👤 Anna Bugrova

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. Show less

Imperfect first-trimester screening for hypertensive disorders of pregnancy (HDP) means many high-risk women miss the window for preventive aspirin, and the biological heterogeneity of HDPs is overlooked. This study aimed to leverage first-trimester serum proteomics to create a more precise tool for predicting preeclampsia (PE) and differentiating it from other HDPs. A prospective nested case-control study ( Show less

Aim    To develop of a protein panel to identify patients with progressive chronic heart failure with reduced left ventricular ejection fraction (HFrEF) based on proteomic analysis of blood fractions.Material and methods    The study included 81 patients with HFrEF associated with postinfarction myocardial scarring or dilated cardiomyopathy. Patients were enrolled both in a stable period (n=48) and with signs of decompensated heart failure (n=33). Proteomic chromatography-mass-spectrometric analysis of blood plasma and extracellular vesicles (EVs) was performed in all patients. The analysis identified proteins differentially represented between groups in each blood compartment. The effectiveness of using individual proteins and integrated protein panels based on these proteins to identify patients with progressive HFrEF was assessed.Results    Twelve plasma proteins and one BB fraction protein were detected, the concentration of which significantly differed between the groups with and without decompensated HFrEF. Individual protein concentrations demonstrated approximately the same quality indicators in identifying patients with decompensated HF as the classical HF marker, the N-terminal fragment of pro-brain natriuretic peptide (NT-proBNP). Accordingly, we developed two integrated panels including the concentrations of NT-proBNP and several plasma or BB fraction proteins. The plasma panel included five proteins (APOE, LPA, C7, GPLD1, and TF), and the BB panel included two proteins (APOC4, FGB); the proteins are designated in accordance with their genes in the UniProt database. The plasma protein panel demonstrated the highest efficiency in identifying patients with decompensated HF, with a sensitivity of 78.8% and a specificity of 87.5%.Conclusion    The study resulted in the development of a plasma protein panel that can identify patients with progressive chronic HFrEF. This panel is more effective than previously described or currently used biomarkers. However, further research is needed to implement this protein panel into clinical practice. Show less

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. Show less

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 Show less

The development of blood tests for the early detection of individual predisposition to socially significant diseases remains a pressing issue. 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. 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). 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. Show less

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. Show less

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. 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. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I. Show less