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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.
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We report the proteomic datasets on the mouse macrophage cell line PMJ2R infected with tick-borne encephalitis virus (TBEV) for two and six days. Data were acquired using shotgun ultra-high resolution mass spectrometry. Peptide identifications were done using the Mascot version 2.4 (Matrix Science), and quantification was performed by a label-free approach. Protein profiles of early (two days) and late (six days) stages of infection were compared between each other and the respective control samples. Protein profiles of infected and control samples differed in the number of identified proteins and their relative abundances. Proteins detected in the TBEV-infected cells were involved in various processes related to the infection, including defense response against the virus, regulation of viral process, negative regulation of viral genome replication, RNA binding, or innate immune response. Also, proteins specific for the early and late stages of infection were identified.
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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).
Assuntos
Proteínas Sanguíneas/análise , Carcinoma de Células Renais/sangue , Neoplasias Renais/sangue , Proteoma/análise , Biomarcadores Tumorais/sangue , Cromatografia Líquida , Feminino , Humanos , Masculino , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
In the present study, the possibility of hepatitis C virus core antigen (HCVcoreAg) detection in buffer solution, using atomic force microscope chip (AFM-chip) with immobilized aptamers, has been demonstrated. The target protein was detected in 1mL of solution at concentrations from 10-10Ð to 10-13Ð. The registration of aptamer/antigen complexes on the chip surface was carried out by atomic force microscopy (AFM). The further mass-spectrometric (MS) identification of AFM-registered objects on the chip surface allowed reliable identification of HCVcoreAg target protein in the complexes. Aptamers, which were designed for therapeutic purposes, have been shown to be effective in HCVcoreAg detection as probe molecules.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Microscopia de Força Atômica/métodos , Proteínas do Core Viral/análiseRESUMO
A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.
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Citocromos b5/química , Espectrometria de Massas/métodos , Microscopia de Força Atômica/métodos , Proteoma/química , Campos Eletromagnéticos , Humanos , Microscopia de Força Atômica/instrumentaçãoRESUMO
GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020[1]), and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin) and denatured (lysozyme, α-lactalbumin and pepsin) proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.
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A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.
Assuntos
Substituição de Aminoácidos , Antígenos da Hepatite C/química , Antígenos da Hepatite C/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Proteínas do Core Viral/química , Proteínas do Core Viral/isolamento & purificação , Cromatografia de Afinidade , Humanos , Ligação Proteica , Soro/químicaRESUMO
A method for detection of cancer-associated protein D-NFATc1 in serum using nanowire (NW) biosensor based on field-effect nanotransistor is developed. Field-effect nanotransistor was fabricated on the basis of «silicon-on-insulator¼ structures. For the biospecific detection of target protein, the NW surface was modified with aptamers against the target protein. Using the 3 um-NW enabled to obtain stable source-drain characteristics and to register D-NFATc1 in serum at concentration of 2.5 x 1014 M in the mode of drain-source current vs. gate voltage characteristics measurements. Data collection in the mode of drain-source current vs. gate voltage characteristics measurements was carried out with the use of high-speed data collection system running TURBO NBS software.
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Técnicas Biossensoriais , Fatores de Transcrição NFATC/sangue , Nanofios , Proteínas de Neoplasias/sangue , Software , Transistores Eletrônicos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
The nanowire (NW) detection is one of fast-acting and high-sensitive methods allowing to reveal potentially relevant protein molecules. A NW biosensor based on the silicon-on-insulator (SOI)-structures was used for biospecific label-free detection of NFAT 1 (D-NFAT 1) oncomarker in real time. For this purpose, SOI-nanowires (NWs) were modified with aptamers against NFAT 1 used as molecular probes. It was shown that using this biosensor it is possible to reach the sensitivity of ~10(-15) M. This sensitivity was comparable with that of the NW biosensor with immobilized antibodies used as macromolecular probes. The results demonstrate promising approaches used to form the sensor elements for high-sensitive disease diagnostics.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Fatores de Transcrição NFATC/análise , Nanofios/química , Proteínas de Neoplasias/análise , Anticorpos Monoclonais/química , Aptâmeros de Nucleotídeos/síntese química , Técnicas Biossensoriais/economia , Técnicas Eletroquímicas , Humanos , Limite de Detecção , Dióxido de Silício/química , Soluções , Succinimidas/químicaRESUMO
A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.
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Aptâmeros de Nucleotídeos/química , Proteína gp120 do Envelope de HIV/análise , Ácidos Nucleicos Imobilizados/química , Microscopia de Força Atômica/métodos , Anticorpos Imobilizados/química , Aptâmeros de Nucleotídeos/análise , Proteína gp120 do Envelope de HIV/imunologia , Microscopia de Força Atômica/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Achievement of the concentration detection limit for proteins at the level of the reverse Avogadro number determines the modern development of proteomics. In this review, the possibility of approximating the reverse Avogadro number by using nanotechnological methods (AFM-based fishing with mechanical and electrical stimulation, nanowire detectors, and other methods) are discussed. The ability of AFM to detect, count, visualize and characterize physico-chemical properties of proteins at concentrations up to 10(-17)-10(-18) M is demonstrated. The combination of AFM-fishing with mass-spectrometry allows the identification of proteins not only in pure solutions, but also in multi-component biological fluids (serum). The possibilities to improve the biospecific fishing efficiency by use of SOMAmers in both AFM and nanowire systems are discussed. The paper also provides criteria for evaluation of the sensitivity of fishing-based detection systems. The fishing efficiency depending on the detection system parameters is estimated. The practical implementation of protein fishing depending on the ratio of the sample solution volume and the surface of the detection system is discussed. The advantages and disadvantages of today's promising nanotechnological protein detection methods implemented on the basis of these schemes.