[Analysis of phosphatidylcholines alterations in human glioblastoma multiform tissues ex vivo]. / Analiz izmenenii sostava fosfatidilkholinov v glioblastomakh cheloveka ex vivo.

Significant metabolism alteration is accompanying the cell malignization process. Energy metabolism disturbance leads to the activation of de novo synthesis and beta-oxidation processes of lipids and fatty acids in a cancer cell, which becomes an indicator of pathological processes inside the cell. The majority of studies dealing with lipid metabolism alterations in glial tumors are performed using the cell lines in vitro or animal models. However, such conditions do not entirely represent the physiological conditions of cell growth or possible cells natural variability. This work presents the results of the data obtained by applying ambient mass spectrometry to human glioblastoma multiform tissues. By analyzing a relatively large cohort of primary and secondary glioblastoma samples, we identify the alterations in cells lipid composition, which accompanied the development of grade IV brain tumors. We demonstrate that primary glioblastomas, as well as ones developed from astrocytomas, are enriched with mono- and diunsaturated phosphatidylcholines (PC 26:1, 30:2, 32:1, 32:2, 34:1, 34:2). Simultaneously, the saturated and polyunsaturated phosphatidylcholines and phosphatidylethanolamines decrease. These alterations are obviously linked to the availability of the polyunsaturated fatty acids and activation of the de novo lipid synthesis and beta-oxidation pathways under the anaerobic conditions in the tumor core.

The Nature of Resistance of the Coagulation Factor XIII Structure to Hypochlorite-Induced Oxidation.

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 role of lipids in the classification of astrocytoma and glioblastoma using MS tumor profiling]. / Rol' lipidov pri klassifikatsii astrotsitomy i glioblastomy pri pomoshchi mass-spektrometricheskogo profilirovaniia opukholei.

Express MS identification of biological tissues has become a much more accessible research method due to the application of direct specimen ionization at atmospheric pressure. In contrast to traditional methods of analysis employing GC-MS methods for determining the molecular composition of the analyzed objects it eliminates the influence of mutual ion suppression. Despite significant progress in the field of direct MS of biological tissues, the question of mass spectrometric profile attribution to a certain type of tissue still remains open. The use of modern machine learning methods and protocols (e.g., "random forests") enables us to trace possible relationships between the components of the sample MS profile and the result of brain tumor tissue classification (astrocytoma or glioblastoma). It has been shown that the most pronounced differences in the mass spectrometric profiles of these tumors are due to their lipid composition. Detection of statistically significant differences in lipid profiles of astrocytoma and glioblastoma may be used to perform an express test during surgery and inform the neurosurgeon what type of malignant tissue he is working with. The ability to accurately determine the boundaries of the neoplastic growth significantly improves the quality of both surgical intervention and postoperative rehabilitation, as well as the duration and quality of life of patients.

Metrics for evaluating the stability and reproducibility of mass spectra.

In this work, we demonstrate a new approach for assessing the stability and reproducibility of mass spectra obtained via ambient ionization methods. This method is suitable for both comparing experiments during which only one mass spectrum is measured and for evaluating the internal homogeneity of mass spectra collected over a period of time. The approach uses Pearson's r coefficient and the cosine measure to compare the spectra. It is based on the visualization of dissimilarities between measurements, thus leading to the analysis of dissimilarity patterns. The cosine measure and correlations are compared to obtain better metrics for spectra homogeneity. The method filters out unreliable scans to prevent the analyzed sample from being wrongly characterized. The applicability of the method is demonstrated on a set of brain tumor samples. The developed method could be employed in neurosurgical applications, where mass spectrometry is used to monitor the intraoperative tumor border.

[The role of lipid metabolism disorders, atypical isoforms of protein kinase C, and mutational status of cytosolic and mitochondrial forms of isocitrate dehydrogenase in carcinogenesis of glial tumors]. / Rol' narushenii lipidnogo obmena, atipicheskikh izoform proteinkinazy S i mutatsionnogo statusa tsitozol'noi i mitokhondrial'noi form izotsitratdegidrogenazy v kantserogeneze glial'nykh opukholei.

The relationship between molecular genetic and metabolic disorders is one of the challenges of modern oncology. In this review, we consider lipid metabolism and its changes as one of the factors of oncogenesis of glial tumors. Also, we demonstrate that the genome and the metabolome are interconnected by a large number of links, and the metabolic pathways, during their reorganization, are able to drastically affect the genetic structure of the cell and, in particular, cause its tumor transformation. Our own observations and analysis of the literature data allow us to conclude that mass spectrometry is a highly accurate current method for assessing metabolic disorders at the cellular level. The use of mass spectrometry during surgery allows the neurosurgeon to obtain real-time data on the level of specific molecular markers in the resected tissue, thereby bringing intraoperative navigation techniques to the molecular level. The generation of molecular fingerprints for each tumor significantly complements the available neuroimaging, molecular genetic, and immunohistochemical data.

High-resolution mass spectra processing for the identification of different pathological tissue types of brain tumors.

The purpose of the work is to demonstrate the possibilities of identifying the different types of pathological tissue identification directly through tissue mass spectrometry. Glioblastoma parts dissected during neurosurgical operation were investigated. Tumor fragments were investigated by the immunohistochemistry method and were identified as necrotic tissue with necrotized vessels, necrotic tissue with tumor stain, tumor with necrosis (tumor tissue as major), tumor, necrotized tumor (necrotic tissues as major), parts of tumor cells, boundary brain tissue, and brain tissue hyperplasia. The technique of classification of tumor tissues based on mass spectrometric profile data processing is suggested in this paper. Classifiers dividing the researched sample to the corresponding tissue type were created as a result of the processing. Classifiers of necrotic and tumor tissues are shown to yield a combined result when the tissue is heterogeneous and consists of both tumor cells and necrotic tissue.

Is myelin basic protein a potential biomarker of brain cancer?

Myelin basic protein is a potential biomarker for the central nervous system diseases in which the myelin sheath is destroyed. Using pseudo-selected reaction monitoring and the method of standard additions, we have measured the myelin basic protein level in the cerebrospinal fluid of patients with neurotrauma (n = 6), chronic neurodegenerative diseases (n = 2) and brain cancer (n = 5). Myelin basic protein was detected only in four out of five cerebrospinal fluid samples of patients with brain cancer. The cerebrospinal fluid myelin basic protein level ranged from 3.7 to 8.8 ng ml-1. We suggest that monitoring of myelin basic protein in cerebrospinal fluid can serve as a diagnostic test for the brain cancer.

Evaluation of plasma peptides extraction methods by high-resolution mass spectrometry.

Monitoring of peptides offers a promising approach for the discovery of novel biomarkers, which might be valuable for detection, treatment and prevention of large variety of diseases. Development of highly effective methods for plasma peptide extraction remains an important task. In the current study, we applied different types of plasma peptide extraction approaches to reveal efficient methods which would provide the highest peptide yield. We used different combinations of plasma treatment with acetonitrile and/or urea/guanidine, protein precipitation by acetone, gel-filtration, ultrafiltration, and two types of solid phase extraction. The extracted peptides were analyzed by LC-MS/MS. The obtained results suggest that several methods, including differential solubilization, organic precipitation, as well as some variants of ultrafiltration and solid phase extraction, provide effective plasma peptide enrichment convenient for further LC-MS/MS analysis. Alas, most of the identified peptides were extracted by only one of the applied methods. Hence, it seems reasonable to consider several methods to increase the possibility of novel biomarker discovery.

[Features of the urine peptidome under the condition of hypertensive pathologies of pregnancy]. / Osobennosti peptidoma mochi pri gipertenzivnykh patologiiakh beremennykh.

In order to find a peptide panel to differentiate close hypertensive conditions a case-control study was designed for 64 women from 4 groups: preeclampsia (PE), chronic hypertension superimposed with PE, chronic hypertension, and healthy individuals. Chromatography coupled with mass-spectrometry and subsequent bioinformatic analysis showed several patterns in the changes of the urine peptidome. There were 36 peptides common for four groups. Twenty two of them 22 belonged to alpha-1-chain of collagen I, nine peptides were from alpha-1-chain of collagen III, two from alpha-2-chain of collagen I, one from alpha-1/2-chain of collagen I, one from alpha-1-chain of collagen I/XVIII and one from uromodulin. Patients with hypertensive disorders had 34 common peptides: 12 from alpha-1-chain of collagen I, 10 from fibrinogen alpha-chain, eight from alpha-1-chain of collagen III, and 4 per other types of collagen. Comparative analysis revealed 12 peptides, which could be used as a diagnostic panel for confident discrimination of pregnant women with various hypertensive disorders.

[Secondary Structure of Aß(1-16) Complexes with Zinc: A Study in the Gas Phase Using Deuterium/Hydrogen Exchange and Ultra-High-Resolution Mass Spectrometry].

Complexes of peptide fragment 1-16 of beta-amyloid with transition metals play an important role in the development of a broad class of neurodegenerative diseases, which determines the interest in investigating the structures of these complexes. In this work, we have applied the method of the deuterium/hydrogen exchange in combination with ultra-high-resolution mass spectrometry to study conformational changes in (1-16) beta-amyloid peptide induced by binding of zinc(II) atoms. The efficiency of the deuterium/hydrogen exchange depended on the number of zinc atoms bound to the peptide and on the temperature of the ionization source region. Deuterium/hydrogen exchange reactions have been performed directly in the ionization source. The number of exchanges decreased considerably with an increasing numbers of zinc atoms. The relationship has been described with a damped exponential curve, which indicated that the binding of zinc atoms altered the conformation of the peptide ion by making it less open, which limits the access to inner areas of the molecule.

Oxidation-induced modification of the fibrinogen polypeptide chains.

By using the mass-spectrometry method, the oxidative modifications of the fibrinogen Aα, Bß, and γ polypeptide chains induced by its oxidation have been studied. The αC-region has been proven to be the most vulnerable target for the oxidizer (ozone) as compared with the other structural elements of the Aα chain. The Bß chain mapping shows that the oxidative sites are localized within all the structural elements of the chain in which the ß-nodule exhibits high susceptibility to oxidation. The γ chains are the least vulnerable to the oxidizer action. The results obtained demonstrate convincingly that the self-assembly centers dealing with interactions of knob "A": hole "a" are not involved in oxidative modification. It is concluded that the numerous oxidative sites revealed are mainly responsible for inhibiting lateral aggregation of protofibrils. The part of amino acid residues subjected to oxidation is supposed to carry out the antioxidant function.

[Analysis of contribution of protein phosphorylation in the development of the diseases]. / Opredelenie roli fosforilirovaniia belkov v razvitii zabolevanii.

In recent decades, studies in the molecular origins of socially significant diseases have made a big step forward with the development and using of high-performance methods in genomics and proteomics. Numerous studies in the framework of the global program "Human Proteome" were aimed at the identification of all possible proteins in various cell cultures and tissues, including cancer. One of the objectives was to identify biomarkers - proteins with high specificity to certain pathologies. However, in many cases, it is shown that the development of the disease is not associated with the appearance of new proteins, but depends on the level of gene expression or forming of proteoforms - splice variants, single amino acid substitutions (SAP variants), and post-translational modifications (PTM) of proteins. PTM may play a key role in the development of pathology because they activate a variety of regulatory or structural proteins in the majority of cell physiological processes. Phosphorylation is among the most significant of these protein modifications.This review will describe methods for analysis of protein phosphorylation used in the studies of such diseases as cancer and neurodegenerative diseases, as well as examples of cases when the modified proteins are involved directly to their development, and screening such significant PTM is used for the diagnosis and choice of treatment.

An untargeted approach for the analysis of the urine peptidome of women with preeclampsia.

Preeclampsia (PE) is a pregnancy complication characterized by high blood pressure and proteinuria. The disorder usually occurs after the 20th week of pregnancy and gets worse over time. PE increases the risk of poor outcomes for both the mother and the baby. In the study we applied LC-MS/MS method for the analysis of the urine peptidome of women with PE. Samples were prepared using size-exclusion chromatography method which gives more than twice peptides identities if compared with solid phase extraction. Thirty urine samples from women with mild and severe preeclampsia and the control group were analyzed. In total 1786 peptides were identified using complementary search engines (Mascot, MaxQuant and PEAKS). A high level of agreement in peptide identification was observed with previously published data. Label-free data comparison resulted in 35 peptides which reliably distinguished a particular PE group (severe or mild) from controls. Our results revealed unique identifications (correlate to alpha-1-antitrypsin, collagen alpha-1(I) chain, collagen alpha-1 (III) chain, and uromodulin, for instance) that can potentially serve as early indicators of PE.

[Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin ß-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the ß-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

[In vitro protein phosphorylation as a template for SRM method development].

Phosphorylation is one of the most common posttranslational modification (PTM) of proteins. Main challenge of phosphoprotein detection is their low abundance comparing to abundance of unmodified proteins. The method of selected reactions monitoring (SRM) allows to perform very sensitive and selective analysis of desired PTMs. Using myelin basic protein (MBP) as a model we have developed a method for phosphoprotein detection by SRM. The method is based on obtaining of phosphoproteins in a reconstituted kinase system and following usage these phosphorylated protein as a template for the development of the SRM method. The developed method was successfully applied for detection of phosphopeptides of myelin basic protein in the samples of human brain glioma.

Changes of protein profile of human urine after long-term orbital flights.

We analyzed protein profile of urine samples obtained from 7 Russian cosmonauts (age 35-51 years), participants of space flights on the International Space Station lasting for 169-199 days. Gradient chromatography with linear increase of eluent proportion was carried out in a system consisting of an Agilent 1100 chromatograph (Agilent Technologies Inc.) and a hybrid mass-spectrometer LTQ-FT Ultra (Thermo). The obtained results help to understand changes in the human body induced by space flight factors.

[Identification of transthyretin posttranslational modifications 1n human blood using mass-spectrometric methods].

Transthyretin, one of the major plasma proteins, has a number of posttranslational modifications and mutations, some of which are associated with the development of severe diseases, for instance, familial amyloid neuropathy and Alzheimer's disease. In order to investigate the role of modified forms in the development of these diseases a complex analytical platform, based on two mass-spectrometric approaches (bottom-up and op-down) has been developed. The high efficiency of this method was shown using 10 plasma samples obtained from patients with Alzheimer's disease and healthy individuals.

Use of models of biomacromolecule separation in AMT database generation for shotgun proteomics.

Generation of a complex proteome database requires use of powerful analytical methods capable of following rapid changes in the proteome due to changing physiological and pathological states of the organism under study. One of the promising technologies with this regard is the use of so-called Accurate Mass and Time (AMT) tag peptide databases. Generation of an AMT database for a complex proteome requires combined efforts by many research groups and laboratories, but the chromatography data resulting from these efforts are tied to the particular experimental conditions and, in general, are not transferable from one platform to another. In this work, we consider an approach to solve this problem that is based on the generation of a universal scale for the chromatography data using a multiple-point normalization method. The method follows from the concept of linear correlation between chromatography data obtained over a wide range of separation parameters. The method is further tested for tryptic peptide mixtures with experimental data collected from mutual studies by different independent research groups using different separation protocols and mass spectrometry data processing tools.

[Investigation of urine proteome in normal humans].

Recent advances in genomics and informatics have given birth to such area in molecular biology as proteomics. Proteomics is currently tasked with the full proteoma mapping of various biological substrates (e.g. body liquids, cells and tissues) both in the norm and pathology, and discovery of biomarkers of pathologies including tumors. The review presents analysis of the urine proteoma literature over the last five years covering the methods, strategy and results of the investigations.

Tandem fourier transform mass spectrometry studies of surface-induced dissociation of benzene monomer and dimer ions on a self-assembled fluorinated alkanethiolate monolayer surface.

A new instrument configuration based on a Finnigan FTMS-2000 platform has been applied to the study of surface-induced dissociation (SID) in this research. Benzene monomer ions C(6)H(6)(+) and dimer ions (C(6)H(6))(2)(+) were impacted on a fluorinated self-assembled monolayer surface at collision energies ranging from 1 to 70 eV. Benzene cations were chosen for this study because the fragmentation characteristics of the molecular cation are well known and its SID has been thoroughly investigated. SID spectra obtained by FTMS-SID are very similar to those reported in the literature for the same surface but exhibit much higher mass resolution. A comparison study of collision-induced dissociation (CID) and SID of benzene molecular cations was performed utilizing the same ICR cell and ion detection protocol. It is demonstrated that SID provides both much higher energy deposition and a narrower internal energy distribution than CID. The present instrument geometry and experimental protocol demonstrate much higher efficiencies than previous SID studies by FTMS and much higher mass resolution than previous SID studies using other types of mass analyzers.