The search for potential hypotensive peptides in the amino acid sequence of human renalase and their identification in proteolytic fragments of this protein.

Renalase (RNLS) is a secretory protein discovered in 2005. It plays an important role in the regulation of blood pressure. Studies by two independent laboratories have shown that administration of purified recombinant RNLS reduced blood pressure in experimental animals. However, the mechanisms of the antihypertensive effect of RNLS still remain unclear, especially in the context of the shift in the catalytic paradigm of this protein. In addition, there is growing evidence that endogenous plasma/serum RNLS, detected by enzyme immunoassay, is not an intact protein secreted into the extracellular space, and exogenous recombinant RNLS is effectively cleaved during short-term incubation with human plasma samples. This suggests that the antihypertensive effect of RNLS may be due to peptides formed during proteolytic processing. Based on the results of a bioinformatics analysis of potential RNLS cleavage sites (Fedchenko et al., Medical Hypotheses, 2022; DOI: 10.1016/j.mehy.2022.110895), a number of short peptides have been identified in the RNLS sequence that show similarity to fragments of known peptide inhibitors of angiotensin-converting enzyme. Some of them were found as a part of larger RNLS peptides, formed during RNLS cleavage by chymotrypsin and, and to a lesser extent, by trypsin.

[Characteristics of behavioral reactions and the profile of brain isatin-binding proteins of rats with the rotenone-induced experimental parkinsonism]. / Osobennosti povedencheskikh reaktsii i profilia izatin-sviazyvaiushchikh belkov mozga u krys s indutsirovannym rotenonom éksperimental'nym parkinsonizmom.

The neurotoxins rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (МPTP) are used for modeling Parkinson's disease in animals (PD). They induce the mitochondrial respiratory chain dysfunction, which leads to the dopaminergic (DA) neuron degeneration. The advantage of the rotenone model consists in ability of rotenone to cause neurodegeneration showing symptoms and molecular biological characteristics similar to those of PD. Isatin (indoldione-2,3) is an endogenous regulator found in tissues and biological fluids of humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. In this work we have investigated behavioral reactions and profiles of brain isatin-binding proteins of rats with Parkinson's syndrome (PS) in comparison with the corresponding parameters of MPTP-induced Parkinsonism in mice. Systemic injection of rotenone caused severe PS comparable with the effect of MPTP injection. It was accompanied by significant body weight loss, death, oligokinesia, muscular rigidity, and postural instability of animals. In spite of the same pathogenic basis of PS caused by rotenone and MPTP, the molecular mechanisms of their action differ. In the case of rotenone-induced PS, the pool of isatin-binding proteins common of the control rats and the rats with PS (146) significantly exceeded the pool of the common proteins of control mice and mice with PS induced by MPTP, whether right after neurotoxin injection (27), or (all the more) in a week after the MPTP injection (14). The comparison of isatin-binding proteins specific of the animals with MPTP-induced PS and with the rotenone-induced PS (as compared with the control animals) revealed total absence of proteins common of these two models of PD. It is to be noted that both neurotoxins particularly affected the proteins participating in the signal transmission and enzyme activity regulation. The changes of the profile of isatin-binding proteins in response to the injection of rotenone suggest that the neuroprotector isatin could also influence positively in the case of the rotenone model of PD.

[The key role of the regulatory 19S subunit in changes in the brain proteasome subproteome induced by the neuroprotector isatin]. / Kliuchevaia rol' reguliatornoi 19S subchastitsy v izmenenii subproteoma proteasom mozga, indutsirovannom neiroprotektorom izatinom.

Isatin (indole-2,3-dione) is an endogenous regulator exhibiting various effects mediated by numerous isatin-binding proteins localized in different compartments of cells of the brain and peripheral tissues. It attenuates manifestations of experimental parkinsonism induced by administration of the MPTP neurotoxin and reduces the movement disorders characteristic of this disease. The molecular mechanisms of the neuroprotective action of isatin include its direct interaction with proteasomes, intracellular supramolecular complexes responsible for the targeted elimination of proteins. Incubation of fractions of 26S and 20S rabbit brain proteasomes, containing the whole spectrum of proteasomal subunits, as well as a number of proteasome-associated proteins, with isatin (100 µM) had a significant impact on the profile of released proteins. In the case of 26S proteasomes containing, in addition to the core part (20S proteasome), 19S regulatory subparticles, incubation with isatin resulted in a more than threefold increase in the number of dissociated proteins. In the case of 20S proteasomes (containing only the 20S core particle), incubation with isatin resulted in a significant decrease in the number of dissociated proteins compared to the control. Our results indicate an important role of the regulatory 19S subunit components in the formation of the proteasome subproteome and the sensitivity of these supramolecular complexes to isatin.

[Comparative analysis of proteins associated with 26S and 20S proteasomes isolated from rabbit brain and liver]. / Sravnitel'nyi analiz belkov, assotsiirovannykh s 26S i 20S proteasomami mozga i pecheni krolika.

We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.

[Changes in the mitochondrial subproteome of mouse brain Rpn13-binding proteins induced by the neurotoxin MPTP and the neuroprotector isatin]. / Izmenenie mitokhondrial'nogo subproteoma Rpn13-sviazyvaiushchikh belkov mozga myshi pod deistviem neirotoksina MFTP i neiroprotektora izatina.

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.

CD44-Associated Tn Antigen as a New Biomarker of Tumor Cells with Aberrant Glycosylation.

Tn antigen is a tumor-associated antigen that appears on cancer cells as a result of aberrant O-glycosylation. The most studied form of Tn antigen is found in mucins, in particular, in mucin 1 (MUC1). Antibodies against this form of Tn antigen are used to diagnose tumors, as well as to generate T-killers with a chimeric receptor. Some carcinomas do not carry MUC1 and antibodies of a different specificity are required to detect Tn antigen on these cells. In our work, we searched for anti-Tn antibodies without preliminary assumptions about the proteins that may be carriers of the Tn antigen. For this purpose, we obtained several pairs of isogenic cell lines with the wild type and knockout of the Cosmc gene, which is essential for correct protein O-glycosylation. Using the created lines as immunogens, we generated a monoclonal antibody AKC3, which reacted with the Cosmc-deficient A549 lung adenocarcinoma cells and did not bind to the wild-type cells. Using mass spectrometry, as well as co-immunoprecipitation, it was shown that the AKC3 antibody recognized the Tn antigen in the context of CD44 protein - a protein important for tumor growth. The AKC3 antibody can be used for tumor diagnosis, and to generate T cells with a chimeric receptor for treatment of tumors that do not express mucins.

[Qualitative difference of mitochondrial subproteoms of brain RPN10- and RPN13-binding proteins]. / Kachestvennoe razlichie mitokhondrial'nykh subproteomov RPN10- i RPN13-sviazyvaiushchikh belkov mozga.

Good evidence exists that the ubiquitin-proteasome system (UPS) plays an important role in degradation of mitochondrial proteins and membrane proteins associated with mitochondria (MAM proteins). Mitochondria contain all components of the ubiquitin-conjugating system, which are necessary for the attachment of ubiquitin molecules to target proteins, subjected to subsequent degradation in proteasomes. An important stage in the delivery of proteins for proteolytic degradation in proteasomes is their interaction with ubiquitin receptors located on the regulatory subunit (19S) of the proteasome: the Rpn10 or Rpn13 subunit. These subunits make basically the same contribution to the subsequent translocation of target proteins to the core part of the proteasome. A comparative study of mouse brain mitochondrial subproteomes bound to Rpn10 and Rpn13 subunits revealed a high specificity of the repertoire of Rpn10 and Rpn13-binding proteins. Moreover, proteins, for which mitochondrial localization or association with mitochondrial membranes was previously shown, prevailed in the case of using the Rpn13 subunit as an affinity ligand (Rpn13-binding proteins). This suggests that Rpn10 and Rpn13 play different roles in the degradation of mitochondrial proteins and MAM.

Proteome data of serum samples from patients with schizophrenia.

Schizophrenia is a complex chronic disease. The molecular determinants and neuropathology of schizophrenia are multifaceted; an important role in the pathogenesis is played by the dysregulation of molecular and epigenetic mechanisms. However, the molecular mechanisms of the development of the disease have not yet been studied. An important task is the accumulation and systematization of "OMICS"-knowledge of the molecular profiles (transcriptome, proteome, metabolome) of blood specific to pathology. Thereby the development and improvement of mass spectrometric methods for the detection of biological molecules has become increasingly important in biomedical research. In the field of applied problems in biomedical research, the most prevalent issue involves the identification of serological protein markers associated with the development of schizophrenia, which account for the diseases that cause the a life-shortening illness, disability, decreased of functioning and quality of life and wellbeing or health status. OMICS approaches are designed to detect genes (genomics), mRNA (transcriptomics), proteins (proteomics) and metabolites (metabolomics) in a specific biological sample. We report the proteomic datasets on the serum samples from patients with schizophrenia (series "SCZ") and healthy volunteers (series "CNT"). Data were acquired using shotgun ultra-high resolution mass spectrometry.

[The effect of a neuroprotective dose of isatin or deprenyl to mice on the profile of brain isatin-binding proteins]. / Izmenenie profilia izatin-sviazyvaiushchikh belkov mozga myshei pri odnokratnom vvedenii neiroprotektornoi dozy izatina ili deprenila.

Isatin (indol-2,3-dione), an endogenous biofactor found in the brain, peripheral tissues and biological body fluids of humans and animals, exhibits a wide range of biological and pharmacological activities. They are realized via interaction with numerous isatin-binding proteins. Some of these proteins identified during proteomic profiling of the brain are involved in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl (selegiline), a pharmacological agent used for treatment of Parkinson's disease. In this study, we have investigated the effect of a single dose administration of isatin (100 mg/kg) and deprenyl (10 mg/kg) to mice on the profile of the brain isatin-binding proteins. Comparative proteomic analysis of brain isatin-binding proteins of mice treated with isatin or deprenyl resulted in identification of a representative group of proteins (n=200) sensitive to the administration of these substances. The change in the profile of isatin-binding proteins may be obviously attributed to accumulation of isatin and deprenyl in the brain and their interaction with target proteins; this prevents protein binding to the affinity sorbent. Thus identified brain isatin-binding proteins of the control animals obviously represent specific targets that interact directly with isatin (and also with deprenyl) in vivo. Isatin or deprenyl administered to animals interact with these proteins and thus inhibit their binding to the affinity sorbent (immobilized isatin analogue).

Ubiquitin Subproteome of Brain Mitochondria and Its Changes Induced by Experimental Parkinsonism and Action of Neuroprotectors.

The review summarizes the data of our research and published studies on the ubiquitination of brain mitochondrial proteins and its changes during the development of experimental parkinsonism and administration of the neuroprotector isatin (indole-2,3-dione) with special attention to the mitochondrial ubiquitin-conjugating system and location of ubiquitinated proteins in these organelles. Incubation of brain mitochondrial fraction with biotinylated ubiquitin in vitro resulted in the incorporation of biotinylated ubiquitin in both mitochondrial and mitochondria-associated proteins. According to the interactome analysis, the identified non-ubiquitinated proteins are able to form tight complexes with ubiquitinated proteins or their partners and components of mitochondrial membranes, in which interactions of ubiquitin chains with the ubiquitin-binding protein domains play an important role. The studies of endogenous ubiquitination in the total brain mitochondrial fraction of C57Bl mice performed in different laboratories have shown that mitochondrial proteins represent about 30% of all ubiquitinated proteins. However, comparison of brain subproteomes of mitochondrial ubiquitinated proteins reported in the literature revealed significant differences both in their composition and involvement of identified ubiquitinated proteins in biological processes listed in the Gene Ontology database. The development of experimental parkinsonism in C57Bl mice induced by a single-dose administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in a decrease in the total number of mitochondrial ubiquitinated proteins and increase in the number of oxidized mitochondrial proteins containing the ubiquitin signature (K-ε-GG). Comparison of ubiquitinated proteins associated with the mouse brain mitochondrial fraction and mouse brain mitochondrial proteins bound to the proteasome ubiquitin receptor (Rpn10 subunit) did not reveal any common proteins. This suggests that ubiquitination of brain mitochondrial proteins is not directly related to their degradation in the proteasomes. Proteomic profiling of brain isatin-binding proteins identified enzymes involved in the ubiquitin-conjugating system functioning. Mapping of the identified isatin-binding proteins to known metabolic pathways indicates their participation in the parkin (E3 ubiquitin ligase)-associated pathway (CH000000947). The functional links involving brain mitochondrial ubiquitinated proteins were found only in the group of animals with the MPTP-induced parkinsonism, but not in animals treated with MPTP/isatin or isatin only. This suggests that the neuroprotective effect of isatin may be associated with the impaired functional relationships of proteins targeted to subsequent degradation.

[Identification of the Molecular Partners of Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) That Are Involved in Human Lymphocyte Activation].

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein that is found in lymphocytes and is tightly associated with the phosphatase CD45. The function of LPAP is still unknown. Studies of the LPAP interactome may reveal new details of how C45 and lymphocyte signaling in general are regulated. LPAP binding partners were sought using coimmunoprecipitation coupled with mass spectrometry, stabilization of protein complexes with chemical crosslinkers, and Blue Native electrophoresis. In addition to CD45, several proteins were identified as LPAP partners, including CD71, CD98, cytoskeletal proteins, the amino acid transporter SLC1A4, and the cell signaling component HS1. It was confirmed that more than 70% of LPAP molecules were bound with CD45 in a 1 : 1 complex. The effect of CD45 on LPAP was studied in CEM and Jurkat cells with a CD45 knockout. The LPAP levels in the cells were 10% of the level in wild-type cells. In the absence of CD45, LPAP phosphorylation at Ser-153 and Ser-163 was not affected, whereas phosphorylation at Ser-99 and Ser-172 decreased significantly. Based on the results, CD45 was assumed to play a role in regulating LPAP expression and phosphorylation status.

Comparative Analysis of Blood Plasma Proteome in Patients with Renal Cell Carcinoma.

Comparative mass spectrometric analysis of protein composition was carried out in 36 blood plasma specimens from patients with renal cell carcinoma and 20 specimens from donors. Analysis of protein composition of plasma specimens devoid of the major protein fractions showed a 20-50% higher level of protein identifications in patient' specimens. Specimens of the control and experimental series were similar by protein composition, 70-80% identifications in experimental and control series coinciding. High similarity of biological processes with participation of the proteins identified in both series was observed. The greater part of proteins in both series were located extracellularly and were exosomal (specimens from renal cancer patients) or vesicular (specimens from healthy volunteers).

Application of Adeno-Associated Virus Vectors for Engineering SCF-Containing Extracellular Vesicles of Mesenchymal Stromal Cells.

Mesenchymal stromal cells from rat adipose tissue were transduced with adeno-associated viral (AAV) vector encoding stem cell factor SCF that stimulates proliferation of cardiac c-kit+ cells and improved cardiac function and survival of animals after myocardial infarction. Extracellular vesicles isolated from the medium conditioned by mesenchymal stromal cells by ultracentrifugation were characterized by Western blotting, transmission electron microscopy, nanoparticle tracking analysis, immunostaining, and mass spectrometry analysis. Using proteomic analysis, we identified transgenic SCF in extracellular vesicles released by AAV-modified mesenchymal stromal cells and detected some proteins specific of extracellular vesicles secreted by transduced cells. Extracellular vesicles from AAV-transduced mesenchymal stromal cells could be used for delivery of transgenic proteins as they were readily endocytosed by both cardiosphere-derived cells and cardiac-progenitor cells.

Comprehensive Analysis of Carbohydrate-Active Enzymes from the Filamentous Fungus Scytalidium candidum 3C.

Complete enzymatic degradation of plant polysaccharides is a result of combined action of various carbohydrate-active enzymes (CAZymes). In this paper, we demonstrate the potential of the filamentous fungus Scytalidium candidum 3C for processing of plant biomass. Structural annotation of the improved assembly of S. candidum 3C genome and functional annotation of CAZymes revealed putative gene sequences encoding such proteins. A total of 190 CAZyme-encoding genes were identified, including 104 glycoside hydrolases, 52 glycosyltransferases, 28 oxidative enzymes, and 6 carbohydrate esterases. In addition, 14 carbohydrate-binding modules were found. Glycoside hydrolases secreted during the growth of S. candidum 3C in three media were analyzed with a variety of substrates. Mass spectrometry analysis of the fungal culture liquid revealed the presence of peptides identical to 36 glycoside hydrolases, three proteins without known enzymatic function belonging to the same group of families, and 11 oxidative enzymes. The activity of endo-hemicellulases was determined using specially synthesized substrates in which the glycosidic bond between monosaccharide residues was replaced by a thio-linkage. During analysis of the CAZyme profile of S. candidum 3C, four ß-xylanases from the GH10 family and two ß-glucanases from the GH7 and GH55 families were detected, partially purified, and identified.

[The effect of deprenyl and isatin administration to mice on the proteomic profile of liver isatin-binding proteins]. / Vliianiia vvedeniia deprenila i izatina mysham na profil' izatin-sviazyvaiushchikh belkov pecheni.

Isatin (indol-2,3-dione) is an endogenous indole found in the brain, peripheral tissues and biological body fluids of humans and animals. Its wide spectrum of biological activity is realized via interaction with numerous isatin-binding proteins; these include proteins playing an important role in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl, a pharmacological agent used for treatment of Parkinson's disease. In this study, the effects of the course of deprenyl (1 mg/kg) and isatin (20 mg/kg) administration for 21 days on the profile of the isatin-binding proteins of the liver of mice have been investigated. Proteomic profiling of liver isatin-binding proteins of control mice by means of 5-aminocaproylisatin as an affinity ligand resulted in identification of 105 proteins. Treatment of animals with a low dose of isatin slightly decreased (up to 91), while injections of deprenyl slightly increased (up to 120) the total number of isatin-binding proteins. 75 proteins were common for all three groups; they represented from 62.5% (in deprenyl treated mice) and 71% (in control mice), to 82% (isatin treated mice) of the total number of identified liver isatin-binding proteins. Proteomic analysis of the isatin-binding proteins of mice treated with isatin (20 mg/kg) or deprenyl (1 mg/kg) for 21 days revealed a representative group of proteins (n=30) that were sensitive to the administration of these substances. Taking into account the previously obtained results, it is reasonable to suggest that the change in the profile of isatin-binding proteins may be attributed to accumulation of isatin and deprenyl in the liver and interaction with target proteins prevents their subsequent binding to the affinity sorbent. In this context, the identified isatin-binding liver proteins of control animals that do not bind to the affinity sorbent (immobilized isatin analogue) after treatment of animals with either deprenyl or isatin appear to be specific targets directly interacting with isatin in vivo.

[Quantitative targeted screening of proteins associated with lung adenocarcinoma cancer by the method of selected reaction monitoring]. / Napravlennyi kolichestvennyi skrining markerov adenokartsinomy legkogo metodom monitoringa vybrannykh reaktsii.

In the present study, we applied selected reaction monitoring (SRM) to a group of proteins that were previously reported to be associated with lung cancer (Novikova S.E. et al. (2017) Biomeditsinskaya khimiya, 63, 181-210. [1]). Measurements were performed on 59 plasma samples. These samples included: 23 samples of plasma of patients diagnosed with lung adenocarcinoma (LAC), 11 samples of plasma of patients diagnosed with squamous cell lung carcinoma (SqCC), 25 samples of donors with no previous history of oncological diseases, and one pooled sample from each of the above group. As a result of the SRM measurements 52 proteins were detected at least in one individual plasma sample. Statistical analysis showed that there were two groups confidently differentiated by the concentration value of 8 proteins wherein 5 proteins displayed increased level (P00738, P26639, P21926, P08603, P51149) in LAC group and 3 proteins (P51884, O15162, Q8N2K0) indicated diminishing the concentration level towards the control level. Data on protein concentrations obtained for LAC and SqCC did not distinguish the samples by statistical clustering analysis. These potential biomarkers can be used for further development of methods for early diagnostics of lung cancer.

A new method for quantitative determination of renalase based on mass spectrometric determination of a proteotypic peptide labelled with stable isotopes.

RATIONALE: Renalase is a recently discovered kidney secretory protein, which is considered as an important component involved in blood pressure regulation. Although altered levels of renalase have been detected in plasma and urine of patients with various kidney diseases, there is certain inconsistency of changes in the renalase levels reported by different laboratories. The latter is obviously associated with the use of the ELISA as the only available approach for quantitative analysis of renalase. Thus there is a clear need for the development of antibody-independent approaches for renalase quantification. METHODS: We have developed a new method for quantitative determination of human renalase, which is based on mass spectrometric detection of a proteotypic peptide containing С-terminal 13 C15 N-labelled lysine. It corresponds to a tryptic peptide of human renalase, which has been previously detected in most mass spectrometric determinations of this protein. RESULTS: Using the labelled peptide H-EGDCNFVAPQGISSIIK-OH, corresponding to positions 100-116 of the human renalase sequence, as an internal standard and recombinant human renalase we have generated a calibration curve, which covered the concentration range 0.005-50 ng/mL with a limit of quantitation of 5 pg/mL. Using this calibration curve we were able to detect urinary renalase only after enrichment of initial urinary samples by ammonium sulfate precipitation (but not in untreated urine). CONCLUSIONS: Results of our study indicate that quantitative determination of renalase based on mass spectrometric detection of a proteotypic peptide labelled with stable isotopes gives significantly lower values of this protein in human urine than those reported in the literature and based on the ELISA.

Isolation of Large Amounts of Highly Pure Mitochondria for "Omics" Studies.

Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.

Quantitative target proteomics of chromosome 13 human blood plasma proteins.

Quantitative proteomic analysis of 50 blood plasma samples of healthy volunteers who underwent a comprehensive medical examination and were found eligible for space flights was performed. As a result of directed mass spectrometric analysis, signals for 128 proteins, which accounted for nearly 40% of the total number of chromosome 13 gene products, were detected. The analysis of interindividual variation of concentrations of chromosome 13 proteins showed the presence of a pool comprising 41 proteins with a low variation (CV < 30%), which can potentially be used as biomarkers.

Identification of Peptide AEDG in the Polypeptide Complex of the Pineal Gland.

The polypeptide complex of the epiphysis and the peptide AEDG, constructed on the basis of its amino acid analysis, exert similar biological effects. Both bioregulators normalize melatonin synthesis in the pineal gland, functioning of the brain, eye retina, cardiovascular, endocrine, and immune systems; they also act as antioxidants, stress-protectors, and geroprotectors. Within the epiphysis polypeptide complex, free amino acids (3.26%), dipeptides (23.19%), tripeptides (50.72%), tetrapeptides (22.10%), and pentapeptides (0.72%) were revealed by mass spectrometry and HPLC. Peptide AEDG was detected among the tetrapeptides of the epiphysis polypeptide complex by selective reaction monitoring method. The biological effects of the epiphysis polypeptide complex are determined by the effect of its component AEDG.