rotors, time-consuming and labor-intensive protocol, low yield considering initial biofluid volume and low throughput, development of new EV isolation approaches is still ongoing. Here we compare three methods for isolating the most studied EV subtype, small extracellular vesicles (sEVs), from human plasma: ultracentrifugation (UC), express asymmetric depth filtration (ExADFi), and anti-CD9 immunoaffinity capture (AS-CD9) with focus on their Raman and proteomic profiles. For all three methods, purity and quality of the sEV isolation were assessed based on the level of contamination of the sEV fraction with major plasma proteins such as albumin and apolipoproteins (APOA1, APOH, APOA4, APOC2, APOC1, and APOC4). UC showed the highest ratio of protein to nanoparticle concentration. AS-CD9 and ExADFi provided comparable to UC purity and levels of non-vesicular contaminants with AS-CD9 requiring minimal time and labor. ExADFi showed characteristics including purity of the sEV samples, yield, and isolation time that is between the UC and AS-CD9 methods. Raman spectroscopy provided more details about characteristics of the isolated sEVs and confirmed differences observed in the proteomic profiles. The findings demonstrate that the AS-CD9 and ExADFi methods could be appropriate substitutes of the classical UC-based isolation method and be chosen depending on the final requirements and use of the purified sEVs such as further functional and biomarker studies.

The aim of this study was to examine the hepatoprotective activity of multicomponent mixtures of natural origin in the BALB/C mouse model (n = 59), with acute direct toxic liver injury (DTLI) induced by the administration of streptozotocin (STZ) (100 mg/kg) in combination with a high-fat and high-fructose diet (HFFD). The hepatoprotective activity of activated hydrolytic lignin (Bp-Cx-1), methanolic fraction of Bp-Cx-1 (Bp-Cx-M) and isoflavones from kudzu Pueraria lobata roots (IFL) were evaluated on molecular level using mass spectrometry (MS)-based omics technologies. Untargeted label-free DIA quantitation resulted in 7214 protein groups identification (FDR 1%) after filtering across 40 liver tissue extracts. All treatment groups were closer to the control samples on the liver proteomic landscape compared to the untreated DTLI group, with the best results shown for the Bp-Cx-M and IFL groups. In order to identify differences between specific groups, we applied the post hoc Dunn's test and used Hedges' g as the effect size metric, revealing 64 proteins that tended to return to their normal level after treatment. In-depth proteomic liver tissue analysis enabled us not only to reveal the main pathways such as inflammation and oxidative stress, which are in a good agreement with DTLI and non-alcoholic liver disease pathophysiology, but also to evaluate hepatoprotective activity of multicomponent mixtures of natural origin containing polyphenols and mostly associated with protein metabolism (e.g., PSMD7, HCFC1) and deubiquitination pathways (e.g., UCHL3). It is worth noting that the Bp-Cx-1 isolated methanol fraction (Bp-Cx-M) demonstrated a pronounced increased hepatoprotective activity compared to the parent material due to the enrichment with active components such as polyphenols. Consistent with the proteomic findings of restored ubiquitin-proteasome function, assessment by comet assay revealed that treatments with Bp-Cx-M and IFL significantly reduced DNA damage by 50% compared to the untreated DTLI group. The developed MS-based multi-omics approach may be implemented for the robust and high-throughput screening method during assessment of new hepatoprotective agents of synthetic or natural origin.

Human papillomavirus (HPV)-associated cervical lesions remain a significant disease burden and minimally invasive blood-based biomarkers could complement cytology and HPV testing. This study aimed to characterize the proteomic composition of plasma-derived CD9+ small extracellular vesicles (sEVs) across the morphological spectrum of HPV-associated cervical disease, from histologically normal (NILM) through low-grade (LSIL) and high-grade (HSIL) lesions to invasive squamous cell carcinoma (SCC). Plasma samples from 34 women (NILM, LSIL, HSIL, SCC) were pooled per group, and CD9+ sEVs were isolated using an electrochemically controlled immunoaffinity capture method, followed by nanoparticle tracking analysis, transmission electron microscopy, Western blotting, and label-free LC-MS/MS proteomic profiling. The core sEV proteome comprised 258 shared proteins. LSIL showed the most pronounced changes with broad enrichment of complement and coagulation components and acute-phase reactants alongside depletion of immunoglobulin chains and complement C1r-like protein (C1RL). HSIL exhibited few differential proteins, dominated by neutrophil degranulation and retinoid metabolism pathways. SCC demonstrated extensive cargo depletion (22 downregulated proteins) and a nearly sevenfold upregulation of C1RL. Five proteins (including immunoglobulin chains and GPLD1) correlated positively with lesion severity. Pathway analysis consistently implicated platelet activation, lipoprotein remodeling, and insulin-like growth factor signaling. We conclude that plasma CD9+ sEVs carry stage-specific proteomic signatures distinguishing HPV-associated cervical lesions, with C1RL emerging as a candidate biphasic marker warranting further validation.

Pancreatic cancer (PC) is the sixth leading cause of cancer-related deaths worldwide. Patients with pancreatic ductal adenocarcinoma (PDAC), the most common type of PC, have a 5-year survival rate of approximately 10%. This low survival rate is mainly attributed to late-stage diagnoses and the lack of robust screening methods. Several serum proteins have been proposed as potential PDAC biomarkers, but they have not been introduced into clinical practice due to their low sensitivity and specificity. Therefore, the identification of new PDAC biomarkers remains highly important, and multiple reaction monitoring (MRM), a highly accurate mass spectrometry (MS) technique, can be used for this purpose. Using MRM MS analysis, we estimated the concentrations of 103 proteins in peripheral blood plasma from 132 participants: patients with newly diagnosed PDAC at different stages and healthy individuals. We identified six proteins that were differentially presented between healthy controls and patients with PDAC at all stages (adjusted p-value < 0.01), and that were associated with survival rates for 23 months. A developed cross-validated model based on these six proteins showed an average accuracy of 90% in distinguishing between early-stage PDAC and healthy controls (AUC = 0.933). However, further research is needed to implement this model in clinical practice.

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.

The aim of this study was to examine the hepatoprotective activity of multicomponent mixtures of natural origin in the BALB/C mouse model (59 female mice), with non-alcoholic fatty liver disease (NAFLD) induced by the administration of streptozotocin (STZ) in combination with a high-fat, high-fructose diet. The hepatoprotective activity of activated hydrolytic lignin (BP-Cx-1), metanolic fraction of BP-Cx-1 (BP-Cx-M) and isoflavones from kudzu Pueraria lobata roots (IFL) was evaluated using mass spectrometry (MS)-based omics technologies. Untargeted label-free DIA quantitation resulted in 8088 protein groups identification (FDR 1%) in 40 liver tissue extracts. All treatment NAFLD groups were closer to the control samples on the liver proteomic landscape with the best results shown for BP-Cx-1-m and ISF groups. This corroborated well with metabolomic fingerprinting by FTICR MS. In order to identify differences between specific groups, we applied the post-hoc Dunn’s Test and used Hedges' g as the Effect Size metric and 64 proteins that tended to return to their normal level after treatment were revealed. Pathophysiologically specific proteomics changes occurring during NAFLD were mostly associated with metabolism of the proteins (PSMD7, HCFC1) and deubiquitination pathways (UCHL3). It is worth noting that BP-Cx-1 isolated methanol fraction (Bp-Cx-m) demonstrated the pronounced increased hepatoprotective activity due to the enrichment with active components such as polyphenols. Additionally, it was shown that treatments with Bp-Cx-M and IFL significantly reduced DNA damage by 50% compared to the NAFLD untreated group. Finally, MS-based multiomics approach demonstrated its power as an advanced screening method for the assessment of the hepatoprotective activity and molecular mechanisms of action of complex multicomponent mixtures of natural origin.

Early recognition of a risk of Alzheimer's disease (AD) remains a global challenge, and blood proteomic markers are of particular interest for wide-scale diagnostic use. Quantitative multiple reaction monitoring (MRM) approach demonstrates good reproducibility in the characteristic changes in the levels of reported candidate biomarkers (CBs) in different cohorts in AD. Following up on our previous study, we performed a joint analysis of 331 blood plasma samples from two different clinical cohorts of participants, comprising a total of 95 samples from patients with AD, 136 samples from patients with mild cognitive impairment (MCI), and 100 samples from controls. The obtained results confirm the significance of 37 CBs. A logistic regression-based algorithm was used to build protein classifiers, and a total of 21 important proteins were selected, 13 of which (ORM1, APOA4, LBP, HP, FN1, BCHE, APOE, PZP, A1BG, TF, SERPINA7, TTR, and F12) formed a universal panel that demonstrated strong classification performance in distinguishing AD patients from controls (ROC-AUC = 0.90) and in separating stable and progressing patients with MCI (ROC-AUC = 0.81). Overall, the analysis confirms the high potential of the MRM method for validating CBs in independent cohorts.

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.

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.

The aim of this study was to investigate the molecular composition of follicular fluid (FF) extracellular vesicles (EVs) in women of different reproductive ages and its possible relationship to sperm fertilizing ability. FF EVs were obtained by differential centrifugation. The concentration and size distribution of FF EVs were analyzed by nanoparticle tracking analysis. The lipidome and proteome were analyzed by liquid chromatography–mass spectrometry. The isolated FF EVs had a variety of shapes and sizes; their concentration and size distribution did not differ significantly between the age groups. In women younger than 35 years, the concentration of vesicular progesterone was 6.6 times higher than in women older than 35 years, and the total levels of the main lipid classes were increased in younger women. A proteomic analysis revealed that not only FF EV-specific proteins, but also proteins involved in sperm activation were present. New data were obtained on the composition of FF EVs, confirming their importance as molecular indicators of age-related changes in the female reproductive system. In addition, these results shed light on the possible interaction between the FF EVs of women in different age groups and male germ cells. Therefore, studying the transcriptomic and metabolomic profile of FF EVs may be a crucial approach to evaluate the efficacy of ART.

The study of urinary peptidome is an important area of research, which concerns the characterization of endogenous peptides, as well as the identification of biomarkers for a wide range of socially significant diseases. First of all, this relates to renal and genitourinary pathologies and/or pathologies associated with proteinuria, such as kidney diseases, bladder, prostate and ovarian cancers, diabetic nephropathy, and pre-eclampsia. Unlike proteins, peptides do not require proteolytic hydrolysis, can be analyzed in their native form and can provide certain information about occurring (patho)physiological processes. Mass spectrometry (MS)-based approaches are the most unbiased and sensitive instruments with high multiplexing capacity and provided most of the current information about endogenous urine peptides. However, despite the large number of urine peptidomic studies, there are certain issues related to the insufficient comparability of their results due to the lack of consistent approaches to their interpretation. Also the development of a custom project-specific protein library for endogenous peptides search and identification is another important point that should be noted in the context of high-throughput peptidomic analysis. Here we propose the custom-specific urinary protein database and the grouping of endogenous urinary peptides with overlapping sequences as useful tools, which can facilitate the acquisition and analysis of LC-MS peptidomic data, as well as the comparison of results of different studies, which should facilitate their more efficient further application.

The molecular mechanisms of organismal adaptation to spaceflight (SF) conditions are not fully investigated and remain relevant for study. Newly emerging multi-omics technologies may provide information at the molecular level regarding physiological changes caused by SF factors. The aim of the study was to apply blood proteome analysis using dried blood spots (DBS) micro-sampling technique to study at the molecular level the physiological response to SF conditions for 7 cosmonauts during 169–199 days long-duration missions (one of the cosmonauts participated in a year-long mission). The level of 119 blood proteins was quantified in DBS samples by targeted mass spectrometry based proteomics with a validated MRM assay using stable isotope-labeled peptide standards (SIS). To identify proteins whose concentrations are sensitive to the high physiological stress of landing and takeoff the Mann-Whitney and Cohen's d-size effect tests were used. As a result, 10 proteins (p-value < 0.05 and Cohen's d size > 0.8) were found to be significant. In order to determine additional stress marker proteins, the dynamic changes in protein concentration during SF were analyzed to find proteins whose concentrations change monotonously after stress exposure (takeoff or landing). A simple exponential decay model for protein concentration relaxation was proposed which allows to reveal additional 14 proteins. Intersections of both protein panels mentioned above resulted in a final list of 19 blood proteins that are related to SF factors such as takeoff and landing. The panel includes 2 plasma proteins (Lumican and Insulin-like protein binding growth factor 3) that were previously mentioned in other studies to be related with SF. As far as we know this is the first DBS proteomic study for monitoring adaptive reactions of the organism to long-term SF.

Objectives 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.Methods 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.Results 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.Conclusions 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.

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.

The work is devoted to the study of the composition of protein concentrates from amaranth grains (Amaranthus hypochondriacus L.), Voronezh variety. Amaranth protein concentrates were obtained by the alkaline extraction of proteins, the neutralization of the solution, followed by ultrafiltration; by separating the starch fraction with amylolytic enzymes; alkaline extraction of proteins and their precipitation at pH 4.5. The conditions for protein extraction were selected, followed by gas chromatography-mass spectrometric analysis and their identification. It has been shown that proteins from amaranth grain were more efficiently extracted with a buffer containing urea at protein concentrations in solution of 1.7, 1.9, and 2.9 mg/cm3respectively, while the buffer with detergents was more effective for the extraction of low molecular weight proteins at protein concentrations in solution of 4.9, 2.9, and 9.0 mg/cm3 respectively. As a result of HPLC-MS/MS analysis followed by a search and identification in the UNIPROT database, it was established that the main protein of amaranth grain is 11S-globulin, which is a reserve protein of amaranth seeds. In amaranth concentrates, 14 unique proteins characteristic only for A.hipochondriacus L., as well as proteins that were not characteristic to this species, but were reliably identified. Based on the results of semi-quantitative analysis of the peptide profile of amaranth grain protein concentrates, a high frequency of occurrence of the main 11S-globulin proteins was established in all samples. The frequency of occurrence of other proteins in samples obtained by different methods differed significantly, which was associated with the peculiarities of protein isolation from amaranth grain. The obtained results can be used for the production of plant protein concentrates with a set protein composition.

Intrauterine growth restriction (IUGR) remains a significant concern in modern obstetrics, linked to high neonatal health problems and even death, as well as childhood disability, affecting adult quality of life. The role of maternal and fetus adaptation during adverse pregnancy is still not completely understood. This study aimed to investigate the disturbance in biological processes associated with isolated IUGR via blood plasma proteomics. The levels of 125 maternal plasma proteins were quantified by liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) with corresponding stable isotope-labeled peptide standards (SIS). Thirteen potential markers of IUGR (Gelsolin, Alpha-2-macroglobulin, Apolipoprotein A-IV, Apolipoprotein B-100, Apolipoprotein(a), Adiponectin, Complement C5, Apolipoprotein D, Alpha-1B-glycoprotein, Serum albumin, Fibronectin, Glutathione peroxidase 3, Lipopolysaccharide-binding protein) were found to be inter-connected in a protein–protein network. These proteins are involved in plasma lipoprotein assembly, remodeling, and clearance; lipid metabolism, especially cholesterol and phospholipids; hemostasis, including platelet degranulation; and immune system regulation. Additionally, 18 proteins were specific to a particular type of IUGR (early or late). Distinct patterns in the coagulation and fibrinolysis systems were observed between isolated early- and late-onset IUGR. Our findings highlight the complex interplay of immune and coagulation factors in IUGR and the differences between early- and late-onset IUGR and other placenta-related conditions like PE. Understanding these mechanisms is crucial for developing targeted interventions and improving outcomes for pregnancies affected by IUGR.

It is suggested that activated CD44+ cells play a profibrogenic role in the pathogenesis of active glomerulopathies. Complement activation is also involved in renal fibrogenesis. The aim of the study was to evaluate the role of the activation of CD44+ cells in the kidney tissue and complement components' filtration to the urine as factors of renal tissue fibrosis in patients with glomerulopathies. In total, 60 patients with active glomerulopathies were included in our study: 29 patients with focal segmental glomerulosclerosis (FSGS), 10 patients with minimal change disease (MCD), 10 patients with membranous nephropathy (MN), and 11 patients with IgA nephropathy. The immunohistochemical peroxidase method was used to study the expression of CD44+ in kidney biopsies. Components of complement were analyzed in urine by the multiple reaction monitoring (MRM) approach using liquid chromatography. Strong CD44 expression was noted predominantly in PEC and mesangial cells (MC) in patients with FSGS, and to a lesser extent, in patients with MN and IgA nephropathy, and it was absent in patients with MCD. Expression of profibrogenic CD44+ in glomeruli correlated with the levels of proteinuria and complement C2, C3, and C9 components, and CFB and CFI in urine. The CD44+ expression scores in the renal interstitium correlated with the level of C3 and C9 components of complement in the urine and the area of tubulo-interstitial fibrosis. The strongest expression of CD44+ was found in the glomeruli (MC, PEC, and podocytes) of patients with FSGS compared with other glomerulopathies. The CD44 expression score in the glomeruli and interstitium is associated with high levels of complement components in the urine and renal fibrosis.

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).