Method of detecting o-glycosylated proteins in cell homogenates prepared for proteomic and phosphoproteomic analysis

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of detecting O-glycosylated proteins in cell homogenates that are prepared for proteomic and phosphoproteomic analysis. The disclosed invention can be used to perform proteomic and phosphoproteomic analysis. The method involves performing two-dimensional electrophoresis, followed by identification of spots using MALDI-TOF spectroscopy and phosphoproteomic techniques. The cell homogenates are desalinated by gel-penetrating chromatography or dialysis. The cell homogenates are subjected to glycosylation based on a β-elimination principle in a 0.05 M NaOH solution which contains 38 mg/ml NaBH4 for 16 hours at +45°C, followed by addition of cyanine dye JC-1 in concentration of 10-6 M. The cell homogenates are incubated for 15 minutes at room temperature. The homogenates are concentrated by precipitation with 50% acetone, subjected to two-dimensional electrophoresis to form electrophoregrams which are analysed for fluorescence when illuminated on a blue light transilluminator with an amber light filter, which visually appears in form of strips which are fluorescent in the dark. Said strips are extracted from the gel and used to perform proteomic or phosphoproteomic analysis. Further analysis of intensity and arrangement of the extracted strips is performed by comparing silver nitrate-coloured electrophoregrams of homogenates before and after a deglycosylation procedure.

EFFECT: disclosed invention enables to identify proteins which change their composition or degree of O-glycosylation as a result of any physiological action on the cell.

5 dwg

 

The technical field of the invention: the invention relates to the field of biotechnology, in particular to the development of the method of preparation of cellular material for proteomic and phosphoproteomic analysis using the J-aggregates.

Art: now for the molecular study of the proteome of cells actively used proteomic and phosphoproteomic technology, thanks to which it becomes possible to assess the status of the signaling pathways of the cell, including those exposed to viral infections, and to identify signaling proteins involved in the response to any viral infection, regardless of taxonomic affiliation of the virus. Such proteins are the most promising cellular target for development of antiviral therapy, however, for the analysis of proteomics methods and phosphoproteomic required the development of methods of pretreatment of cell homogenates used for this analysis.

As is well known, N - and O-linked oligosaccharides are major structural components of many proteins, and the diversity of oligosaccharide structures, namely the change in the structure and different degrees of saturation of sites of glycosylation in glycoproteins contribute to the heterogeneity of the mass, which causes significant signs of difficulties and in the analysis of proteins using different proteomic and phosphoproteomic methods. Because O-linked oligosaccharides are usually less mass than N-linked glikana, but are more numerous and more heterogeneous in structure, preparation of biological material for proteomic and phosphoproteomic analysis should include deglycosylation O-glycoproteins. Methods deglycosylation can be divided into two groups: enzymatic and chemical. For enzymatic deglycosylation use of endo - and ectoparasites that hydrolyzing O-glycosidic linkage between the monosaccharide residues of oligosaccharides, and is able to cleave the bond between the carbohydrate part and the serine/threonine protein part of the molecule. Unlike deglycosylation N-glycoproteins, which is carried out with the use of certain N-glycanase, and the reaction takes place completely in the case of O-glycosidic bonds oligosaccharide chain can be removed only with a mixture of field of glycosidase inhibition, each of which is characterized by a narrow specificity. A similar method is used in prO-LINK Extender™ Kit for Complex O-Linked Glycans" firm ProZyme Inc., USA (http://www.prozyme.com). There are also sets of enzymes for simultaneous deglycosylation N - and O-linked glycoproteins: set "GlycoPro™ Enzymatic Deglycosylation Kit for N-Linked & Simple O-Linked Glycans", firms ProZyme Inc., USA and set Enzymatic Protein Deglycosylation Kit" by Sigma-Aldrich Co. LLC, USA http://www.sigmaaldrich.com). In atente US 20060269980 proposed Protocol enzymatic deglycosylation N - and O-linked with oligosaccharides of glycoproteins using a set field of glycosidase inhibition (Prasa F, Endo H, Endo F, O-glycosidase and neuraminidase) with subsequent detection of the hydrolysis products of mass spectrometric methods, including the method of SELDI mass spectrometry for affine chips [1].

In the patent US 20100190146 for deglycosylation N - and O-linked glycoproteins and subsequent analysis of the profile derived glycogenes proposed microfluidic device. In the construction of the device includes deglycosylated column solid phase on an inert carrier, which is attached deglycosylated enzymes for specific removal of oligosaccharides from the surface glycoproteins. Assessment completion of the reaction and identification products deglycosylation possible using methods of mass spectrometry and protein microarrays [2].

The main advantages of enzymatic methods are more lenient conditions and the absence of nonspecific side reactions. The disadvantages of this group of methods can be attributed to the high cost of analysis, the need to comply with strict storage conditions are set, the possibility of obtaining low yield of the desired product because of steric hindrance, and the complexity of identifying deglycosylated proteins due to complete removal of oligosaccharide chains of glycoproteins.

For chemical deglycosylation glycoproteins use modificy the consistent method controllable periodic destruction of oxidation, β-elimination, as well as methods with anhydrous triftormetilfullerenov (TFMS) and anhydrous hydrazine. When controllable periodic destruction oxidation rupture of C-C bonds in the glycols with the formation of aldehyde groups. There are two versions controllable periodic destruction oxidation: the method of Smith is to restore oxidized oligosaccharide with sodium borohydride, followed by hydrolysis of the obtained polyol; it is possible to conduct stepwise degradation of oligosaccharide) and method Barry (is selective cleavage of oxidized oligosaccharide under the action of phenylhydrazine; fission occurs as acetylenic and glycosidic linkages). The advantages of controllable periodic destruction of oxidation can be attributed quantitative cleavage picolinic groups and reaction processes in microvolumes, while the disadvantages of the oxidation of some amino acids, which creates additional difficulties for further analysis of the properties of the protein [3].

Hydrazines glycoproteins, despite the possibility of simultaneous removal of N - and O-linked oligosaccharides leads to complete degradation of the protein part due to selective attack hydrazine amide bonds and the formation of hydrazides of the corresponding amino acids, and therefore are not applicable in those cases when you want to preserve the structure of the protein for further analysis. These met the d used in GlycoRelease™ Glycan Hydrazinolysis Kit" by ProZyme Inc., USA.

Method using triftormetilfullerenov acid (TFMS) is mixed with anisole allows nonspecific destroy N - and O-linked oligosaccharides of glycoproteins without noticeable degradation of protein components. However, even in the optimized version of the method with TFMS is very time consuming, and leads to incomplete removal of sugar: can be associated with glycoprotein monosaccharides, for the removal of which requires disclosure of pyranose rings by processing periodates sodium and repeat test with TFMS or β-elimination to obtain a fully deglycosylated glycoprotein [4]. A similar method is used in sets "GlycoFree™ Chemical Deglycosylation Kit" by ProZyme Inc., USA and GlycoProfile™ IV Chemical Deglycosylation Kit TFMS Deglycosylation System" by Sigma-Aldrich Co. LLC, USA.

Deglycosylation on the mechanism of β-elimination by processing of glycoproteins in mild alkaline conditions allows for preferential destruction of O-linked oligosaccharides, namely to split O-glikana as oligoneuriella. Carrying out the reaction of β-elimination in the presence of a reducing agent avoids degradation delocalising relations, which makes this method almost without flaws. A similar method is used in GlycoProfile™ β-Elimination Kit" by Sigma-Aldrich Co. LLC, USA.

<> After deglycosylation necessary to determine the pattern of proteins, including figuring out which of these proteins were initially glycosylated, since such information will allow us to more fully characterize the object under study. Depending on the chosen method of deglycosylation there are various options identification pattern deglycosylation and initially deglycosylated proteins. For example, after oxidation periodates sodium is the disclosure of the pyranose ring, associated with the glycoprotein monosaccharides, and formed aldehyde groups, which can be identified by a photocolorimetric method based on the reduction of chloride 2,3,5-triphenyltetrazolium aldehyde groups with the formation of red dye formazan. The colour intensity of the resulting solution to determine photoelectric colorimeter with a green filter or on the spectrophotometer at a wavelength of 546 nm. The method allows to determine the total number of glycoproteins oxidized by periodate sodium, by assessing the content of aldehyde groups on the calibration curve using control solutions of glycoproteins. This method of detection is used in the set (Glycoprotein Carbohydrate Estimation Kit" of Thermo Fisher Scientific Inc., USA (http://www.thermoscientific.com)). To the advantages which this method can be considered fairly accurate and well-reproducible results and the ability to detect small amounts of aldehyde groups, the main disadvantage is the possibility of determining the degree of deglycosylation only in whole and not for each specific protein.

For visualization of the pattern partially deglycosylated glycoproteins with monosaccharide residues are common reactions with fluorescently labeled derivatives of amines, hydrazine and hydroxylamine in the presence of a reducing agent. Among the most widely used reagents can be distinguished dancigers and cancellationtoken [5], various Biotin hydrazides [6], and various aromatic amines (for example, 2-aminopyridine [7]).

In the patent application WO 0228841 also offered a new range of reactive dyes for fluorescent labeling of biomolecules containing aldehyde functional group, which, including, suitable for fluorescent labeling of aldehydes, formed after oxidation periodates sodium monosaccharide residues of glycoproteins [8].

After the reaction the formed adduct stabilized by treatment with borohydride or cyanoborohydride sodium and next methods of separation chromatography, electrophoresis or precipitation carry out a direct or indirect detection of labeled products [9]. In the case of Biotin-peroxidase method, the detection is carried out after immobilization on nitrocellulose the first membrane by means of showing biotinylated monosaccharide residues of glycoproteins in the system of streptavidin-alkaline phosphatase.

After deglycosylation on the mechanism of β-elimination oligosaccharide is cleaved from the protein component together with hydrogen carbon atom of serine/threonine that also allows for further modification to identify the pattern deglycosylation glycoproteins. For marking deglycosylation similarly glycoproteins often use dithiotreitol (DTT) and Biotin pentylamine (BAP), electrophilic accession which occurs at the double bond, forming a covalent bond between the proton and one of the carbon atoms of the double bond [10]. In the case of processing deglycosylated glycoprotein DTT form a stable sulfide adducts, which can be identified by mass spectrometry [11]. In turn, the use of Biotin pentylamine allows selective biotinylation deglycosylation glycoproteins with further enrichment using affinity chromatography and identification by methods of liquid chromatography and mass spectrometry. Also possible to visualize biotinylated monosaccharide residues of glycoproteins after transfer to the membrane using streptavidin and alkaline phosphatase [10].

The technical result of the invention, achieved in the implementation of the method according to Faure is ule, is to identify proteins that change the composition or the degree of their O-glycosylation in the result of any physiological effect on the cell. Identification is carried out visually in the form of the choice spots on two-dimensional electrophoregrams, painted silver nitrate, which can then be extracted and used for protein identification using MALDI-TOF, phosphoproteome analysis or other highly sensitive method of determining the sequence of a protein.

Disclosure of the invention: the invention is a method of preparing biological material for proteomic and phosphoproteome analysis by deglycosylation glycoproteins followed by the introduction of covalent labels and visualization of the pattern originally deglycosylation and deglycosylated proteins in the preprocessed cell homogenate using J-aggregates.

The structural formula of the dye JC-1, used as covalent labels and the basis for the formation of J-aggregates is shown in figure 1. Sequential scheme of conducting deglycosylated proteins with the introduction of covalent modification and visualization of the pattern deglycosylation and deglycosylated proteins in the preprocessed cell homogenate using J-aggregates is shown in Figure 2.

The main aspect given to the CSOs of the invention is the use of cyanine dyes, in particular dye JC-1, for covalent modification of O-deglycosylation on the mechanism of β-elimination of glycoproteins and get labeled product. The scheme of reactions of deglycosylation glycoproteins and subsequent labeling deglycosylated protein product cyanine dye, indicating the possible mechanisms of the formation of covalent bonds is shown in Figure 3.

Another aspect of this invention is the non-covalent tagging of primary modified dye deglycosylated proteins, in particular dye JC-1, high concentrations of the same dye by forming supramolecular J-aggregates. The packing model J-aggregates based on the covalently-modified dye deglycosylation glycoproteins, at high concentration of cyanine dye JC-1 is shown in Figure 4.

The method is based on the ability of cyanine dyes to the formation of supramolecular structures, namely complex polymolecular associates - J-aggregates. The formation of J-aggregates is the result of adsorption of the dye on the surface and is realized by self-Assembly in a buffer solution at high concentration of the dye in the presence of metal ions or polymers [12]. It is known that the formation of J-aggregates thiacarbocyanine dye inducedots the addition of gelatin and other biologically active molecules with positively charged groups, which by the electrostatic interaction associated anions dye and there is a further self-Assembly of J-aggregates [13]. When using thiacarbocyanine dyes for efficient formation of J-aggregates necessary condition is the presence of monovalent cations, the addition of which leads to the reduction of electrostatic repulsion between dye molecules and, thus, accelerates the aggregation processes [14]. Unlike thiacarbocyanine dyes JC-1 dye is a lipophilic cation 5,5', 6,6'-tetrachloro-1,1,3,3'-tetramethylbenzidine-carbocyanine, which is also widely used as fluorescent probes for biomedical applications, including to assess the membrane potential of mitochondria, due to its almost a definite change in the spectral characteristics depending on the concentration of the dye and formed patterns [12, 15]. At low concentrations of the dye JC-1 in the spectrum of luminescence (λvasb=490 nm) is observed intense band monomers at a wavelength of 530 nm, whereas increasing the concentration of JC-1 leads to a shift of the peak in the long wavelength region of the spectrum up to 595 nm, with increasing concentration of the dye peak also narrowed, reflecting not only on the formation and increase the AI of size J-aggregates JC-1, but about improving their structure [12, 15]. For example, after conducting electrophoretic analysis of the pattern of all proteins and staining plates PAG silver nitrate secondary staining modified dye deglycosylation glycoproteins leads to the formation of J-aggregates, the formation of which is determined by the method of fluorescent detection using transilluminator blue color with light amber color Safe Imager™ 2.0 company Life Technologies, USA http://www.lifetechnologies.com or UVIblue company Uvitec, Cambridge, UK (www.uvitec..uk and visually manifested in the form of glow-in-the-dark bands corresponding deglycosylated glycoproteins. Gel documentation molecular chains formed J-aggregates on the basis of the covalently-modified dye JC-1 deglycosylation glycoproteins performed using digital camera Panasonic Lumix DMC-GF2C in a dark room. Such simple visual detection of the pattern originally deglycosylated proteins and covalently modified deglycosylation glycoproteins makes the developed method of analysis is applicable for the preparation of biological material for proteomic and phosphoproteomic analysis.

Examples of the analysis of the results obtained after carrying out two-dimensional electrophoresis according to O Farrell deglycosylation and delicas the isolation of proteins in the homogenate of the cells, infected model strain of tick-borne encephalitis virus EK-328 or variant of the M strain EK-328, before deglycosylation glycoproteins, and after deglycosylation and covalent modification dye JC-1 without processing plate PAG and treated with a solution of the same dye in a high concentration with the formation of J-aggregates is shown in Figure 5.

A brief description of graphics:

Figure 1. The structural formula of the dye JC-1, used as covalent labels and as a basis for the formation of J-aggregates.

Figure 2. Sequential scheme preobresti homogenates of cells infected model strains of viruses, through deglycosylation glycoproteins followed by the introduction of covalent labels and visualization pattern deglycosylation and deglycosylated proteins in the preprocessed cell homogenate using J-aggregates.

Figure 3. The scheme of reactions of deglycosylation glycoproteins on the mechanism of β-elimination and subsequent labeling deglycosylated protein product cyanine dye JC-1, indicating the possible mechanisms of the formation of covalent bonds.

Figure 4. The packing model J-aggregates based on the covalently-modified dye deglycosylation glycoproteins, the high concentration of C is innovage dye JC-1.

Figure 5. Electrophoretic analysis of the pattern deglycosylation and deglycosylated proteins in the homogenate of cells spew infected model strain of tick-borne encephalitis virus EK-328 (1-3) or option M strain EK-328 (4-6): before deglycosylation glycoproteins (1, 4), after deglycosylation and covalent modification of glycoproteins dye JC-1 without processing plate page (2, 5) and treated with a solution of cyanine dye JC-1 with the formation of ordered molecular chains J-aggregates (3, 6). Proteins separated by method 20-electrophoresis according to O Farrell and stained for total protein silver nitrate using a set of "PageSilver™ Silver Staining Kit. Arrows indicate bands corresponding to deglycosylated the glycoproteins.

The implementation of the invention based On the method O-deglycosylation glycoproteins on the mechanism of β-elimination [10, 11] was developed and optimized a method for identifying a pattern primarily deglycosylation and deglycosylated proteins in the preprocessed cell homogenate through covalent binding deglycosylation glycoproteins with cyanine dye JC-1 [12], electrophilic accession which, as in the case of DTT and VAR, occurs at the double bond [10]. Before and after deglycosylation glycoproteins on the mechanism of β-elimination with Cova is entei modification and tagging deglycosylated proteins cyanine dye JC-1 was carried out 2D-electrophoresis according to O Farrell with subsequent visualization of the pattern originally deglycosylation and modified dye deglycosylated proteins by staining gels with silver nitrate using a set of "PageSilver™ Silver Staining Kit" of Thermo Fisher Scientific Inc., USA (http://www.fermentas.com). After the pattern is identified all proteins by assessing gel-documented two-dimensional images of electrophoregram method of scanning scanner Epson expression 1680 or using digital camera Panasonic Lumix DMC-GF2C to confirm manifestations on the plate PAG bands, corresponding exactly deglycosylation the glycoproteins, the gel was placed in a horizontal electrophoresis chamber, and staining was conducted initially labeled deglycosylated proteins by adding a cyanine dye JC-1 in a high concentration with the formation of ordered molecular chains of J-aggregates.

Covalent modification deglycosylated proteins can be carried out using other cyanine dyes, including oxa - and tatiannah dyes [16, 17], are capable of forming supramolecular J-aggregates.

Re-identification using transilluminator blue color with light amber color Safe Imager™ 2.0 (Life Technologies, USA) or UVIblue ("Uvitec, Cambridge, UK) and digital camera Panasonic Lumix DMC-GF2C in a dark room with the subsequent imposition of the obtained gel-documented from the interests of enables rapid and convenient method for the visual detection to determine the pattern originally deglycosylated proteins and covalently modified deglycosylated proteins, what makes this method ideal for use in the preparation of biological material for proteomic and phosphoproteomic analysis.

Further, the invention is illustrated in the examples that show the application of the proposed method for the preparation of biological material for proteomic and phosphoproteomic analysis based on the preprocessing of homogenates of cells, including infected model by viral strains, namely deglycosylation contained glycoproteins and subsequent introduction of covalent labels with visualization of the pattern deglycosylation and deglycosylated proteins using J-aggregates JC-1. It should be understood that the examples are only for illustration and are not intended to limit the scope of the claims expressed in the claims. On the basis of the present description, the person skilled in the art will be able to easily offer their own versions and modifications of the invention, without departing from the General concept of the present invention and without the involvement of private inventive activity, so it should be clear that such variations and modifications will also be in the scope of claims of the present invention.

Example 1. Preparation of lysate of cells infected by a model strain of tick-borne encephalitis virus EK-328 or variations is that M, obtained from strain EK-328.

The suspension of cells of the embryo kidney pig spew after infection by a strain of tick-borne encephalitis virus EK-328 or variant of the M strain EK-328, and processing are thoroughly mixed on a rotator and selected aliquots following bytes:

When the titer of 1:512-0,25 ml,

When the titer of 1:256-0,5 ml,

When the titer of 1:128-1 ml.

To the cell suspension in DMEM was added 100 mg of glass beads with a diameter of 0.8 mm, 10 μl per ml of a cocktail of inhibitors of proteolysis "Halt Protease Inhibitor Cocktail ("Thermo Fisher Scientific Inc., USA) and subjected to intensive processing on the shaker VP1-2: 10 episodes of 30 seconds for each drug, maintaining the temperature of the preparation of about 0°C by incubation in an ice bath. Then the suspensions add the detergent Triton X-100 to a final concentration of 1% and incubated on ice with periodic shaking for 3 hours.

The suspension is decanted, clarify by centrifugation at 2000 g for 30 min and frozen in liquid nitrogen for further use.

Example 2. Pretreatment of cell homogenates: hold O-deglycosylation glycoproteins on the mechanism of β-elimination with subsequent covalent modification deglycosylated proteins cyanine dye JC-1.

As the buffer solution for the cell homogenate using a buffer solution with a slightly alkaline pH of 7.2 on the Snov, 100 mm Tris-HCl with the addition of 100 mm NaCl. A sample volume of 400 ml pre-concentrate on the cell to tangential ultrafiltration ("Amicon, USA). The primary concentrate cialiswhat against buffer solution Tris-HCl 100 mm, NaCl 100 mm during the days at +4°C: 75 ml of sample is applied 3 times 25 ml of a gel-filtration column with Sepharose 6 FastFlow (elution buffer contains 100 mm Tris-HCl and 100 mm NaCl, pH 7,2). Conditions are selected in such a way that most of the impurities of protein and nonprotein nature is not able to contact the sorbent. Viral protein comes out of the speakers in the free volume, is equal to ~ 66% of the total volume of the column. For further work using 3 fractions of the first peak.

The fractions of the first peak of viral material by volume of 0.5 ml, add 0.5 ml of 0.1 M NaOH and 38 mg NaBH4. The mixture was thoroughly stirred and heated 16 hours at +45°C. thereafter, to the mixture is added 10 μl of 10-4M cyanine dye JC-1, dissolved in a binary solution of acetone-Tris-HCl (C=0.05 M, pH=8,5), and incubated for 15 min at room temperature (dye JC-1 provided by the laboratory processes photosensitivity Institute of biochemical physics behalf of N.M. Emanuel Academy of Sciences (IBCP RAS)). After incubation, the viral preparation is subjected to a final purification by the method of cation exchange chromatography on Sepharose 6 Fast Flow at a pH of 7.2; purified protein elute from the column buffer solution with a content of 10 mm Tris-HCl and 100 MM NaCl.

Example 5. Analysis of proteins from primary and preprocessed viral drugs homogenate using 20-electrophoresis.

To study the protein composition of the preparations obtained before and after O-deglycosylation glycoproteins on the mechanism of β-elimination and subsequent tagging deglycosylated proteins cyanine dye JC-1, using 2D-electrophoresis according to O Farrell. For this purpose, aliquots of the chromatographic fractions with a volume of 500 µl, with a total protein content of about 100 mcg, precipitated by adding 100% acetone (1:1) for 30 min at +4°C and subsequent centrifugation in a tabletop centrifuge at 14000 rpm for 15 minutes the precipitate absoluut, washed 1 time with 100 μl of 50% aqueous acetone, and solubilizers, Progresa at 99°C for 10 min in 50 ál of buffer on the basis of 9 M urea containing as a reductant 1 M thiourea, to denature the samples.

Received the denatured sample is centrifuged in a tabletop centrifuge at maximum speed for 10 min, after which the clarified supernatant is used for isfocusable in the tube. Fractionation in the first direction is isoelectrofocusing (IEF) in glass tubes (2,4×180 mm)filled with 4% SDS page, cooked on the 9M urea solution containing 2% Triton X-100 and 2% of socialfollow (pH 5-7, 5-8 and 3.5-10 in a ratio of 4/1). Protein extract (100 to 150 µl) was applied on the acid edge of the gel and spend IEF device company BioRad (USA), before reaching 2400/hour total for each column of the page. Then column page with separated during IEF protein is used as the starting zone of the fractionation in the second direction, which is carried out by electrophoresis in slabs PAG (200×200×1 mm) with a linear concentration gradient of acrylamide 7.5 to 25% and in the presence of 0.1% SDS to the device for vertical electrophoresis firm Helicon (Russia). With the edges of each plate to form a pocket for the application of protein markers. For visualization of proteins plates PAG paint silver nitrate using a set of "PageSilver™ Silver Staining Kit according to the manufacturer's instructions ("hrm Fisher Scientific Inc., USA).

Gel documentation obtained two-dimensional electrophoregrams carried out by scanning at scanner Epson expression 1680" or by using a digital camera the Panasonic Lumix DMC-GF2C. Overlay the gel-documented images obtained after conducting electrophoretic analysis of the pattern of primary deglycosylated proteins (Figure 5 (1, 4)) and the pattern of all proteins (Figure 5 (2, 5)) allows to identify possible pattern originally deglycosylation and covalently modified deglycosylated proteins without confirmation iiproperty artifactory shown on plate PAG bands, presumably it is appropriate deglycosylation the glycoproteins.

Example 4. Identification pattern deglycosylation and deglycosylated proteins purified from the preprocessed homogenate of cells infected by a model strain of tick-borne encephalitis virus EK-328 or variant of M, obtained from strain EK-328.

To uniquely identify a pattern originally deglycosylation and deglycosylated proteins from the preprocessed drug viral homogenate after conducting electrophoretic analysis of all proteins and staining the gel with silver nitrate conduct secondary staining modified dye deglycosylation glycoproteins. For this painted nitrate silver plate PAG placed in the camera for horizontal electrophoresis multiSUB MAXI ("leaver Scientific Ltd., UK, www.cleaverscientific.com)add 1200 ml of 10-5M cyanine dye JC-1 in a binary solution of acetone-Tris-Hcl (C=0.05 M, pH=8,5), also containing pre-dissolved surfactant, pyridinium bromide at a concentration of 10-3M, adding which leads to the formation of a shell of surfactant molecules around the J-aggregates, leading to a more efficient structuring, and incubated for 10 min at room temperature. After incubation, the solution for staining drained, gel prom is provide cooled to +4°C PBS solution and document the formation of molecular chains of J-aggregates on the basis of the covalently-modified dye deglycosylation glycoproteins using transilluminator blue color with light amber color Safe Imager™ 2.0 (Life Technologies, USA) or UVIblue ("Uvitec, Cambridge, UK) and digital camera Panasonic Lumix DMC-GF2C in a dark room (Figure 5 (3, 6)).

Overlay the gel-documented images obtained after conducting electrophoretic analysis of the pattern of primary deglycosylated proteins (Figure 5 (1, 4)) and the pattern of all proteins (Figure 5 (2, 5)) after staining the gel with silver nitrate, as well as the pattern of the modified dye deglycosylation glycoproteins after secondary staining (Figure 5 (3, 6)), which leads to the formation of J-aggregates can uniquely identify a pattern originally deglycosylation and covalently modified deglycosylated proteins.

List of used sources

1. Gibbs B.F. Complete chemical and enzymatic treatment of phosphorylated and glycosylated proteins on protein chip arrays. Priority date 11.05.2005. Patent US 2006269980 (A1).

2. Bynum M.A., Grimm R., K.P. Killeen, Robotti C.M. Microfluidic Glycan Analysis. Priority date 29.01.2009. Patent US 20100190146 (A1).

3. Vinogradov A.A., Yamskov I.A. Deglycosylation glycoproteins. Bioorganic chemistry. - 1998. - So 24. No. 11. - S-815.

4. Edge A.S. Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function. - 2003. Biochemical Journal. - V. 376. - P. 339-350.

5. Ingham K.C., Brew S.A. Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and alpha 1-acid glycoprotein. Biochimica et Biophysica Acta. - 1981.- V. 670. No. 2. - P. 181-189.

6. De Bank P.., Kellam Century, Kendall D.A., K.M. Shakesheff Surface engineering of living myoblasts via selective periodate oxidation. Biotechnology and Bioengineering. - 2003. - V. 81. No. 7. - P. 800-808.

7. Shilova N.V., O.E. Galanina, Rubina A.Y., Butvilovskaya V.I., Huflejt M.E., Chambers J., Roucoux, A., Bovin N.V. 2-Aminopyridine-a label for bridging of oligosaccharides HPLC profiling and glycoarray printing. Glycoconjugate Journal. - 2008. - V. 25. No. 1. - P. 11-14.

8. Haugland R.P., Steinberg T.N., Patton W.P., Zhenjun D. Reagents for labeling biomolecules having aldehyde or ketone moieties. Priority date 02.10.2000. Patent application WO 0228841 A2.

9. Sinkeldam R.W., Greco N.J., Tor Y. Fluorescent analogs of biomolecular building blocks: design, properties, and applications. Chemical Reviews. - 2010. - V. 110. No. 5. - P. 2579-2619.

10. Wells L., K. Vosseller, Cole R.N., Cronshaw J.M., M.J. Matunis, Hart G.W. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Molecular & Cellular Proteomics. - 2002. - V. 1. No. 10. - P. 791-804.

11. Murrey H.E., Hsieh-Wilson L.C. The chemical neurobiology of carbohydrates. Chemical Reviews. - 2008. - V. 108. No. 5. - P. 1708-1731.

12. Sorokin A.V. Optical properties and structure of J-aggregates of dye JC-1 in solutions. Biophysical journal. - 2008. - So 21. No. 2. - S-121.

13. Slavnova T.D., Gorner N., A.K. Chibisov J-aggregation of anionic ethyl meso-thiacarbocyanine dyes induced by binding to proteins. Journal of Physical Chemistry. - 2007. - V. 111. No. 33. - P. 10023-10031.

14. Yao H., Isohashi T., Kimura K. Electrolyte-induced mesoscopic aggregation of thiacarbocyanine dye in aqueous solution: counterion size specificity. Journal of Physical Chemistry. - 2007. - V. 111. No. 25. - P. 7176-7183.

15. Reers, M., Smith T.W., Chen L.B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry. - 1991. - V. 30. - No. 18. - P. 4480-4486.

16. Yamaguchi A., N. Kometani, Y. Yonezawa Luminescence properties of the mixed J-aggregate of oxacyanine dye and thiacyanine dye. Formation of a persistence-type aggegate. Journal of Physical Chemistry. - 2005. - V. 109. No. 4. - P. 1408-1414.

17. Achyuthan K.E., Lu L., G.P. Lopez, D.G. Whitten Supramolecular photochemical self-assemblies for fluorescence "turn on" and "turn off assays for chem-bio-helices. Photochemical &Photobiological Sciences. - 2006.- V. 5. No. 10.- P. 931-937.

The method of detection of O-glycosylated proteins of cell homogenates prepared to proteomics and phosphoproteome analysis by two-dimensional electrophoresis followed by identification of spots spectroscopy MALDI-TOF or phosphoproteomic, characterized in that thoroughly desalted by gel-chromatography or dialysis cell homogenates are deglycosylation on the principle of β-elimination in a solution of 0.05 M NaOH containing 38 mg/ml NaBH4,for 16 hours at 45°C followed by the addition of cyanine dye JC-1 at a concentration of 10-6M and incubation for 15 min at room temperature, concentrated by precipitation with 50% acetone, subjected to two-dimensional electrophoresis with the formation of electrophoregram, analyzed by fluorescence when exposed to UV-transilluminator blue color with light amber color, visually manifested in the form of glowing in the dark bands, which can be extracted from the gel and used for proteomic or phosphoproteome analysis, additional analysis of the intensity and location of extractable by the OS can be performed by comparing the painted silver nitrate electrophoregram homogenates before and after the procedure deglycosylation.



 

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SUBSTANCE: method for the prediction of the length of abscess formation accompanying a sequestration phase of acute pancreatitis involves the DNA recovery from peripheral venous blood, the polymorphism analysis of +250 A/G Ltα, and if observing the genotypes +250 GG or +250 AG Ltα, a risk of early abscess formation accompanying the sequestration phase of acute pancreatitis is predicted.

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1 tbl, 2 dwg

FIELD: medicine.

SUBSTANCE: predicting in vitro an ability of a cartilage cell population to produce a stable hyaline cartilage in vivo of the cell population ensures determining an expression of a number of marker genes comprising a positive marker FRZB (Frizzled-like protein I), a negative marker ALK1 (Activine A Receptor, Type II-Like Kinase) and one or more markers specified in a group consisting of a negative marker PEDF (Pigment epithelium derived factor), a positive marker COL11 (Collagen, Type XI AI), a positive marker COL2 (Collagen, Type II. Alpha I) and a positive marker FGFR3 (Fibroblast growth factor receptor 3). The expression levels of each specific marker are presented by a numerical value. The numerical values are combined into a cumulative value, wherein the cumulative value shows an ability to produce the stable cartilage.

EFFECT: effective prediction of the ability of the cartilage cell population to produce the stable hyaline cartilage in vivo.

6 cl, 4 dwg, 11 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: there are performed two rounds of a polymerase chain reaction with the primers B37 - B853r and B123 - B320r to detect a fragment of a segment B coding a RNA-dependent RNA-polymerase of a pancreatic infectious necrosis virus that is followed by an electrophoretic measurement of a size of an amplified fragment of a nucleotide sequence. If the electrophoregram shows the fragments of the length of 860 and/or 240 pn in the analysed sample, the pancreatic infectious necrosis virus is diagnosed in salmon.

EFFECT: invention is applicable for the diagnostic purposes in scientific research institutions, veterinary laboratories, and fish farms.

2 dwg, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood; polymorphism of the tumour necrosis factor α gene -308G/A is analysed, and if observing the allele -308A TNFα, a high risk of the development of angiopathy of the lower extremities in the patients suffering type 2 diabetes mellitus is predicted.

EFFECT: obtaining the new criteria for assessing the risk of the development of diabetic angiopathy of the lower extremities in the patients suffering type 2 diabetes mellitus.

1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood; polymorphism of a tumour necrosis factor α gene is analysed, and if observing the genotypes -308 AA or -308 GA TNFα, a risk of the late onset of a reactive phase of acute pancreatitis is predicted.

EFFECT: obtaining the new criteria for assessing the time of the reactive phase of acute pancreatitis.

2 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention represents a method for assessing the water resistance of an antiperspirant, involving: a) sampling participants; b) fulfilling the requirement not to use any products or using the products containing no antiperspirant in the axillary region for a specific period of time; c) cleansing the axillary regions of each participant, d) applying a required amount of the antiperspirant product on one axillary region and a placebo product on the other axillary region of each participant; e) performing the stage d) until the required number of applications is completed provided more than one application is preferred; f) the last application is followed by a water test comprising a rotary motion of the participants in the swimming pool and/or swimming for a period of time of the activity in the swimming pool at a depth adequate to wet the axillary regions; g) performing a perspiration test; and h) stating if the antiperspirant shows at least the standard antiperspirant activity.

EFFECT: method improvement.

9 cl

FIELD: biotechnologies.

SUBSTANCE: invention represents method for revealing c.-53-2A>G mutation in SLC26A5 gene accompanied with the development of non-syndrome-based autsomal-recessive deafness. Method includes DNA separation from lymphocytes of peripheral blood by method of phenolic-chloroform extraction. There performed is a polymerase chain reaction with the possibility to perform fluorescence analysis at final point. Two areas of SLC26A5 gene are simultaneously amplificated in mixture of two pairs of sequences of oligonucleotides with fluorescent mark: CACCACAAAGAAGAGATG, TCAGCATGATCCATAGTAC, FAM- agtgtCacTagGggaaaa-BHQ-1, VIC-agtgtCacCagGggaaaa-BHQ-2, flanking the area with possible mutation of c.-53-2A>G in SLC26A5 gene.

EFFECT: invention allows obtaining accurate, objective clinical diagnosis of genetic autsomal-recessive deafness.

1 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: blood serum of the younger patient suffering chronic prostatitis is examined for total testosterone, sex hormone-binding globulin to calculate a free testosterone index; high-density lipoproteins and triacylglycerides are determined, and an atherogenic index is calculated by formula. If the atherogenic index is <3.7, a high risk of the early development of atherosclerosis is predicted.

EFFECT: using the declared method enables the more accurate prediction of the early development of atherosclerosis in the patients with chronic prostatitis.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: what is presented is a diagnostic technique for patient's disposition to Stargardt macular degeneration. Patient's skin fibroblasts are cultured and prepared by viral constructs carrying the genes Oct4, Sox2 and Klf4 under the control of CMV promotor. That is followed by the targeted fibroblast differentiation into retinal cells. Coding RNA of the gene ABCA4 is recovered from the retinal cells. If observing the delection in exon 39-41 of the gene ABCA4, patient's disposition to Stargardt macular degeneration is diagnosed.

EFFECT: invention provides the effective diagnosing of patient's disposition to Stargardt macular degeneration.

3 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to assessing the functional status of the endothelium (FSE) of experimental animals after the reconstructive surgeries of an abdominal aorta. Substance of the method consists in the fact that the FSE is assessed by means of the initial analysis of biochemical blood assay, including nitrogen oxide (II), superoxide dismutase (SOD), malondialdehyde (MDA); that is followed by abdominal aorta alloplasty; the biochemical control is performed 6 month later; the animals are removed from the experiment; the histological examination of the aorta alloplasty region is assisted by the computed morphometry, and the correlation is calculated. If observing an increase of NO metabolites, the development of intimal thickening is considered to be more manifested that it does not excess the physiological values specific for the arterial wall recovery following the surgical management, while a decrease of NO metabolites shows the development of intimal hyperplasia; an increase of SOD activity and MDA level provides stating the active development of arterial intimal thickening.

EFFECT: more effective FSE assessment in the animals after the reconstructive surgeries of the abdominal aorta.

3 tbl, 5 dwg, 1 ex

Anti-mif antibodies // 2509777

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a detection method of proteins in amyloid state, in which a specimen of lysate of yeast culture or tissue of a mammal is obtained, ionic detergent is added to the specimen, proteins are concentrated in an amyloid shape on a cellulose acetate membrane, and they are detected by means of aptomeres, their conjugates or antibodies specific to amyloid shape of proteins. Besides, a set for detection of proteins in amyloid state is proposed.

EFFECT: invention can be used in medicine for diagnostics of amyloid diseases.

9 cl, 6 dwg, 7 ex

FIELD: biotechnologies.

SUBSTANCE: method involves preparation of a solution for dilution of serum, preparation of serum of donors for negative control with further dilution by means of a dilution solution; besides, serum does not include antibodies to toxoplasma, cytomegalovirus, viruses of herpes simplex and rubella, HBsAg, antibodies to HIV-1,2, to hepatitis C virus, preparation of serum of donors for positive control with its further dilution with a dilution solution; besides, serum includes antibodies to toxoplasma, cytomegalovirus, viruses of herpes simplex and rubella; introduction of liquid control specimens to pits of a board, lyophilic drying of positive and negative control, marking of positive control in red, and negative control in green.

EFFECT: invention allows obtaining general-purpose control specimens for all test systems for determination of an infection of TORCH group, which are incorporated in pits of a working board, which are user-friendly and safe.

1 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely gynaecology and may be used for individual selection of the preparations containing the probiotic lactic bacterial strains for effective intravaginal therapy. For this purpose, vaginal epithelial cells are recovered from the patient, released from the accompanying microflora. That is followed by preparing an epithelial cell suspension in a culture medium, and mixed with a suspension of thermally-activated probiotic lactic bacterial strains. Then, the suspension is incubated; the epithelial cell culture fluid filtrate is prepared and added to the suspension of the tested probiotic lactic bacterial strains in ratio 1:7. Concurrently, a reference of the mixture of the epithelial cell culture medium and the suspension of the tested probiotic lactic bacterial strains in ratio 1:7 is prepared. The test and reference samples are incubated, measured for optical density; and a degree of biomass increase in the test sample is related to that in the reference. The preparation containing the probiotic lactic bacterial strains the biomass increase of which under the influence of patient's vaginal epithelial cells is stimulated most is selected form the effective intravaginal therapy.

EFFECT: invention enables the individual selection of the preparations containing the probiotic lactic bacterial strains for the effective intravaginal therapy.

3 ex, 1 tbl, 2 dwg

FIELD: chemistry.

SUBSTANCE: group of inventions relates to composition of an analysing sensor reagent, which is adapted to facilitate determination of concentration of an analyte in a liquid sample, methods of determining concentration of an analyte in a liquid sample and a method of depositing the composition of the analysing sensor reagent onto a substrate by screen printing. The composition of the analysing sensor reagent contains 1-4.0 wt % glucose oxidase enzyme, 15-20 wt % ferricyanide mediator, 3.6-6.0 wt % hydroxyethyl cellulose polymer and 0.2-1.6 wt % smectite clay. The method of determining concentration of an analyte in a liquid sample involves making an electrochemical analysing sensor which consists of counter electrode and a working electrode, a region for receiving liquid and said analysing sensor reagent, and determining concentration of the analyte in less 35 s. Another method of determining concentration of an analyte in a liquid sample involves pricking the finger of the human subject in order to collect a liquid sample, placing the liquid sample containing one analyte into the analysing sensor, bringing the liquid sample into contact with the analysing sensor reagent and determining concentration of the analyte in the liquid sample. The method of depositing said composition of an analysing sensor reagent onto a substrate by screen printing involves making a mesh screen consisting of a first part with a light-sensitive emulsion and a second part without a light-sensitive emulsion; feeding the analysing sensor reagent onto the mesh screen, and bringing the reagent into contact with the substrate through the second part of the mesh screen.

EFFECT: high sensor stability, easy application of the reagent, shorter overall duration of analysis and high adhesion of the reagent to the substrate.

14 cl, 11 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: method of cultivation of microalgae for biofuel purpose comprises two stages of algolisation. At the first stage the algolisation is carried out by primary inoculum of the culture, preferably Chlorella vulgaris BIN, obtained in the photobioreactor synchronously or time-shifted, of multifunctional indoor pools with translucent railing. The total volume of the said pools is from 1/30 to 1/15 of the total amount of open water reservoirs. The secondary inoculum is grown with a bulk density of 109-1011 cells/l, at that the cultivation is started in spring with an average daily temperature of water in the pools in the range of 12-18°C. The second stage of cultivation the microalgae is started by selection from the pools of secondary inoculum at a temperature of water in the open water reservoirs of 12-18°C, and continue its supply to the open water reservoirs to reach in them of the bulk density of microalgae 2·108-109. The secondary inoculum is partially taken from the pool as a finished product in spring and autumn with the water temperature in them is in the range of 8-12°C, at that in multifunctional pools the equal amount of water with dissolved biogens is added. The resulting microalgae contain 33.9 % cellulose, 51.0 % proteins, 7.3% fats.

EFFECT: increased productivity of microalgae cultivation method.

3 cl, 1 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: claimed is method of determining analite concentration in sample, which contains: application of input signal to sample, with input signal containing, at least, 3 working cycles within 10 seconds, with each working cycle including excitation impulse and relaxation; measurement of output signal, sensitive to measured substance, within 300 milliseconds from the beginning of excitation impulse of, at least, one working cycle; and determination of analite concentration in sample in response to measured output signal. Also described are portable measuring device and biosensor system for determination of analite concentration in sample, as well as method of reducing systematic error in determination of analite concentration in sample.

EFFECT: increased accuracy and reliability of analysis.

64 cl, 2 tbl, 6 dwg

FIELD: agriculture.

SUBSTANCE: method includes geodetic measurements of the land plot area, three-dimensional measurement of the land plot, based on the measurement of the coordinate component of the resource parameters in different parts of this plot. Resource soil parameters of land plot are determined for each time period of operation taking into account the discrete disposal of part of the resources that were available at the beginning of the measurement period. In determining the resource parameters of the soil its biological activity is additionally measured on the stream of direct solar radiation reaching the horizontal surface of the soil.

EFFECT: method enables to improve the accuracy of measurement the resource parameters of the particular land plot.

1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: method provides sampling of the material under study. Inoculation of the material under study on nutrient medium sabouraud followed by incubation on this medium at 37°C for 24 hours. A quantitative assessment of growth of yeast-like fungi of the genus Candida, and with a value exceeding 10*1 CFU/swab the candidiasis of upper respiratory tract is diagnosed. The obtained colonies are examined under the microscope and the isolated colonies of yeast-like fungi of the genus Candida are separated from these colonies. The isolated colonies of yeast-like fungi of the genus Candida are suspended in five test tubes containing liquid medium sabouraud with the phenol red indicator, where the discs with carbohydrates are added, and in the first test tube - a disc containing maltose, in the second - sucrose, the third - lactose, the fourth-galactose, the fifth - trehalose, and are incubated at 37°C for 24 hours, followed by assessment of colour change of the indicator. The colour change of the indicator to yellow is taken as one, lack of colour change is taken as zero, the sum of the values obtained in assessment of the indicator colour in five test tubes is calculated. If the value of the sum is 0 the candidiasis of upper respiratory tract caused by Candida kruzei is diagnosed, if it is 1 - the candidiasis of the upper respiratory tract caused by Candida glabrata is diagnosed, if it is 3 - the candidiasis of the upper respiratory tract caused by Candida albicans is diagnosed, if it is 4 - the candidiasis of the upper respiratory tract caused by Candida tropicalis is diagnosed, if it is 5 - the rest.

EFFECT: invention enables to identify several types of yeast-like fungi of the genus Candida, causing candidiasis, and enables to take into account the quantitative growth of yeast-like fungi of the genus Candida, according to which the presence of candidiasis is judged.

6 ex

FIELD: biotechnology.

SUBSTANCE: as an object for indirect estimation apparently healthy leaves of the plant Peireskia aculeate are used, on which sterile surface the notches are made, the infecting doses of the strains Burkholderia pseudomallei and Burkholderia mallei under study in different concentrations are applied, placed on a wet swab and incubated at 32°C for 24-48 h. The extent of their cytopathogenicity is evaluated visually according to the damage to the leaf blade - maceration, ulceration and blackening. Simultaneously the level of virulence of strains of pathogens for laboratory animals is determined, and the extent of cytopathogenicity for P. aculeata is correlated with virulence for laboratory animals.

EFFECT: invention provides high sensitivity, reproducibility, versatility, ease and accessibility of the study.

5 tbl, 4 ex

FIELD: organic chemistry, medicine, oncology.

SUBSTANCE: invention relates to new macrocyclic compounds of the formula: , wherein A, D, D1, G, Z, Z1, E, X, T, Q, Y, Y1, J, J1, U, U1 and n have values given in the description. Also, invention relates to a method for identification of agents that induce the continuous mitotic block in cell after temporary exposition of cell with this agent. Compounds elicit anticancer and antimitotic (mitosis-blocking) activity. Invention provides preparing new macrocyclic compounds eliciting strong anticancer effect.

EFFECT: improved identifying method, valuable medicinal properties of compounds.

21 cl, 2 tbl, 501 ex

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