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https://doi.org/10.33647/2074-5982-15-2-12-23
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BIOLOGICALLY ACTIVE COMPONENTS OF ANTLERS EXTRACTS (CERVUS NIPPON) AND RED DEER (CERVUS ELAPHUS) PEPTIDE-
PROTEIN NATURE
Vladislav N. Karkischenko, Maxim S. Dulya*, Daniil V. Khvostov, Ruslan A. Ageldinov, Stanislav L. Lyublinskiy, Nikolay N. Karkischenko
Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia 143442, Russian Federation, Moscow region, Krasnogorsk district, Settlement Svetlye Gory, building 1
A complex study of the protein composition of the biologically active components of the extracts of the velvet deer antlers (VDA, include Cervus nippon and Cervus elaphus) using two-dimensional gel electrophoresis (2D-PAGE), size-exclusion chromatography (SEC), and peptide mapping with high-performance liquid chromatography with mass spectrometric detection (HPLC-MS) with the use of fermentation by trypsin was done. A molecular-mass distribution characteristic for protein extracts of VDA has been established. Optimal conditions for extraction, chromatographic separation and relative quantitative determination of the main components have been determined. The results of the identification of the most significant (major and minor) protein components in the extracts of the studied objects are described in detail in accordance with the algorithm of the search program Spectrum Mill MS Proteomics Workbench and the protein database Uniprot. The data of protein profiling are clustered according to molecular and biological functions. The connections of the identified proteins with possible mechanisms of biological action and targets, which can be affected by the protein components of the studied objects, are presented. Based on the results of the study, conclusions about the multicomponent protein composition of extracts of VDA were drawn. The marker protein components in the studied extracts are suggested and the possible interrelationships of the detected proteins in the extracts with biological effects are indicated.
Keywords: 2D-PAGE, HPLC-MS, IEF, peptide mapping, SEC, VDA Conflict of interest: the authors declare no conflict of interest.
For citation: Karkischenko V.N., Dulya M.S., Khvostov D.V., Ageldinov R.A., Lyublinskiy S.L., Karkischenko N.N. Biologically Active Components of Antlers Extracts (Cervus Nippon) and Red Deer (Cervus Elaphus) Peptide-Protein Nature. Journal Biomed. 2019;15(2):12-23. https://doi.org/10.33647/2074-5982-15-2-12-23
Submitted 29.04.2019 Revised 27.05.2019 Published 10.06.2019
List of abbreviations: VDA — velvet deer antlers, 2D-PAGE — two-dimensional polyacrylamide gel electrophoresis, SEC — size-exclusion chromatography, HPLC-MS — high-performance liquid chro-matography with mass spectrometric detection, IEF — isoelectric focusing, RP — regulatory peptides, SDS-PAGE — sodium dodecyl sulfate polyacrylamide gel electrophoresis, PPARGC1A — peroxisome proliferator-activated receptor gamma coactivator 1-alpha, Wnt — acronym from "Wingless/Integrated", CNR1 — cannabinoid Receptor 1, PFL — pyruvate formate-lyase.
БИОЛОГИЧЕСКИ АКТИВНЫЕ КОМПОНЕНТЫ ЭКСТРАКТОВ ПАНТОВ ПЯТНИСТОГО И БЛАГОРОДНОГО ОЛЕНЕЙ ПЕПТИДНО-
БЕЛКОВОЙ ПРИРОДЫ
В.Н. Каркищенко, М.С. Дуля*, Д.В. Хвостов, Р.А. Агельдинов, С.Л. Люблинский, Н.Н. Каркищенко
ФГБУН «Научный центр биомедицинских технологий Федерального медико-биологического агентства России» 143442, Российская Федерация, Московская обл., Красногорский р-н, п. Светлые горы, владение 1
Проведены исследования белкового состава биологически активных компонентов экстрактов пантов пятнистого и благородного оленей (ШЮ и ПБО) методами двумерного гель-электрофореза (2D ПААГ), гель-размерной эксклюзионной хроматографии (ГРХ) и пептидным картированием методом высокоэффективной жидкостной хроматографии с масс-спектрометрическим детектированием (ВЭЖХ-МС) высокого разрешения с применением ферментативного расщепления трипсином. Установлено молекулярно-массовое распределение, характерное для белковых экстрактов ППО. Найдены оптимальные условия экстракции, хроматографического разделения и относительного количественного определения главных компонентов. Подробно представлены результаты идентификации наиболее значимых (мажорных и минорных) белковых компонентов в экстрактах изучаемых объектов в соответствии с алгоритмом поисковой программы Spectrum Mill MS Proteomics Workbench и белковой базой данных Uniprot. Данные белкового профилирования кластеризованы по молекулярным и биологическим функциям. Отображены связи идентифицированных белков с возможными механизмами биологического действия и мишенями, на которые могут оказывать воздействие белковые компоненты изучаемых объектов. По результатам исследования сделаны выводы о многокомпонентности белкового состава экстрактов ППО, содержащие перспективные для более детального изучения компоненты. Предложены маркерные белковые компоненты в составе изучаемых экстрактов и указаны возможные взаимосвязи обнаруженных белков в составе экстрактов с биологическими эффектами.
Ключевые слова: 2D ПААГ, ВЭЖХ-МС, ГРХ, ИЭФ, пептидное картирование, ППО Конфликт интересов: авторы заявили об отсутствии конфликта интересов.
Для цитирования: Каркищенко В.Н., Дуля М.С., Хвостов Д.В., Агельдинов Р. А., Люблинский С.Л., Каркищенко Н.Н. Биологически активные компоненты экстрактов пантов пятнистого и благородного оленей пептидно-белковой природы. Биомедицина. 2019;15(2):12-23. https://doi.org/10.33647/2074-5982-15-2-12-23
Поступила 29.04.2019
Принята после доработки 27.05.2019
Опубликована 10.06.2019
Список сокращений: ППО — панты пятнистого оленя, 2D ПААГ — двумерный гель-электрофорез, ГРХ — гель-размерная эксклюзионная хроматография, ВЭЖХ-МС — высокоэффективная жидкостная хроматография с масс-спектрометрическим детектированием, ИЭФ — изоэлектрическая фокусировка, РП — регуляторные пептиды, SDS-PAGE — электрофорез белков в полиакриламидном геле, PPARGC1A — рецептор пероксисом, активируемый пролифератором гамма-коактиватор 1-альфа, Wnt — акроним от «Wingless/Integrated», CNR1 — рецептор каннабиноидов 1-го типа, PFL — пируват формиат лиаза.
Introduction
VDA is a part of the body, shaped like horns, but consisting of spongy cartilaginous tissue of Cervus Nippon. They are penetrated by a large number of blood vessels and have an intensive blood supply. When antlers reach the size of normal horns (on the 120-150th day from the beginning of growth), the process of their ossification begins. It is believed that during this period they are most biologically significant. Blood taken from a vein of an animal at the time of cutting also has activity similar to VDA. The expressed therapeutic properties of VDA are justified by the fact that during the growth of the horns of a young deer, his body actually produces up to 25 kg of bone tissue. Such growth rates are characteristic only for this family. This process requires strong support from all functional systems of the body and high concentrations of substances of a regulatory and protective nature, accordingly.
The use of drugs based on VDA and their blood in traditional medicine has more than two thousand years' history and is known in the past among many nations of the world. However, it is most widely spread as a branch of traditional medicine, and as a culture of maintaining a healthy lifestyle in China, the countries of Southeast Asia, Tibet and, to a lesser extent, among the peoples of southern Siberia and the Far North. At the beginning of XII, in China, for the first time, deer began to be domesticated in order to obtain VDA and their blood for the preparation of medicines [3].
It is known that medical preparations containing VDA products in their composition include a large number of substances of a regulatory nature: kinins, amino acids, phospho-lipids, growth factors involved in maintaining the activity and stability of the processes of an-abolism and catabolism in the body, regulating the processes of inflammation, regeneration and immunity. The main functional difference between VDA and plant adaptogens is that the latter increase the body's resistance, limiting the harmful effects of external factors, while
the first drugs increase the capacity of the body's own resistance [3].
Structural and functional information about peptides of plant and animal origin is important for system biology. It can also be used in various application areas: in agriculture, in the manufacture of dietary products, pharmaceuticals, functional foods [1, 6].
The relevance of research
Modern tools of peptidomics and proteomics in biomedical research are aimed at analyzing and developing new active substances, drugs, searching for indicator peptides or proteins and their identification, as well as developing algorithms for identifying and subsequently using effective combinations of various substances of protein-peptide nature for the diagnosis, effective therapies and their use as functional foods [1, 6].
It is paradoxical that, despite the long period of use of extracts and by-products from the tissues of deer-like animals, the composition of their active components remains poorly understood, components associated with a wide spectrum of biological activity of the studied tissues are not reliably identified.
It is known that the composition of extracts from VDA includes fatty acids, waxes, aromatic and steroid compounds, cholesterol esters, as well as micro and macronutrients, free amino acids, oligo and polysaccharides, derivatives of the uracil series, the xanthine group.
Within the framework of the research cycle, the authors' special attention is attracted by the peptide-protein composition of the tissues under the study. Many types of biological activity of a substance and preparations based on extracts and by-products of the studied tissues can be associated with the peptide-protein composition.
The studied objects are multicomponent protein-peptide complexes. The use of a number of methods of sequential sample preparation, separation by 2D electrophoresis, frac-tionation by chromatography allows obtaining
data on the nature (composition and relative amount) of the protein profile with visualization of the protein pool of the studied extracts.
The purpose of the study was to decipher the protein profile and identify the biologically active compounds of the peptide-protein fraction of antlers from Cervus nippon and Cervus elaphus. The tasks were to develop methods for sample preparation of extracts of the tissues under different conditions, selection of conditions for further protein profiling and their identification in natural raw materials by the methods of 2D-PAGE, SEC and HPLC-MS after enzymatic hydrolysis by trypsin.
Materials and Methods
The samples of extracts from antlers of the sika deer (VDA) (Cervus nippon) and antlers of the red deer (ARD) (Cervus elaphus) were received from the collection of the bioana-lytical research laboratory (SCBMT FMBA of Russia), obtained under the technology of water-alcohol extraction and subsequent lyo-philization. Materials
In our research, solvents and reagents of analytical degree of purity were used. Solutions for sample preparation were prepared using deionized water. To assess the presence of compounds of peptide or protein nature, an optional go spectrophotometer (Thermo scientific, USA) with 96-well cuvettes 10 mm long ("Corning", USA) used by spectroscopic method. The degree of proteolysis was assessed using a diode-matrix detector 1260 PAH UG Max-light cell connected to a high-performance liquid chromatograph 1260 infinity II of the laboratory of the OGRN pump equipped with a built-in degasser, equipped with an automatic sample entry system Multisampler 1260, and a unit for thermostating the chromatographic column ("Agilent technologies", USA).
HPLC-MS/MS HR analysis was performed using liquid chromatograph model 1290 infinity II "Agilent technologies", equipped with
pump 2-channel degasser, equipped with automatic input samples Multisampler 1290 with mass spectrometric detector QTOF 6545XT with ESI-ionisation at atmospheric pressure ("Agilent technologies", USA), the server station computer ("HP", USA), chromatographic column columns with different 300SB-C18, length 250 mm and internal diameter 2.1 mm, the size of the sorbent is 5.0 ^m with a pore size of 300 A and it is optimal for peptide mapping with a length of 150 mm and an internal diameter of 2.1 mm, the size of the sorbent is 2.7 ^m ("Agilent", USA).
2D page electrophoresis was performed using a ready-made kit ReadyPrep 2-nd starter kit, strips ReadyStrip IPG strip, 11 cm, pH 4-7 gels criterion TGX any KD dye bioSafe Coomassie stain, the results were detected on the Gel system Doctor EZ system using the software image Lab bio-rad ("Biorad", USA).
SEC analysis was performed on a chromatographic system of medium pressure to detect NGS ("Bio-rad", USA), equipped with a multi-wave spectrophotometric detector, a pH sensor of eluent, a conductometric cell and a chromatographic column for high-resolution SEC analysis Enrich SEC-650, as calibration standards with known molecular masses, gel filtration was used-standard ("Bio-rad", USA).
To identify proteins, a database of poly-peptide sequences was used on the Internet (www.ebi.uniprot.org) and the program for the collection and processing of spectrum data Mill MS proteomics workbench ("Agilent", USA). Methods
Total protein determination was performed by the Bradford protein assay without prior protein precipitation in accordance with the general pharmacopoeial monograph (1.2.3.0012.15, "Protein Determination") of the RFSP. The lyophilisate of the extract was pre-dissolved in purified water to a concentration of 150 mg/ml in a volume of solvent. The amount of protein was determined by the cal-
ibration curve for a solution of bovine serum albumin.
All procedures of two-dimensional separation by electrophoresis of proteins were performed according to the protocol included in the kit. The amount of the sample was taken at the rate of 250 mg of protein per 185 ^l of denaturing solution. In two-dimensional electrophoresis, the first stage of separation is isoelectric focusing. Proteins are separated by electrophoresis based on their pl, the pH value at which the protein does not carry a net charge. The second separation stage is SDS-PAGE, where proteins already separated by IEF are further separated by their size. Gels were previously washed with distilled water and filled with a 200-300 ml paint solution and left overnight for better staining at room temperature. The next day, the gels were washed with distilled water in triplicate and the results were detected on the Gel Doc EZ System.
The molecular weight protein distribution of the product was carried out on a high pressure liquid isocratic chromatograph with a spectro-photometric flow detector with a wavelength of 280 nm.
Analysis on an Agilent Technologies 1260 Infinity II liquid chromatograph was performed in AutoMSMS mode. AdvanceBio Peptide Mapping Chromatographic Column, precursor ZORBAX Extend-C18 NarrowBore Guard Column were used. Elution was carried out with a mixture consisting of components A and B in the gradient mode: up to 0.5 min — 5% B, from 0.5 to 15.0 min increased to 35% B, from 17.0 — to 95% and held for 13 min, from 30.01 minutes — returns to initial conditions. The column equilibration time for initial conditions is 5 minutes. Component A was a 0.1% solution of formic acid in deionized water, component B was a 0.1% solution of formic acid and 10% deionized water in acetonitrile. The flow rate was 400 ^l/min, the analysis time was 30 minutes. Conditions of mass spectrometric detection are shown in Table 1.
Results and discussion
Total protein determination
Percentage of total protein concentration according to the Bradford method studied extracts demonstrates that the largest amount of protein (30%) is in the VDA sample, and the
Table 1. Conditions for mass spectrometric detection
Таблица 1. Условия масс-спектрометрического детектирования
Parameter name Value
Mass spectrometry detector QTOF 6545XT
Ionization source Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI)
Resolution 60,000
Type of collisional dissociation cell Collisional dissociation cell
Ionization energy (30 ± 15)%
Detectable charge state 2-5
Scan mode AutoMSMS
Detected mass range 100-1700
Polarity of detected Ions Positive ion detection
Spray voltage 3,5 kV
Fragmenter voltage 175 V
Flow rate of desiccant dryer gas 13 l/min
Nebulizer gas pressure 35 psig
Spray temperature 250°C
smallest (~20%) in ARD . The coefficient of variation does not exceed 10%.
2D PAGE
Using the method of 2D PAGE held comparative proteome analysis was done on basis results received of samples VDA and ARD. Identification by molecular mass and meaning pI was held with using Image Lab Bio-rad software (Figs 1 and 2).
In the VDA sample, more than 30 marker plots were counted. By the pI values, 3 groups of proteins can be distinguished: 1) pI=4.1.
Molecular weight: 12.1, 23.8, 39.5, 50.2 and 66.4 kDa; 2) pI=5.0-6.0. Molecular weights: 18-20 kDa and 70-105 kDa; 3) pI=7.0. This group shows a large accumulation of proteins with a mass about of 15 kDa, which may indicate a significant amount of low molecular weight proteins.
The second sample shows the best separation with the display of about 90 marker plots. According to the IEF more groups can be distinguished than in the first sample, but the molecular mass distribution remains very similar.
Fig. 1. 2D PAGE of ARD sample stained with Coomassie. Рис. 1. 2D-электрофорез ПБО, окрашенного кумасси.
Fig. 2. 2D PAGE of VDA sample stained with Coomassie. Рис. 2. 2D-электрофорез ППО, окрашенного кумасси.
Observed masses in the low area of 11-15, 20 and 25 kDa. In the field of medium weights, this range is from 50 to 75 kDa. No area with large molecular masses of proteins were detected.
The large dynamic range of concentrations of the studied extracts, as well as the wide variability of the physicochemical properties of the components in their composition, necessitates the use of multistage separation technologies with predominant use of SEC, 2D PAGE, specialized chromatographic chips, and all their possible combinations with technological methods (solid phase extraction, precipitation, exhaustion, etc.).
Size-exclusion chromatography
VDA sample demonstrated similar profile at low molecular weight areas as well as ARD sample. Also the meanings received from 2D PAGE result are confirmed by SEC at more high areas of molecular mass distribution. Received chromatograms are presented in pictures 3 and 4 and relevant values of molecular masses are demonstrated in the Table 2.
HPLC-MS
Peptide mass spectrometry is currently rapidly improving as the methodological basis of proteomics, both in terms of qualitative and quantitative analysis [2]. Unlike proteomics, based on controlled protein cleavage into pep-tides with "predictable" terminal amino acids, peptide — mass spectrometry is aimed at analyzing native peptides.
According to the results of previous analyzes, indicating a closely related component protein composition of the extracts and based on the most pronounced results for subsequent analysis, VDA extracts were used in this step. The result of the decoding of the protein profile of the fraction from the VDA and their classification became the annotated groups of proteins presented below (Figs 5 and 6). HPLC-MS analysis after enzymatic hydrolysis with trypsin demonstrated the identification of a large number of protein components in both fractions. For a more convenient perception, the obtained data was analyzed in the UniProt
0 4 8 12 16 20 24 28 32 36 40
Volume (mi) >•
Fig. 3. SEC water solution of the ARD extract. Рис. 3. ГРХ водного раствора экстракта ПБО.
0 4 8 12 16 20 24 28 32 36 40
Volume (ml) >
Fig. 4. SEC water solution of the VDA extract. Рис. 4. ГРХ водного раствора экстракта ППО.
Note: * — In these calculations, when building a calibration curve, the coefficient of determination is 0.99. Примечание: * — В данных расчетах при построении калибровочной кривой коэффициент детерминации равен 0,99.
Table 2. The results of SEC from VDA and ARD extracts* Таблица 2. Результаты ГРХ по экстрактам ПБО и ППО*
Molecular weight distribution in ARD extract
Peak number The volume of output, ml Column volume, ml Column dead volume, ml Kav, distribution coefficient Mol. weight, Da The contribution of the peak, %
1 13.14 23.56 8 0.33 166725 20.02
2 14.33 23.56 8 0.41 75638 12.27
3 15.69 23.56 8 0.49 30651 33.41
4 18.02 23.56 8 0.64 6522 7.91
5 20.07 23.56 8 0.78 1671 7.57
6 23.09 23.56 8 0.97 225 4.32
Molecular weight distribution in VDA extract
Peak number The volume of output, ml Column volume, ml Column dead volume, ml Kav, distribution coefficient MW, Da The contribution of the peak, %
1 9.82 23.56 8 0.12 1512382 7.87
2 13.2 23.56 8 0.33 160211 20.44
3 14.37 23.56 8 0.41 73655 12.5
4 15.69 23.56 8 0.49 30651 41.82
5 18.03 23.56 8 0.64 6478 9.7
6 20.1 23.56 8 0.78 1638 3.26
7 23.1 23.56 8 0.97 223 2.04
database, which reflected the enormous number of functions and biological roles of protein components from the studied fractions. In total, 435 protein components were found in extracts from VDA, including 75, which were also not experimentally established in the deer antlers. The diagrams demonstrate general directions of functions and processes involving the identified proteins. Table 3 shows the brightest representatives of the studied profiles with the highest index of similarity.
In the classification according to molecular functions of protein components from the VDA fraction, four directions predominate: transferases, hydrolases, DNA/RNA-binding. Proteins that realize transport functions dominate involvement in biological processes.
The results given in Table 3 constitute a representatively selected part of the analyzed protein components of the tissues under study; however, they are among the known
and significant among all. Particular attention is caused identified in the extracts from the VDA: PPARG1A, Wnt protein, CB1, PFL.
Peroxisome proliferative activated receptor, gamma, coactivator 1 (PPARGC1A) is a coactivator of nuclear receptors and other transcription factors that regulate metabolic processes, including biogenesis and respiration of mitochondria, hepatic gluconeogen-esis and switching between types of muscle fiber [7].
Wnt proteins are a large number of secreted molecules involved in cell-to-cell signaling. The influence of the Wnt protein controls many cellular processes in many different organisms. Signal transmission based on the interaction with this molecule is largely associated with tumor genesis, early embryonic mesodermal pattern, brain and kidney morphogenesis, regulation of breast proliferation and Alzheimer's disease [5, 8].
Fig. 5. Groups ofproteins from VDA extracts, distributed by molecular functions.
Рис. 5. Группы белков из экстрактов ППО, распределенные по молекулярным функциям.
Fig. 6. Protein groups from VDA extracts, distributed by biological processes.
Рис. 6. Группы белков из экстрактов, распределенные по биологическим процессам.
Table 3. Molecular mass characteristics of some typical annotated proteins in the composition of the VDA extract Таблица 3. Молекулярно-массовые характеристики некоторых типичных аннотированных белков в составе экстракта ППО
Protein number MS/MS Score Amino acid coverage, % Total spectral intensity MW, Da Protein name
1 55.61 6.8 1.54E+06 66149 Serum albumin (Fragment)
2 48.51 4.9 1.49E+06 69080 ALB
3 43.71 12.3 3.09E+06 42765 HBA
4 42.4 30.3 5.21E+06 15953 Adult beta-globin
5 33.18 23.4 3.26E+06 15824 Hemoglobin subunit beta-3
6 21.95 10.3 2.12E+05 19244 HBB
7 21.47 14.2 3.43E+06 15952 Beta-globin subunit
8 12.44 6.1 1.86E+05 16522 HBG-T2
9 12.60 4.1 3.13E+05 30235 APOA1
10 12.31 2.5 3.90E+04 39576 FETUB (Fragment)
11 10.65 2.8 6.47E+04 32307 Vps23 core domain protein
12 10.36 4.9 6.50E+04 26317 HSD11B1L
13 10.27 2.8 9.22E+04 46667 GABRD
14 10.25 3.8 2.16E+05 34418 MDH1B (Fragment)
15 9.98 2.5 1.04E+05 34364 MRPL2
16 9.68 2.8 4.49E+05 23404 Glycerol-3-phosphate acyltransferase
17 9.22 3.4 2.77E+05 60607 DNA topoisomerase
18 8.95 2.2 1.53E+05 34361 Iron ABC transporter substrate-binding protein
19 8.93 5.9 8.06E+04 26292 GntR family transcriptional regulator
20 8.06 7.5 2.91E+05 16021 BUB3
21 7.80 4.7 9.29E+04 23302 WRN
22 7.47 2.2 9.41E+04 55725 Phosphoglucomutase
23 7.44 5.0 1.46E+05 19653 Histone H4
24 7.40 8.7 7.39E+04 23879 Arginine/serine-rich 7 splicing factor (Fragment)
25 7.23 6.1 6.51E+04 10838 Rhodanese-like domain-containing protein
26 7.20 2.3 1.17E+05 38297 Methionine import ATP-binding protein MetN
27 7.17 3.9 5.85E+05 27897 HAD family hydrolase
28 7.08 3.7 7.83E+04 15634 HTH-type transcriptional regulator LrpC
29 6.72 2.6 8.52E+04 41870 Beta sliding clamp
30 6.45 3.9 1.44E+05 20987 PPEF2 (Fragment)
31 6.19 5.0 1.07E+05 35138.7 Protein Wnt
32 4.02 1.3 3.03E+04 83831 Formate C-acetyltransferase
33 3.82 3.6 3.93E+03 37282.6 Сannabinoid receptor 1 (Fragment)
34 3.0 4.2 4.86E+05 25684.1 PPARGC1A
The cannabinoid receptor type 1 (CB1) is a cannabinoid G-protein coupled receptor. The CB1 receptor is expressed predominantly in the peripheral and central nervous systems and is activated by: endocannabinoids, a group of retrograde neurotransmitters; plant phytocann-abinoids, such as tetrahydrocannabinol and its synthetic analogues [4].
Pyruvate formate-lyase (PFL) or formate C-acetyltransferase is an enzyme in the trans-ferase family that helps regulate anaerobic glucose metabolism. It catalyzes the reversible conversion of pyruvate and coenzyme-A to formate and acetyl-coenzyme A. This enzyme is involved in the metabolic pathways of pyruvate, propionate and butyrate [9].
Conclusions
Using two-dimensional gel electrophoresis and size-exclision chromatography, protein profiling and molecular-mass distribution data were obtained from proteins in VDA extracts under denaturing conditions.
Using the Bradford method, the content of total water-soluble proteins in the extracts was evaluated. The effect of detergent additives and extraction conditions on the total protein level in lyophilisates was revealed.
The HPLC-MS assay of extract samples identified extensive and representative groups
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INFORMATION ABOUT THE AUTHORS | СВЕДЕНИЯ ОБ АВТОРАХ
Vladislav N. Karkischenko, Dr. Sci. (Med.), Prof., Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia; e-mail: scbmt@yandex.ru
Каркищенко Владислав Николаевич, д.м.н., проф., ФГБУН «Научный центр биомедицинских технологий Федерального медико-биологического агентства России»; e-mail: scbmt@yandex.ru
Maxim S. Dulya*, Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia; e-mail: mdulya@gmail.com
Daniil V. Khvostov, Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia; e-mail: daniil hvostov@mail.ru
Дуля Максим Сергеевич*, ФГБУН «Научный центр биомедицинских технологий Федерального медико-биологического агентства России»; e-mail: mdulya@gmail.com
Хвостов Даниил Владиславович, ФГБУН «Научный центр биомедицинских технологий Федерального медико-биологического агентства России»; e-mail: daniil hvostov@mail.ru
Ruslan A. Ageldinov, Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia; e-mail: ageldinov@gmail.com
Stanislav L. Lyublinskiy, Cand. Sci. (Biol), Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia
Агельдинов Руслан Андреевич, ФГБУН «Научный центр биомедицинских технологий Федерального медико-биологического агентства России»; e-mail: ageldinov@gmail.com
Люблинский Станислав Людвигович, к.б.н., ФГБУН «Научный центр биомедицинских технологий Федерального медико-биологического агентства России»
Nikolay N. Karkischenko, Dr. Sci. (Med.), Prof., Corresponding Member of the Russian Academy of Sciences, Academician of the Russian Academy of Rocket and Artillery Sciences, Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia; e-mail: niknik2808@yandex.ru
Каркищенко Николай Николаевич, д.м.н., проф., чл.-корр. РАН, акад. РАРАН, ФГБУН «Научный центр биомедицинских технологий Федерального медико-биологического агентства России»; e-mail: niknik2808@yandex.ru
* Corresponding author / Автор, ответственный за переписку
