• Domestic science and medicine in the 19th - early 20th centuries Chemistry of the 19th century in medicine
• What are peptides? Types and types of peptides. All about peptides and anti-aging cosmetics with peptides Additional peptides are not formed
• Toxic effect on humans of hazardous chemicals
• Radioautography Method of autoradiography in cytology
• Identification of bacteria by antigenic structure
• Organic matter in wastewater What are organic compounds in water
• Hydrogen index (pH factor)
• What is the pH of water and why is it important to know it?
• Redox potential Redox systems
• Reversible redox system Reversible redox systems

The most characteristic physicochemical properties of proteins are: high viscosity of solutions, slight diffusion, the ability to swell over a wide range, optical activity, mobility in an electric field, low osmotic pressure and high oncotic pressure, the ability to absorb UV rays at 280 nm (this the latter property, due to the presence of aromatic amino acids in proteins, is used to quantify proteins).

Proteins, like amino acids, are amphoteric due to the presence of free NH2 and COOH groups and are characterized, respectively, by all the properties of acids and bases.

Proteins have pronounced hydrophilic properties. Their solutions have a very low osmotic pressure, high viscosity and little diffusivity. Proteins are capable of swelling to a very large extent.

A number of characteristic properties are associated with the colloidal state of proteins, in particular, the phenomenon of light scattering, which underlies the quantitative determination of proteins by nephelometry. This effect is also used in modern methods of microscopy of biological objects. Protein molecules are not able to pass through semi-permeable artificial membranes (cellophane, parchment, collodion), as well as biomembranes of plant and animal tissues, although with organic lesions, such as kidneys, the capsule of the renal glomerulus (Shumlyansky-Bowman) becomes permeable to blood serum albumins, and they appear in the urine.

Protein denaturation Under the influence of various physical and chemical factors, proteins undergo coagulation and precipitate, losing their native properties. Thus, denaturation should be understood as a violation of the general plan - the unique structure of a native protein molecule, leading to the loss of its characteristic properties (solubility, electrophoretic mobility, biological activity, etc.). Most proteins denature when heated with a solution above 50-60 ° C. External manifestations of denaturation are reduced to a loss of solubility, especially at the isoelectric point, an increase in the viscosity of protein solutions, an increase in the amount of free functional SH-rpypp and a change in the nature of X-ray scattering. The most characteristic sign of denaturation is a sharp decrease or complete loss by the protein of its biological activity (catalytic antigenic or hormonal) protein molecules and random and disordered structures are formed.

The book is the first textbook in Russian on the basics of protein and peptide mass spectrometry. The purpose of this publication is to interest young researchers in an informative, beautiful and demanded discipline all over the world, to provide an opportunity to more effectively apply mass spectrometry to solve fundamental and applied scientific problems. The book is written in the format of lectures for beginners, well illustrated and accompanied by a representative list of cited literature.

The publication is intended for students and graduate students of chemical, physicochemical, biological and medical specialties; will be useful to researchers who are already working in the field of protein and peptide research or are interested in this scientific area.

Foreword

Dedicated
professors of the Faculty of Chemistry
Moscow State University M.V. Lomonosov
Alexander Leonidovich Kurts
Kim Petrovich Butin

The most important achievements of mass spectrometry over the past 20 years are associated with the study of natural compounds, including biopolymers. With the advent of electrospray ionization and matrix-assisted laser desorption/ionization techniques, sugars, nucleic acids, proteins, lipids, and other bioorganic macromolecules have become available for mass spectrometry. Undoubtedly, the greatest success has been achieved in the study of proteins. Due to its sensitivity, information content, rapidity, and the ability to work with mixtures, mass spectrometry today is the main method for analyzing these objects that are difficult to study.

Perhaps it can be recognized that modern mass spectrometry has won the competition with the classical method of determining the primary amino acid sequence in peptides according to Edman, since mass spectrometric sequencing turned out to be much faster, more sensitive, more informative, and even cheaper. Fast and reliable determination of the primary structure of proteins, i.e. sequences of amino acid units, in itself already an excellent result. However, mass spectrometry is capable of studying structures of more complex orders (from 2 to 4), including non-covalent interactions of proteins with the appearance of superprotein formations, determining the type and place of post-translational modifications, working with glycoproteins, lipoproteins, phosphoproteins, etc. Mass spectrometry has become indispensable in medicine, as it is able to quickly and reliably diagnose cardiovascular, genetic and oncological diseases. It was the successes of mass spectrometry that led to the formation at the end of the last century of a new scientific direction - proteomics. The method also plays an important role in metabolomics.

Unfortunately, in the Russian-language literature there have not yet been textbooks or monographs on this most important and multifaceted topic in its application. Russia drastically lags behind developed countries both in the study of this discipline and in the use of its achievements. Mass spectrometers working in Russia have to rely on English editions of books, original articles and reviews. In 2012, the book "Principles of mass spectrometry as applied to biomolecules" was published in Russian, edited by J. Laskin and H. Lifshitz (translated from English, Publishing House "Technosfera"), which is a collection of articles by leading experts in the field mass spectrometry in application to biology. The book is intended for advanced readers. She provides

a good, in many respects, remote opportunity to get acquainted with modern achievements in the field of mass spectrometry of biomolecules, since most of the methods described in it are not yet used in our country.

The book "Fundamentals of Mass Spectrometry of Proteins and Peptides" offered to readers is the first textbook in Russian that outlines the basics of mass spectrometry of proteins and peptides. The book is written in the format of lectures for beginners, illustrated with a large number of drawings, spectra, diagrams, and is accompanied by a representative list of cited literature. It is designed for students and graduate students of chemical, physicochemical, biological and medical specialties; will be useful to researchers who are already working in the field of protein and peptide research or who are interested in this scientific area.

The purpose of publishing such a book is to interest young researchers in an informative, very beautiful and demanded discipline all over the world, to provide an opportunity to more effectively apply mass spectrometry to solve their own fundamental and applied scientific problems.

The focus of the textbook is on the methods of ionization of proteins and peptides, the processes of fragmentation of these compounds in the gas phase. The issues of tandem mass spectrometry and the existing methods for initiating fragmentation are considered in sufficient detail. This section is very important in modern mass spectrometry. It is useful for researchers working with any chemical compounds and biopolymers. Several chapters are devoted to the identification of proteins and peptides. This includes options for automated identification, manual interpretation of spectra, a description of certain difficulties of mass spectrometric sequencing and options for overcoming them. The advantages and disadvantages of two main approaches to establishing the chemical structure of proteins are considered: "top-down" and "bottom-up" mass spectrometry. A separate chapter is devoted to questions of quantitative analysis.

The book is dedicated to Alexander Leonidovich Kurtz and Kim Petrovich Butin, two friends, wonderful professors of the Faculty of Chemistry at Moscow State University named after M.V. Lomonosov, who are very close to us in human terms, who directly participated in our chemical and humanitarian education. We have always highly appreciated the chemical erudition and amazing personal qualities of these scientists. It was communication with these people that inspired us at the very beginning of the 21st century to start research in the field of peptide mass spectrometry.

A.T. Lebedev
K.A. Artemenko
T.Yu. Samghin

2 LITERATURE REVIEW.

2.1 Mass spectrometry in proteomics.

2.1.1 General principles.

2.1.2 Proteomic analysis using mass spectrometry.

2.1.3 Identification of proteins by the peptide mass imprint method.

2.1.4 Protein identification by peptide fragmentation fingerprinting.

2.2 Interpretation of the results of mass-spectrometric identification of proteins.

2.2.1 Determining the list of identified proteins.

2.2.2 Identification of highly homologous proteins.

2.2.3 Databases of protein amino acid sequences.

2.3 Mass spectrometric analysis of single gene products.

2.3.1 Proteotyping and population proteomics.

2.3.2 Identification of protein microheterogeneity using the top-down method.

2.3.3 Identification of genetically determined protein polymorphism by the "bottom-up" method.

2.3.4 Databases of protein and gene polymorphisms.

2.3.5 Mass spectrometric data repositories.

3 MATERIALS AND METHODS.

3.1 Materials.

3.1.1 Mass spectrometry data for human liver microsomal fraction proteins.

3.1.2 Control set of mass spectra "Aurum Dataset".

3.1.3 Mass spectrometry data from the PRIDE proteomic repository.

3.1.4 Databases of amino acid sequences of human proteins.

3.1.5 Data on possible human protein polymorphisms.

3.2 Methods.

3.2.1 Web server for protein identification by mass spectra.

3.2.2 Batch processing of mass spectra using the peptide mass imprint method.

3.2.3 Batch processing of tandem mass spectra.

3.2.4 One-dimensional proteomic mapping.

3.2.5 Software implementation of the iterative algorithm for identifying the SAP.

3.2.6 Validation of the SAP identification algorithm.

4 RESULTS AND DISCUSSION.

4.1 Increasing coverage of amino acid sequences by identified peptides.

4.1.1 Identification of proteins in gel sections.

4.1.2 One-dimensional proteomic maps and their properties.

4.1.3 Detection of highly homologous proteins of the cytochrome P450 superfamily by increasing the degree of coverage of amino acid sequences by identified peptides.

4.2 Identification of PDA in proteins of the cytochrome P450 superfamily.

4.3 SDA identification algorithm.

4.3.1 Iterative scheme for processing tandem mass spectra.

4.3.2 Sensitivity and specificity of the SDA identification algorithm.

4.4 Application of an iterative algorithm for the detection of PDA in the mass spectrometric data of the PRIDE proteomic repository.

4.4.1 Initial data used to identify PDA.

4.4.2 Identification of peptides and proteins using mass spectrometric data downloaded from the PRIDE repository.

4.4.3 Identification of single amino acid polymorphisms.

4.5 Analysis of identified PDAs.

4.5.1 Analysis of PDA-containing peptides.

4.5.2 Association of identified PDAs with human diseases.

Recommended list of dissertations

• Post-translational regulation of cytochrome P450 subfamily 2B 2013, Doctor of Biological Sciences Zgoda, Victor Gavrilovich

• Mass spectrometric determination of the activity and content of cytochromes P450 2013, candidate of biological sciences Moskaleva, Natalya Evgenievna

• Structural and functional mapping of proteins of cytochrome P450-containing monooxygenase systems 2002, Doctor of Biological Sciences Kolesanova, Ekaterina Fedorovna

• Method for Recognition of Amino Acid Sequences in Peptide Mass Spectra for Problems of Proteomics 2007, candidate of technical sciences Lyutvinsky, Yaroslav Igorevich

• Universal Scale of Chromatographic Retention Times of Biomacromolecules in Problems of "Quick-Fire" Proteomics 2011, Candidate of Physical and Mathematical Sciences Prydatchenko, Marina Leonidovna

Introduction to the thesis (part of the abstract) on the topic "Analysis of mass spectra of peptide fragments for the identification of genetically determined protein polymorphism"

Similar theses in the specialty "Mathematical biology, bioinformatics", 03.01.09 HAC code

• Transcriptomic-proteomic approach for the analysis of proteoforms of the HepG2 cell line 2018, Candidate of Biological Sciences Kiseleva, Olga Igorevna

• Transcriptome and proteome of chromosome 18: extrapolation of analysis results to human genomes and model objects 2017, Doctor of Biological Sciences Ponomarenko, Elena Alexandrovna

• Evaluation of the plasticity of the blood plasma proteome of a healthy person under extreme conditions of vital activity 2011, candidate of biological sciences Trifonova, Oksana Petrovna

• Search and identification of potential biomarkers of ovarian cancer in human serum 2015, candidate of biological sciences Arapidi, Georgy Pavlovich

• Photodynamic Analysis of Thylakoid Membrane Protein Complexes Using High-Resolution Mass Spectrometry 2011, candidate of chemical sciences Galetsky, Dmitry Nikolaevich

Dissertation conclusion on the topic "Mathematical biology, bioinformatics", Chernobrovkin, Alexey Leonidovich

List of references for dissertation research Candidate of Biological Sciences Chernobrovkin, Alexey Leonidovich, 2012

Please note that the scientific texts presented above are posted for review and obtained through original dissertation text recognition (OCR). In this connection, they may contain errors related to the imperfection of recognition algorithms. There are no such errors in the PDF files of dissertations and abstracts that we deliver.

480 rub. | 150 UAH | $7.5 ", MOUSEOFF, FGCOLOR, "#FFFFCC",BGCOLOR, "#393939");" onMouseOut="return nd();"> Thesis - 480 rubles, shipping 10 minutes 24 hours a day, seven days a week and holidays

Kaisheva Anna Leonidovna Mass-spectrometric identification of proteins and protein complexes on the chips of an atomic force microscope: dissertation... candidate of biological sciences: 03.01.04 / Kaisheva Anna Leonidovna; [Place of protection: Nauch.-issled. in-t biomed. chemistry them. V.N. Orekhovich RAMS].- Moscow, 2010.- 104 p.: ill. RSL OD, 61 10-3/1308

Introduction

Chapter 1 Literature Review 10

1.1. Analysis of scientific and technical groundwork in the field of highly sensitive proteomic technologies

1.2 Characterization of hepatitis C virus 20

1.2.1 Methods for diagnosing hepatitis C 22

1.2.2 Serological protein markers of hepatitis C 25

Chapter 2. Materials and methods 28

2.1 ACM chips 28

2.2 Protein preparations and reagents 29

2.3 AFM analysis 30

2.4 Sample preparation for mass spectrometric analysis 31

2.5 Mass spectrometric analysis 33

2.5.1 MALDI-MS analysis of proteins on the surface of the AFM chip 33

2.5.2 ESI-MS analysis of proteins on the surface of the AFM chip 34

Chapter 3 Results and Discussion 35

3.1 MS - identification of proteins caught by "chemical fishing" on the surface of the AFM chip from the analyte solution

3.2 MS identification of proteins biospecifically captured on the surface of an AFM chip from an analyte solution

3.3 MS identification of proteins on the surface of an AFM chip biospecifically isolated from blood serum samples

Conclusion 83

Literature

Introduction to work

The relevance of the work.

One of the priority areas in modern biochemistry is the creation of effective analytical methods for proteomic analysis, the main task of which is to detect and inventory proteins in the body, study their structure and functions, and identify protein interactions. The solution of this problem will allow creating new systems for diagnosing diseases and their treatment. Standard methods of modern proteomic analysis are based on the separation of multicomponent protein mixtures using chromatography, electrophoresis in combination with mass spectrometric methods (MS) for protein identification. Despite the undoubted advantage of standard MS analysis in terms of speed and reliability of identification of protein molecules, it has significant application limitations due to low

concentration sensitivity of the analysis at the level of 10" "10" M and a high dynamic range of protein content in biological material. At the same time, the vast majority of functional proteins, including biomarkers of such socially significant diseases as viral hepatitis B and C, tumor markers, etc. ., are present in the blood plasma at concentrations of 10" Mi less.

One of the ways to overcome this methodological limitation of the concentration sensitivity of analysis is to use biomolecular detectors, which allow the detection of single molecules and their complexes and theoretically have no concentration sensitivity limitations. Biomolecular detectors include detectors based on nanotechnological devices such as atomic force microscopes (AFM), nanowire detectors, nanopores, and a number of other detectors. The unique sensitivity of AFM detectors makes it possible to visualize individual protein molecules and count their number. When using AFM as a biomolecular detector, it is necessary to use special chips that allow the concentration of biological analyte macromolecules from a large volume of incubation solution on a limited chip surface. The studied protein objects can be concentrated on the chip surface both due to physical or chemical adsorption, and due to biospecific interactions (AFM-biospecific fishing).

However, in practice, the limitation of the use of AFM-based nanodetectors is that, despite the possibility of visualizing individual protein molecules on the chip surface, such detectors are not able to identify them, which is especially important in the study of complex protein mixtures, including biological material. Therefore, the development of an analysis method that complements the capabilities of the AFM method seems to be an urgent task. To date, the only proteomic method that allows unambiguous and reliable identification of protein molecules is MS analysis. In the dissertation work, an approach was developed that combines the high sensitivity of the AFM method and reliable MS identification for the detection of proteins and their complexes from an analyte solution.

Purpose and objectives of the study.

The purpose of this work was the mass spectrometric identification of proteins and protein complexes detected in the biomaterial using atomic force microscopy.

To achieve this goal, the following tasks were solved:

A scheme for the MS identification of proteins caught on the surface of an AFM chip using chemical or biospecific fishing has been developed;

Conditions for enzymatic hydrolysis of proteins on the surface of an AFM chip for subsequent MS identification have been developed;

MS-identification of model proteins on the surface of the AFM chip was carried out;

MS-identification of proteins on the surface of the AFM chip biospecifically isolated from a multicomponent mixture (serum) was carried out.

Scientific novelty of the work .

In the dissertation, a scheme was developed that allows MS identification of proteins and protein complexes caught from a solution or a multicomponent mixture on the surface of an AFM chip. For this, the optimal conditions for sample preparation were selected, including the mode of hydrolysis (temperature, humidity, composition of the trypsinolytic mixture, trypsinolysis time) of protein molecules covalently and non-covalently immobilized on the surface of the AFM chip. The peculiarity of this work was that, compared with the standard proteomic protocols of enzymatic hydrolysis, the preparation of samples for MS analysis was carried out not in solution, but on a limited area.

chip surface. The developed scheme made it possible to effectively carry out MS analysis and identify both individual proteins and protein complexes on the surface of the AFM chip. MS analysis of proteotypic peptides of the studied proteins was carried out using two types of ionization (MALDI and EST) and two types of detectors (TOF and ion trap). The developed scheme for coupling AFM-biospecific fishing and MS was also successfully tested for the detection of protein markers of viral hepatitis C (HCV) (HCVcoreAg and E2) in blood serum samples.

The practical significance of the work .

The results of this work make it possible to create highly sensitive proteomic methods without the use of labels and additional sample preparation procedures for the detection of proteins found in low concentrations in biological material, including in blood serum. An approach based on atomic force microscopy and mass spectrometry has been proposed, which will make it possible to detect and identify protein markers of hepatitis C virus in human blood serum.

The approach can be used in developments aimed at creating new diagnostic chips, searching for biomarkers of a wide range of socially significant diseases.

Approbation of work.

The main results of the study were presented at the "1st, 2nd and 3rd International Nanotechnology Forum" (Moscow, 2008-2010); "IV Congress of the Russian Society of Biochemists and Molecular Biologists", Novosibirsk, 2008; at the International Congress "Human Proteome", Amsterdam, 2008; at the International Congress "Human Proteome", Sydney, 2010.

Publications.

The structure and scope of the dissertation.

The dissertation consists of an introduction, literature review, description of research materials and methods, research results and their discussion, conclusion, conclusions and list of references. The work is presented on 104 pages, illustrated with 33 figures and 4 tables, the list of references consists of 159 titles.

Analysis of scientific and technical groundwork in the field of highly sensitive proteomic technologies

One of the priority areas in modern science is the discovery and elucidation of the role of various types of proteins in the body, as well as understanding the molecular mechanisms that lead to the development of diseases.

Despite continuous improvement in proteomic methods, the number of newly discovered disease biomarkers has remained virtually 1/2 the same over the past decade. This is due to the fact that the concentration limit of detection of traditional proteomic methods does not exceed 10"9 M. At the same time, it is important for proteomics to develop new analytical approaches for identifying proteins in a lower concentration range, in particular, low-copy protein molecules (with a concentration of 10"13 M and less), including biomarkers in biological material. Since it can be assumed that it is in these concentration ranges that the protein markers of most diseases are found.

One of the actively developing areas, which makes it possible to somewhat increase the concentration sensitivity of analysis, is the creation of analytical complexes based on nanochromatographic and nanoelectrophoretic systems compatible with mass spectrometers.

The nanochromatographic system in combination with mass spectrometry and electrospray type ionization made it possible to increase the sensitivity of protein detection by two orders of magnitude compared to high-resolution chromatography (HPLC). The concentration sensitivity limit of such conjugated systems is limited by the sensitivity of the electrophoresis/chromatography stage, and does not exceed 10-12 M for individual proteins (for example, for cytochrome C and bradykinin).

Currently, chromatographic methods have developed into separate independent areas - SELDI MS analysis (surface enhanced laser desorption and ionization/time of flight mass spectrometry), protein fishing methods using magnetic microparticles. In these technologies, the hydrophobic or charged surfaces of SELDI-chips are. or magnetic microparticles, combinations with mass spectrometric analysis, are successfully used: for? detection- and identification as separate types; proteins, and for protein/peptide profiling of blood serum [c, 8; \b, 15]. SEbDIi МЄ is: a powerful approach that allows you to study a biomaterial through the adsorption of biomolecules (proteins, peptides) on a chemically activated? surface (cation / anion exchange chips) followed by mass spectrometric analysis of adsorbed: molecules:. SEEDPMЄ approach is applied; for protein profiling of biomaterial; and recently: it has been used as a “diagnosis using proteomic barcodes” [17].. The essence of such a “barcode diagnosis” is to identify? features of the protein profile of the biological sample; associated with a particular disease: Yes, known; what d at. cancerous diseases, the “proteomic barcode” of the biomaterial is significantly different from that in healthy1 groups” of individuals: Therefore, control over changes in protein; composition of the biomaterial can become the basis for early diagnosis of diseases. On; today, using the SELDI approach? МЄ markers were identified. gastric, ovarian, prostate, and breast cancers: A limitation of this method5 is the inability to identify proteins with high resolution and accuracy, which is especially important in; analysis of multicomponent mixtures such as biological material.

In addition to the problem of low concentration sensitivity of existing analytical systems, a stumbling block for the proteomic analysis of biological material has become a wide dynamic range of protein concentrations, especially in blood serum, which varies from 1(G M up to individual protein molecules. High-copy (major) proteins interfere in such systems detection and identification of low-copy (minor) proteins.

The problem of a wide concentration range of proteins in a biomaterial can be solved by applying methods for depleting blood serum from major protein fractions, methods for separating multicomponent mixtures and nanotechnological methods based on biospecific and chemical fishing of protein molecules of an analyte from complex mixtures on the surface of chips to various biosensors or on an activated surface. magnetic microspheres.

Traditionally, one-dimensional, more often two-dimensional gel electrophoresis is used to separate multicomponent protein mixtures. The principle of protein separation by two-dimensional gel electrophoresis is based on the difference between proteins according to the values ​​of their isoelectric points Hf of molecular weights. In proteomics, these approaches are used for protein mapping of biomaterial (tissue, blood plasma, etc.). The combination of 1D and/or 2D electrophoresis with mass spectrometry allows the identification of separated and visualized proteins. However, the procedure of two-dimensional gel electrophoresis is still not automated, it is rather complicated and time-consuming to perform, it requires a high qualification of the operator, and the analysis results are often poorly reproducible.

More convenient in comparison with two-dimensional electrophoresis, the procedure for separating proteins is high-performance chromatography (HPLC); which is an automated procedure that allows you to remove high-copy proteins from a complex mixture in order to subsequently identify low-copy proteins.

In order to directly identify proteins in complex mixtures, a chromatographic column can be connected to a mass spectrometer. However, intact proteins are practically not amenable to high-quality separation using HPLC, since they denature during analysis (due to low pH values ​​of the medium and high concentration of organic solvents), and also due to the low accuracy of mass spectrometric analysis, therefore, direct identification of most intact proteins , especially with a molecular weight exceeding 10 kDa, is often impossible. Analytical measurement accuracy can be improved by hydrolytic cleavage of proteins to peptide fragments, molecular weight from 700 to 4000 Da using proteases; such as trypsin (bottom-up technology). To achieve a qualitative separation of proteins in a mixture, a combination of several chromatographic procedures is used, the so-called multidimensional chromatography.

Methods for diagnosing hepatitis

Currently, for the protein diagnosis of hepatitis C, test systems for the detection of anti-HCVcore are used. The first ELISA tests detecting the presence of anti-HCVcore antibodies became available in the early 1990s, but they had low sensitivity and selectivity. Later, in the late 1990s, a new generation of anti-HCVcore ELISA tests appeared, which had a fairly high sensitivity of about 95-99% and could detect HCV several months after infection.

For example, in 1996, test systems developed by Vector-Best (Novosibirsk) and Diagnostic Systems (Nizhny Novgorod) appeared on the Russian market to detect antibodies - anti-HCV of the IgM class. The role of IgM antibodies in serodiagnosis has not been sufficiently studied, however, some studies have shown the importance of this marker for the detection of chronic hepatitis C. It has also been established that the correlation between the detection of viral RNA and anti-HCV IgM in patients is 80-95%. To determine the phase of development of viral hepatitis C Afanasyev A.Yu. et al. used a coefficient reflecting the ratio of anti-HCV IgG to anti-HCV IgM in the blood of patients. To date, many enzyme-linked immunosorbent assay (ELISA) systems have been developed that detect circulating antibodies to many epitopes of the hepatitis E virus.

Modern laboratory diagnostics of: viral hepatitis E in most medical institutions in Moscow, is carried out; in accordance with the existing orders of the Ministry of Health of the Russian Federation and the Department of Health: Moscow and is to determine immunoglobulins? class G to hepatitis E virus (anti-HGV IgG) in the blood serum of patients. Identification of this marker makes it possible to judge the presence of a current or past infection.

Disadvantages of the methods; EEISA-based detections, in addition to low sensitivity (more than GO "12 M)j, are also due to false-detection; viral hepatitis E in patients - due to post-infectious immunity,., cross-reactivity of antibodies, as well as insufficient sensitivity in the acute period) phase BFG BI LINKS: THIS is an active search for sensitive, specific, fast and easy-to-perform methods for detecting1 markers of “hepatitis E .

Another group of methods for detecting viral hepatitis in serum: blood is-B_registration; RNA BEI using PCR; Definition of RNA. BFG methods; GAD cannot be used as a primary test for - confirmation or exclusion; diagnosis; but; may be; Useful for confirming the diagnosis: Diagnosis of 1 BFG is by analysis of the 5' non-coding RNA region. However, the results of the analysis vary among different BFG genotypes.

Biological microchips have appeared on the Russian market, which make it possible to conduct - BFG genotyping and - determine an effective antiviral scheme; therapy. This biochip is an oligonucleotide chip for BFG genotyping based on the analysis of the NS5B region. The obtained results indicate the ability of the biochip to identify all 6 HCV genotypes and 36 subtypes, including the most virulent and drug-resistant forms.

On the one hand, PCR analysis methods are supersensitive and allow detecting and amplifying the signal from just one RNA molecule in a sample, but on the other hand, these methods are characterized by false positive results due to random contamination of samples, false negative results due to the high mutability of the virus and a relatively high analysis cost. Even in the same person, HCV RNA levels can periodically change by more than a millifold, leading to false negative results if low? virus replication or if the virus persists in tissues without entering the blood. The results of the quantitative determination of RIG, HCV in different laboratories do not agree well enough.

Of particular value for the early detection of viral hepatitis C Bt biomaterial are HCV protein antigens due to the fact that they appear1 in the blood serum several weeks earlier, even before the development of a full-fledged immune response of the body.

The HCVcoreAg surface antigen of the hepatitis C virus is the main marker of infection with the hepatitis C virus. It is detected 16 weeks before the appearance of antibodies in the blood due to the immune response of the body and before the development of clinical signs, while it is recorded both in the acute and chronic phases diseases. There is only one foreign commercial product (“Ortho Clinical Diagnostics”) for ELISA diagnostics of hepatitis C during the acute phase, based on the detection of HCVcoreAg.

The structural protein HCVcoreAg, consisting of 121 amino acid residues, is located at the N-terminus of the polypeptide and is formed under the influence of cellular proteases. The first proteolytic hydrolysis occurs between residues 191 and 192 (site C1) and leads to the formation of E1 glycoprotein. The second cleavage site (C2) is between amino acids 174 and 191. The corresponding cleavage products are named p21 and p23. Analysis of expression in a number of mammalian cells showed that p21 is the main product, while p23 is found in minor amounts. It is possible that cleavage at the C1 and C2 sites is an interrelated process, since p21 is formed under conditions when hydrolysis at G2 is not observed [D45]. HCVcoreAg is the main RNA-binding protein that appears to form the viral nucleocapsid. The biochemical properties of this protein are still poorly characterized. AFM studies of hepatitis C virus particles made it possible to obtain an image of the HCV capsid.

ASM chips

In the experimental part of the work, two types of AFM chips were used. The first type was used for MS identification of model proteins on the surface of AFM chips. These chips were substrates with functionally active chemical groups (hereinafter referred to as AFM chips with a chemically activated surface), on which the studied molecules were caught and irreversibly immobilized due to covalent bonds, the so-called “chemical fishing” procedure. The second type of AFM chips was used for MS identification on their surface of proteins biospecifically isolated from the analyte solution. Biological probes were previously immobilized on the surface of these chips - in the working areas. Monoclonal antibodies against marker proteins of viral hepatitis B and C (BFB and BFC) or aptamerr against the gpl20 protein and thrombin were used as biological probes. For biospecific-fishing procedures, chips with covalently immobilized probe molecules were incubated. in % analyte solution containing only detectable protein, or blood serum samples

To perform the task of MS identification of model proteins covalently immobilized on the surface of AFM chips of the first type, the following were used in the work: avidin (Agilent, USA), HSA (Agilent, USA), P450 VMZ (kindly provided by Professor A.V. Munro, University of Manchester, UK), thrombin (Sigma, USA), a-FP and anti-a-FP (USBio, USA); To perform the task of MS identification of proteins on the surface of AFM chips of the second type, biospecifically isolated from an analyte solution, monoclonal antibodies (MABs) were used as probe molecules: anti-HCVcore (Virogen, USA), anti-HBVcore (Research Institute of Molecular Diagnostics, Moscow), anti-HBsAg (Aldevron, USA), as target molecules: HBVcoreAg, HCVcoreAg (Virogen, USA) and HBsAg (Aldevron, USA), gpl20 (Sigma, USA), troponin (USBio, USA).

In addition, the following substances were used in the work: acetonitrile, isopropanol, formic acid, distilled water (Merck, USA), trifluoroacetic acid (TFA), ammonium bicarbonate (Sigma, USA), a-cyano-4-hydroxycinnamic acid (HCCA), dihydroxybenzoic acid-(DHB) (Bruker Daltonics, Germany), trypsin (Promega, USA).

Blood serum samples for AFM study were provided by the Department of Infectious Diseases in Children of the Russian State Medical University, Central Research Institute of Epidemiology of Rospotrebnadzor, MNIIEM "n. Gabrichevsky: The presence of hepatitis C virus (HCV) particles in blood serum samples was confirmed using the polymerase chain reaction (PCR) method using the test system "Amplisens HCV Monitor" (Central Research Institute of Epidemiology of the Ministry of Health of the Russian Federation, Moscow).

AFM analysis was carried out at the Laboratory of Nanobiotechnology, IBMC RAMS. The calculation of proteins and antigen/antibody complexes on the surface of the AFM chip was carried out on the basis of the correlation of the heights of the corresponding images of proteins and their complexes, measured using AFM, according to the method described in . Was used by ACM NTEGRA (NT-MDT, Russia). AFM measurements were carried out in the semi-contact mode. NSG10 series cantilevers from NT-MDT were used as probes. The typical radius of curvature of the needles was 10 nm, and the resonant frequency ranged from 190 to 325 kHz. The chip scanning area was 400 µm2. Each measurement was carried out at least 3 times.

Immobilization of proteins and aptamers on the surface of the AFM chip was carried out according to the following procedure.

To a protein solution (0.1 µM) with a volume of 2 µl was added 8 µl of a solution of a mixture of NHS/EDC (v/v=l/l) and thoroughly mixed. The resulting mixture was applied to the surface of the silanized chip and incubated for 2 minutes at room temperature. The chip was then washed twice in a thermoshaker with 1 ml of deionized water at 800 rpm and 37°C. The quality of protein immobilization on the surface of the AFM chip was monitored by atomic force microscopy.

Immobilization of aptamers on the chemically activated surface of the AEM chip was carried out as follows. To a stock solution of DSP at a concentration of 1.2 mM in DMSO/ethanol (v/v=l/l)4 was added a solution of PBS buffer 50 mM (pH 7.4) also in a ratio of 1/1 by volume. The working solution thus obtained was applied to the surface of the AFM chip and incubated for 10 minutes. After that, washing was carried out with a 50% solution of ethanol in water with a volume of 1 ml at 15C for 10 minutes. An aptamer solution with a concentration of 3 JIM was applied to the activated zone of the AFM chip and incubated for 4 minutes with stirring at a speed of 800 rpm. Blocking of unreacted amino groups of the DSP cross-linker was carried out in the presence of 5 mM Tris-HCl solution for 10 minutes at 37°C. The final washing step was carried out twice with a 1 ml aqueous solution for 10 minutes at 25°C.

A trypsinolytic mixture containing a buffer solution of 150 mM NH4HCO3, acetonitrile, 0.5 M guanidine hydrochloride, and glycerol (pH 7.4) was applied to the surface of the AFM chip with immobilized probe molecules. Then, 0.5 μl of modified porcine trypsin solution at a concentration of 0.1 μM was added to the buffer solution. The AFM chip was incubated in a humid environment for 2 hours at a constant temperature of 45C, 0.5 µl of trypsin solution (0.1 µM) was again added to its surface, and the incubation continued for another 12 hours. The trypsinolytic mixture was washed off the surface of the AFM chip with a 10 µl elution solution containing 70% acetonitrile in 0.7% trifluoroacetic acid (TFA). The hydrolyzate thus obtained from the surface of the AFM chip was dried in a vacuum evaporator at 45°C and 4200 rpm. Next, the peptide mixture was dissolved in 10 µl of a 5% formic acid solution or in 10 µl of a 0.7% TFA solution for subsequent MS analysis.

During MS analysis with the MALDI type of ionization, the samples were prepared as follows. Samples dissolved in 0.7% TFA solution with a volume of 10 µl were concentrated and desalted using ZipTip C18 microtips (Millipore, USA) according to the manufacturer's protocol and mixed with a saturated solution of a matrix containing HCCA or DHB in 50% acetonitrile solution with 0 .7% TFA. The resulting mixture was applied to an MTP size MALDI target.

-identification of proteins caught by "chemical fishing" on the surface of an AFM chip from an analyte solution

At this stage of the experimental work, MS spectra were obtained for model proteins chemically immobilized on the surface of AFM chips from an analyte solution. The range of concentrations of the studied proteins in the analyte solution for avidin, HSA, anti-aFP was 10"-10"9 M, troponin, aFP and P450 VMZ - 10"6-10"8 M.

MS analysis was carried out for 6 types of proteins, different in their origin, molecular weight, the number of trypsinolysis sites and their spatial accessibility, the degree of hydrophobicity of the amino acid sequence (the ratio of hydrophobic amino acids to hydrophilic ones), which were covalently immobilized on the surface of the AFM chip from the analyte solution ( Table 1). In these experiments, AFM chips were used, which contained the working and control zones. The working zone was a chemically activated area of ​​the AFM chip surface, on which the model proteins were “chemically fished”; the control zone was the chemically inactive region of the chip surface. The count of visualized captured molecules was recorded using AFM. The experimental data of AFM analysis obtained for the above model proteins, namely the number of molecules caught on the surface of the working area of ​​the AFM chip, are presented in Table 2. protein in analyte solution.

As can be seen from Table 2, the number of molecules registered in the working area of ​​the AFM chip for all presented proteins was -1040 molecules. The sensitivity limit of MS detectors is about 105 molecules. Thus, for the presented model proteins, successful irreversible immobilization on the surface of the AFM chip was carried out, and the number of AFM-registered protein objects was sufficient for subsequent MS identification. At the same time, the minimum recorded concentration of model proteins in the incubation solution was quite low, 10"-10" M.

Mass spectrometric analysis of the samples was performed using MALDI and ESI types of ionization. AFM chip after incubation in the appropriate solution of avidin with a concentration of 10"9 M. Analysis of these spectra made it possible to reliably identify avidin (Gallus Gallus) by its two proteotypic peptides: SSVNDIGDDWK (m/z=618.6) and VGINIFTR (m/z= 460.4). Both peptides had well-defined peaks of their doubly charged ions (MS-spectra). Using AFM-MS analysis of the chemically activated working zone of the AFM-chip after incubation in a solution of the analyte protein with a concentration of 10"8 M, another small protein was detected - troponin I. The MS and MS/MS spectra corresponding to the peptide doubly charged ion 1449 Da are shown in Figure 3. MS analysis of the experimentally obtained spectra made it possible to reliably identify and identify human troponin (gi 2460249) on the surface of the AFM chip with a probability of more than 95% .

Figure 5 shows tandem fragmentation spectra of a globular protein, human serum albumin (HSA), which performs transport functions in blood plasma. The spectra were obtained from the chemically activated working zone of the AFM chip after incubation in an appropriate albumin solution with a concentration of 10"9 M. The analysis of these spectra made it possible to reliably identify human albumin by its two proteotypic peptides: VPQVSTPTLVEVSR (m/z=756.5) and YLYEIAR (m/z=464.3) Both peptides had well defined peaks of their doubly charged ions (MS spectra).

MS/MS spectra of trypsinized objects from the chemically activated surface of the AFM chip incubated in a solution of human serum albumin (C=10 9 M). VPQVSTPTLVEVSR peptide with m/z=756.5 (A), YLYEIAR peptide with m/z=464.3 (B). Experimental conditions: measurements were carried out on an LC/MSD Trap XCT Ultra mass spectrometer (Agilent).

Thus, MS analysis made it possible to identify proteins detected using AFM. Based on the data obtained, a relationship was revealed between the number of identified proteotypic peptides on the surface of the AFM chip and the content of the desired protein in the analyte solution. Such a dependence, for example, for the P450 VMZ and HSA proteins covalently immobilized on the chemically activated surface of the AFM chip, is shown in Figure 6. As can be seen in Figure 6, the higher the protein concentration in the analyte solution (-KG6 M), the greater the number of peptides can be reliably identified both in the case of MALDI-MS and ESI-MS analysis. Significant differences between the number of identified peptides in the concentration range of 10"6-10"9 M among the analyzed proteins in the analyte solution were not observed.

Dependences of the number of identified peptides of analyte molecules on the protein concentration in the incubation solution. (A) - analysis of a mixture of peptides of model proteins HSA, VMZ on mass spectrometers with MALDI-type ionization Bruker Microflex (Bruker Daltonics, Germany) and Autoflex III (Bruker Daltonics, Germany); (B) - analysis of a mixture of peptides of model proteins HSA, VMZ on a mass spectrometer with ESI-type ionization LC/MSD Trap XCT Ultra (Agilent, USA).

The results obtained made it possible to conclude that AFM-MS (MALDI and ESI) makes it possible to detect and identify protein molecules covalently extracted from the analyte solution on the surface of the AFM chip, which differ in their physicochemical properties.

At the same time, in the control zone of the AFM chip (non-activated) after its incubation in the analyte solution, the AFM method did not detect the presence on the chip surface of objects corresponding in height to protein molecules. MS analysis also did not reveal objects of a protein nature. Thus, it was experimentally proved that AFM adequately registers the desired objects - protein molecules of the analyte.

The next stage of this work was the development of an AFM-MS combination scheme for the identification of proteins recovered from a solution of za. through biospecific interactions.

The scheme of mass spectrometric analysis in the case of biospecific AFM fishing of proteins from a solution is shown in Figure 7. According to the above scheme, probe molecules were first immobilized on the surface of the working area of ​​AFM chips, which were monoclonal1 antibodies against protein markers of viral hepatitis B and C or aptamers against proteins of the HIV-1 glycoprotein gpl20 and thrombin, while the surface of the control zone did not contain immobilized probe molecules. The quality control of the immobilization of probe molecules was carried out by AGM-visualization. Then, such a chip was incubated in an analyte solution containing the protein under study. After the stage of washing off nonspecifically adsorbed molecules on the chip surface, and the stage of sample preparation for subsequent mass spectrometric analysis on the AFM chip surface, MS analysis of the AFM-detected proteins was performed.

The experimental part of this section involved two stages of analysis. At the first stage, it was necessary to carry out the MS identification of protein probe molecules covalently immobilized on the AFM chip, and at the second stage, target proteins caught on the corresponding partner molecules from a solution or from blood serum samples due to biospecific interactions. For this purpose, MS analysis of MCA covalently immobilized on the surface of AFM chips against HCV and HBV marker proteins: anti-HCVcore and anti-HBVcore was performed. For mAbs against anti-HCVcore and anti-HBVcore proteins, tandem fragmentation spectra and peptide map spectra were obtained in this work for the first time.

salting out: precipitation with salts of alkali, alkaline earth metals (sodium chloride, magnesium sulfate), ammonium sulfate; at the same time, the primary structure of the protein is not disturbed;

precipitation: use of dewatering agents: alcohol or acetone at low temperatures (about -20°C).

When using these methods, proteins lose their hydration shell and precipitate in solution.

Denaturation- violation of the spatial structure of proteins (the primary structure of the molecule is preserved). It can be reversible (the protein structure is restored after the removal of the denaturing agent) or irreversible (the spatial structure of the molecule is not restored, for example, when proteins are precipitated with concentrated mineral acids, salts of heavy metals).

Protein separation methods Separation of proteins from low molecular weight impurities

Dialysis

A special polymer membrane is used, which has pores of a certain size. Small molecules (low molecular weight impurities) pass through the pores in the membrane, while large molecules (proteins) are retained. Thus, proteins are washed from impurities.

Separation of proteins by molecular weight

Gel chromatography

The chromatographic column is filled with gel granules (Sephadex), which has pores of a certain size. A mixture of proteins is added to the column. Proteins, the size of which is smaller than the size of the Sephadex pores, are retained in the column, as they “get stuck” in the pores, and the rest freely leave the column (Fig. 2.1). The size of a protein depends on its molecular weight.

Rice. 2.1. Separation of proteins by gel filtration

Ultracentrifugation

This method is based on different rates of sedimentation (precipitation) of protein molecules in solutions with different density gradients (sucrose buffer or cesium chloride) (Fig. 2.2).

Rice. 2.2. Separation of proteins by ultracentrifugation

electrophoresis

This method is based on different rates of migration of proteins and peptides in an electric field depending on the charge.

Gels, cellulose acetate, agar can serve as carriers for electrophoresis. The molecules to be separated move in the gel depending on their size: those that are larger will be held back as they pass through the pores of the gel. Smaller molecules will encounter less resistance and therefore move faster. As a result, after electrophoresis, larger molecules will be closer to the start than smaller ones (Fig. 2.3).

Rice. 2.3. Separation of proteins by gel electrophoresis

Proteins can also be separated by electrophoresis by molecular weight. For this use electrophoresis in PAAG in the presence of sodium dodecyl sulfate (SDS-Na).

Isolation of individual proteins

Affinity chromatography

The method is based on the ability of proteins to bind strongly to various molecules by non-covalent bonds. Used to isolate and purify enzymes, immunoglobulins, receptor proteins.

Molecules of substances (ligands), with which certain proteins specifically bind, are covalently combined with particles of an inert substance. A mixture of proteins is added to the column, and the desired protein is firmly attached to the ligand. The remaining proteins freely exit the column. The retained protein can then be washed from the column with a buffer containing the free ligand. This highly sensitive method allows very small amounts of pure protein to be isolated from a cell extract containing hundreds of other proteins.

Isoelectric focusing

The method is based on different IEP values ​​of proteins. Proteins are separated by electrophoresis on a plate with ampholine (this is a substance that has a pre-formed pH gradient in the range from 3 to 10). During electrophoresis, proteins are separated according to the value of their IEP (in IEP, the charge of the protein will be zero, and it will not move in the electric field).

2D electrophoresis

It is a combination of isoelectric focusing and electrophoresis with SDS-Na. First, electrophoresis is carried out in a horizontal direction on a plate with ampholine. Proteins are separated depending on the charge (CEP). Then the plate is treated with a solution of SDS-Na and electrophoresis is carried out in the vertical direction. Proteins are classified based on molecular weight.

Immunoelectrophoresis (Western blot)

An analytical method used to determine specific proteins in a sample (Figure 2.4).

Isolation of proteins from biological material.

Separation of proteins by molecular weight by electrophoresis in PAAG with SDS-Na.

Transfer of proteins from the gel to the polymer plate in order to facilitate further work.

Treatment of the plate with a non-specific protein solution to fill the remaining pores.

Thus, after this stage, a plate was obtained, the pores of which contain separated proteins, and the space between them is filled with a nonspecific protein. Now we need to identify whether among the proteins we are looking for, responsible for some kind of disease. Antibody treatment is used for detection. Under primary antibodies understand antibodies to the desired protein. By secondary antibodies is meant antibodies to primary antibodies. An additional special label (the so-called molecular probe) is added to the composition of secondary antibodies, so that later the results can be visualized. Radioactive phosphate or an enzyme tightly bound to the secondary antibody is used as a label. Binding first to primary and then to secondary antibodies has two goals: to standardize the method and to improve results.

Processing with a solution of primary antibodies  binding occurs in the place of the plate where there is an antigen (the desired protein).

Removal of unbound antibodies (washing).

Treatment with a solution of labeled secondary antibodies for subsequent development.

Removal of unbound secondary antibodies (washing).

Rice. 2.4. Immunoelectrophoresis (Western blot)

In the case of the presence of the desired protein in the biological material, a band appears on the plate, indicating the binding of this protein to the corresponding antibodies.

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