AIM:To identify the features of plasma, platelet hemostasis, and proteomic composition of the blood plasma in patients with acute myocardial infarction (AMI) and healthy volunteers after COVID-19. MATERIAL AND METHODS:The study included patients with AMI who have recently had COVID-19 (AMI-post-COVID, n=56) and patients with AMI who have not recently had COVID-19 (AMI-control, n=141). Healthy volunteers constituted the control groups and were also divided into control-post-COVID (n=32) and control-control (n=71) groups. Previous SARS-CoV-2 infection was determined by anti-N IgG in the blood serum, the level of which persists for 6-10 months after the disease. Hemostasis was evaluated by thromboelastometry (on whole blood), thrombodynamics (on platelet-poor plasma), fibrinolysis, impedance aggregometry, and proteomic analysis. RESULTS:The AMI-post-COVID and AMI-control groups had higher values of thrombus growth rate, size and density based on the data of thromboelastometry and thrombodynamics, as well as increased concentrations of the complement system components, proteins regulating the state of the endothelium, and a number of acute-phase and procoagulant proteins compared to the control groups. Furthermore, in the AMI-post-COVID group, compared to the AMI-control group, the thrombus density was lower, and its lysis rates were higher when measured by the thrombodynamics method on platelet-poor plasma, while the platelet aggregation induced by ADP and thrombin was higher. Also, in the control-post-COVID group, compared to the control-control group, the thrombus formation rate was lower, whereas, in contrast, the thrombus size as measured by the thrombodynamics method and the platelet aggregation induced by arachidonic acid and thrombin were higher. In addition, in the AMI-post-COVID group, compared to the AMI-control group, the concentrations of proteins involved in inflammation and hemostasis were lower. CONCLUSION:Patients with AMI who have recently had COVID-19 are characterized by a less pronounced activation of the immune response compared to patients with AMI who have not had COVID-19. This may be due to long-term chronic inflammation and depletion of components of the immune activation system after SARS-CoV-2 infection. Long-term activation of the hemostasis system in both patients with AMI and healthy volunteers after COVID-19 is primarily due to the platelet component of hemostasis.

This work was devoted to a study of the composition of protein concentrates from amaranth grain (Amaranthus hypochondriacus L.) of variety Voronezh. Amaranth protein concentrates were obtained by alkaline extraction of proteins and neutralization of the solution followed by ultrafiltration, by separating the starch fraction with amylolytic enzymes, and by alkaline extraction of proteins and their precipitation at pH 4.5. Conditions for the extraction of proteins followed by chromatography–mass spectrometric analysis and identification were selected. It was found that proteins from amaranth grain were more effectively extracted with a buffer with urea at protein concentrations of 1.7, 1.9, and 2.9 mg/cm3 in solution, respectively, while a buffer with detergents was more effective for the extraction of low-molecular-weight proteins at protein concentrations of 4.9, 2.9, and 9.0 mg/cm3 in solution, respectively. As a result of HPLC–MS/MS analysis followed by identification and search in the UNIPROT database, it was established that the main protein of amaranth grain is 11S-globulin, which acts as a reserve protein of amaranth seeds. In amaranth concentrates, 14 unique proteins belonging only to A. hipochondriacus L. were identified, and also proteins that did not belong to this species were reliably identified. Based on the results of semiquantitative 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 due to the peculiarities of protein isolation from amaranth grain. The results obtained can be used to prepare plant protein concentrates with a given protein composition.

Accumulation of neurotoxic aggregates of beta-amyloid peptides (Aβ) is a hallmark of Alzheimer's disease (AD) progression. Post-translational modifications (PTMs) increase Aβ aggregation and cytotoxicity, and the content of specific Aβ proteoforms is elevated in senile plaques of AD patients. The pathophysiological mechanisms of aggregate formation and the role of Aβ proteoforms need thorough study both to understand the role played by specific processes in the initiation of neuronal degradation and to find effective preventive means of therapeutic action. The present work investigates the dynamics of accumulation of phosphorylated serine-8 proteoform Aβ (pSer8-Aβ) using the 5xFAD mouse amyloid model. Aβ samples from human cerebrospinal fluid (CSF) and brain were also investigated. Western blot studies using 1E4E11 and 4G8 antibodies showed that accumulation of pSer8-Aβ in mouse brain starts as early as at the age of 3 months and reaches a maximum by the age of 14-17 months, which is generally similar to the dynamics of accumulation of the total pool of Aβ peptides. The pSer8-Aβ level in human CSF in AD patients can reach ~ 1-10% of the total amount of Aβ. Mass spectrometric analysis showed that Aβ phosphorylation by the Ser8, Tyr10, and Ser26 residues in brain tissues, as well as phosphorylation of the APP by Thr719 residue, is possible. These findings support the assumption that pSer8-Aβ proteoforms are involved in amyloidosis in AD. KEYWORDS Beta-amyloid, mass spectrometry, Alzheimer's disease, phosphorylation.

BACKGROUND. Primary focal segmental glomerulosclerosis (FSGS) and membranous nephropathy (MN) are diseases with primary podocyte damage with high proteinuria and nephrotic syndrome. While the mechanisms in primary MN are well understood, the pathogenesis of primary FSGS is still unknown, and therefore, the search for biomarkers that could expand ourunderstanding of its pathogenetic mechanisms. THE AIM: to determine the urine proteomic profile of patients with primary podocytopathies – FSGS in comparison with MN. PATIENTS AND METHODS. The study included 48 patients with a morphologically confirmed diagnosis of CGN occurring with nephrotic syndrome – 32 men and 16 women. In 18 patients, a decrease in glomerular filtration rate < 60 ml/min/1.73 m2 was observed. The histological diagnosis was confirmed by biopsy: 31 patients had FSGS, 17 patients with MN were included as a comparison group. The study of the urinary proteome was carried out by high performance liquid chromatography/mass spectrometry. RESULTS. In patients with FSGS, compared with the MN group, an increased content of 22 different proteins was noted, the most abundant were apolipoprotein A-I, hemopexin, vitronectin, pigment epithelial growth factor, components of the complement system (C3, C4b, factors B and H), retinol – and vitamin D-binding proteins, alpha-2-HS-glycoprotein, histidine-rich glycoprotein, plasma C1 protease inhibitor. In MN, increased urinary excretion of the complement component C2, fibrinogen alpha chain, osteopontin, and the SH3 domain-binding glutamic acid-rich-like protein 3, was detected. CONCLUSION. The proteomic profile of urine in FSGS, compared to MN, reflects the activation of variety of pathological processes – podocyte damage, involvement of parietal epithelial cells, tubulo-interstitial damage, accumulation of extracellular matrix, and complement activation process.

In this study, a method is developed for the simplified preparation of samples for analysis by high performance liquid chromatography and tandem mass spectrometry (HPLC–TMS) for further clinical diagnostic use, including the immunoprecipitation of fibrinogen on magnetic microparticles followed by the enzymatic hydrolysis of the protein. Development of an effective technique that would include the isolation of the target protein from the studied plasma sample or any other physiological fluid with the exclusion of a large number of process steps (for example, numerous stages of reprecipitation and various types of chromatography for the isolation of target proteins) and the use of aggressive conditions (for example, for the elution of proteins from carriers), as well as with a decrease in the total labor intensity and time of preparation of samples for analysis, is necessary for use in proteomic studies.

We proposed a method for the simultaneous rapid determination of four protein cardiomarkers (C‑reactive protein, cystatin C, myoglobin, and D-dimer) in the blood plasma of patients with cardiopathologies. A change in the concentration of each of the proteins separately may indicate specific pathological processes in a human body, and a simultaneous increase in the concentration of all four markers indicates the possible development of acute cardiopathology. Rapid analysis is based on the immunomagnetic affinity separation of target proteins from blood samples using a fluorescent label for each protein under study. The proposed method is automated and optimized for determining physiological and pathological concentrations of target blood markers. An automatic bioanalytical platform is developed based on the proposed method; it enables the rapid determination of cardiomarkers in 17 µL of blood plasma within 14 min.

Introduction. According to research, seminal plasma is a rich source of biomarkers in male infertility including biomarkers of residual spermatogenesis in males with azoospermia. In many cases, extraction of seminal plasma does not require invasive procedures. Therefore, non-invasive test for identification of males with azoospermia and residual focal spermatogenesis can improve selection of patients for microdissection testicular sperm extraction (microTESE) and become a useful tool for patient consultation.The study objective is to evaluate capabilities of proteomic analysis of seminal plasma for identification of males with azoospermia and residual focal spermatogenesis in the testes who have higher chance for sperm extraction using the microTESE procedure.Materials and methods. Samples of seminal plasma of 36 males between 21 and 45 years of age (mean age 33.3 ± ± 3.9 years) were tested for proteomic composition. For proteomic analysis, proteins were hydrolyzed with trypsin. The obtained peptides were analyzed using chromatography-mass spectrometry complex composed of liquid chromatograph nano-HPLC Agilent 1100 and high-resolution mass spectrometer 7Т LTQ-FT Ultra. Proteomic analysis data were compared with results of the microTESE procedure.Results. In this study, proteomic composition of seminal plasma in azoospermia was assessed. Semi-quantitative analysis without markers using bottom-up approach identified 405 different proteins, and 174 of them were detected in all samples. The obtained quantitative parameters were sufficient to identify patients with focal spermatogenesis.Conclusion. Seminal plasma is a potential source of biological markers for prediction of sperm extraction success in patients with azoospermia. The presented results are preliminary, and further studies are needed to confirm the identified protein panel.

Plasminogen is a zymogenic form of plasmin, an enzyme that plays a fundamental role in the dissolution of fibrin clots as well as in many other physiological processes. For the first time, by the method of gas chromatography–mass spectrometry, post-translational modifications in the primary structure of plasminogen treated with physiologically relevant amounts of hydrogen peroxide were identified. It was found that methionine and tryptophan residues located in different structural regions of plasminogen served as targets of the oxidant. Plasminogen oxidation caused a dose-dependent effect in decreasing the fibrinogenolytic activity of plasmin evidenced by the formation of fibrinogen degradation products. The possible antioxidant role of methionines in the oxidative modification of plasminogen is discussed.

Исследовалась окислительная модификация человеческого гемоглобина Hb, обработанного пероксидом водорода. Методом масс-спектрометрии были детектированы окисленные аминокислотные остатки молекулы гемоглобина α Trp14, α Tyr24, α Arg31, α Met32, α Tyr42, α His45, α His72, α Met76, α Pro77, α Lys90, α Cys104, α Tyr140, β His2, β Trp15, β Trp37, β Met55, β Cys93, β Cys112, β Tyr130, β Lys144, β His146. Обсуждается антиоксидантный потенциал молекулы Hb во внутриклеточном пространстве и при его попадании в плазму крови.

The effect of peroxide-induced oxidation of fibrinogen on modification of its primary structure and functional properties was investigated. The oxidation sites were shown to be Met, Trp, and His residues. Using the DLS method, it was found that the oxidative modification of fibrinogen results in the change of microrheological characteristics of fibrin network. The fibrinogen oxidation diminishes its tolerance to plasmin hydrolysis and deteriorates the factor XIIIa ability to stabilize the fibrin gel.

The damage to blood coagulation factor XIII (FXIII) at different stages of its enzymatic activation under the action of various physiological amounts of hypochlorite ion was studied. The results obtained by HPLC-MS/MS, SDS-PAGE, and colorimetry showed that, during the conversion of FXIII to FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased. FXIII oxidized with 150 μM hypochlorite completely retained its enzymatic activity inherent to the intact protein, whereas FXIIIa treated with 50 μM hypochlorite showed sharply reduced enzymatic activity. It was shown that a number of methionine and cysteine residues on the catalytic subunit can perform antioxidant function; additionally, the regulatory subunits of FXIII-B contribute to the antioxidant protection of the catalytic center of the FXIII-A subunit, which, together with the tight packing of the tetrameric structure of the FXIII proenzyme, are the three factors that provide high protein resistance to the oxidizing agent.

The plasma coagulation factor XIII (рFXIII) is a key protein of the blood coagulation systems, the main function of which consists of the enzymatic covalent stabilization of the polymeric structure of fibrin. The protein has a heterotetrameric structure consisting of two catalytic (FXIII-A2) and two regulatory (FXIII-В2) subunits. Using a high resolution mass spectrometry, hypochlorite-induced oxidation of рFXIII molecules and its enzymatic form (FXIIIa) is studied for the first time. It is demonstrated that sulfur and aromatic amino acid residues are the most vulnerable residues to oxidative attack. The mass spectrometry data indicate that oxidized amino acid residues are found in all structural elements of the FXIII-A catalytic subunit in the protein we are studying (except for the activation peptide), while a number of domains remain in native form in the FXIII-В regulatory subunit. When treated FXIIIa with hypochlorite, additional modification sites both in the FXIII- $${\text{A}}_{{\text{2}}}^{*}$$ and in the FXIII-В2 subunits are detected. The data obtained allowed us to postulate that in the process of converting the proenzyme into FXIIIa, new amino acid residues (previously inaccessible to oxidizers) migrate to the surface of the protein globule and become vulnerable targets for oxidizer molecules, while some of the initially surface-exposed amino acid residues move inside the protein, losing their ability to participate in the oxidative modifications. Electrophoresis of reduced samples of covalently cross-linked fibrin detected a decrease in transglutaminase activity of oxidized FXIIIa manifested in the inhibition of the reaction of the formation of γ–γ-dimers. The ability of the рFXIII molecule to resist the oxidative attack due to its antioxidant structural adaptation to the effect of reactive oxygen species is discussed.

The iron-containing protein neuroglobin (Ngb) involved in the transport of oxygen is generally considered the precursor of all animal globins. In this report, we studied the structure of Ngb of the cold-water sponge Halisarca dujardinii. In sponges, the oldest multicellular organisms, the Ngb gene contains three introns. In contrast to human Ngb, its promoter contains a TATA-box, rather than CG-rich motifs. In sponges, Ngb consists of 169 amino acids showing rather low similarity with its mammalian orthologues. It lacks Glu and Arg residues in positions required for prevention of hypoxia-related apoptosis. Nevertheless, Ngb contains both proximal and distal conserved heme-biding histidines. The primary structure of H. dujardinii neuroglobin predicted by sequencing was confirmed by mass-spectrometry analysis of recombinant Ngb expressed in E. coli. The high level of Ngb expression in sponge tissues suggests its possible involvement in the gas metabolism and presumably in other key metabolic processes in H. dujardinii.

Oxidation of fibrinogen with hypochlorite inhibited the fibrin network self-assembly even at the lowest concentration of the oxidant. The analysis of the results of protein electrophoresis at this hypochlorite concentration showed the absence of fragmentation of the protein and covalent cross-linking of its chains. The study of the areas responsible for the conversion of fibrinogen into fibrin by mass spectrometry showed that they are not subject to oxidative damage. However, we identified oxidized amino acid residues, which could affect the protofibril aggregation.

alpha-Crystallin maintains the transparency of the lens by preventing the aggregation of damaged proteins. The aim of our work was to study the chaperone-like activity of native alpha-crystallin in near physiological conditions (temperature, ionic power, pH) using UV-damaged beta(L)-crystallin as the target protein. alpha-Crystallin in concentration depended manner inhibits the aggregation of UV-damaged beta(L)-crystallin. DSC investigation has shown that refolding of denatured UV-damaged beta(L)-crystallin was not observed under incubation with alpha-crystallin. alpha-Crystallin and UV-damaged beta(L)-crystallin form dynamic complexes with masses from 75 to several thousand kDa. The content of UV-damaged beta(L)-crystallin in such complexes increases with the mass of the complex. Complexes containing >10% of UV-damaged beta(L)-crystallin are prone to precipitation whereas those containing <10% of the target protein are relatively stable. Formation of a stable 75 kDa complex is indicative of alpha-crystallin dissociation. We suppose that alpha-crystallin dissociation is the result of an interaction of comparable amounts of the chaperone-like protein and the target protein. In the lens simultaneous damage of such amounts of protein, mainly beta and gamma-crystallins, is impossible. The authors suggest that in the lens rare molecules of the damaged protein interact with undissociated oligomers of alpha-crystallin, and thus preventing aggregation. (C) 2019 Elsevier B.V. All rights reserved.