KR102370468B1 - A composition and a method for removal of residual anionic surfactant, protein-detergent complexes, and micelles, in biological tissues - Google Patents

Abstract

The present invention relates to an anionic surfactant remaining in a biological sample, a byproduct of the surfactant, and/or a composition for removing micelles, and uses thereof. The present invention is a novel cleaning composition capable of removing micelles made of anionic surfactants, proteins or biopolymers bound to surfactants, and/or surfactants remaining in biological samples from which lipids are removed by treatment with anionic surfactants. can be used for development.

Description

A composition, method, and use thereof for removing anionic surfactant remaining in a biological sample, a lipid or protein conjugate bound thereto, and micelles {A composition and a method for removal of residual anionic surfactant, protein-detergent complexes, and micelles, in biological tissues}

The present invention relates to an anionic surfactant remaining in a biological sample, a byproduct of the surfactant, and/or a composition for removing micelles, and uses thereof.

Anionic surfactants, including sodium dodecyl sulfate (SDS) or sodium lauryl sulfate, have a hydrophilic region as a polar anionic sulfate group at one end, and a non-polar straight chain at the other end. It is composed of hydrophobicity in the form of a straight chain. When anionic surfactants are treated in living tissues, the anionic surfactants replicate the hydrophobic structure of the cell membrane, penetrate the membrane, and combine with the membrane phospholipid ichthyosis to form micelles with a single layer. . Accordingly, anionic surfactants are generally used as a method of dissolving proteins and lipids from cell membranes.

As a representative method of separating lipids using anionic surfactants, there is a method of making tissues transparent using electrophoresis. In order to make the entire tissue transparent, the existing method removes the fat present in the tissue using an organic solvent. In this method, the structural support of the tissue is lost due to some fat removed, and biopolymers such as proteins and nucleic acids may be lost. So, first, a hydrogel is put into the biological tissue to form a tissue-hydrating gel complex, and then, sodium dodecyl sulfate, one of the anionic surfactants, is treated to remove only the saturated fat that is not bound to the hydrogel complex. Through this method, it is possible to make tissues transparent while preserving biopolymers such as proteins and nucleic acids. The same is true for proteomic identification studies based on mass spectrometry. In order to separate and fractionate a sample from a living tissue, an anionic surfactant must be used to remove the lipid present in the tissue to form a peptide, which can be used to separate and ionize the peptide using chromatograph.

However, micelles made of sodium dodecyl sulfate or sodium lauryl sulfate, which are not bound to biopolymers, etc., remain in the living tissue without being washed with water or buffer due to the complex structure of the living tissue. In addition, the surfactant remaining in the living tissue interferes with the binding of the antibody with the molecular probe to the antigen protein, which is an artifact in the protein analysis using antigen-antibody fluorescence analysis, antigen-antibody electrophoresis analysis, or mass spectrometry used in protein analysis. causes

Various technologies have been developed to remove anionic surfactants remaining in living tissues, proteins or biopolymers bound to surfactants, and micelles made of surfactants. Prolonged dialysis, anion exchange chromatography, filter aided sample preparation (filter aided sample preparation) FASP), high salt precipitation kits, and SDS specific binding spin cartridges. Although these methods can be used for protein purification by mass spectrometry, they are time consuming, and are not suitable for use in biological samples containing extracorporeal tissues, organs, or parts thereof.

Alternatively, a solution containing a cationic material such as acetone (acetone and trichloroacetic acid (TCA)), ammonium acetate, ammonium sulfate, etc., which is already used in the field of protein purification, is used. However, this material has a strong redox reaction, and when used for protein extraction of a sample extracted by two-dimensional electrophoresis, antigenicity is preserved, but when used in living tissue, antigenicity cannot be preserved. there will be no

In addition, a solution containing Triton X-100, a nonionic surfactant, is used as another method of removing the surfactant remaining in the living tissue. This is because the solution containing Triton X-100 is not only easy to clean, but also has nonionic properties, so it does less damage to biological samples than anionic surfactants such as SDS. However, Triton X-100 is also used for decellularization of whole tissues, and when reacted with ammonium hydroxide, all DNA is removed. In addition, when a solution containing Triton X-100 is treated with anionic surfactants such as sodium dodecyl sulfate, there is a disadvantage that the anionic surfactant does not disappear completely.

Accordingly, the present inventors developed a new cleaning composition that can remove micelles made of anionic surfactants, proteins or biopolymers bound to surfactants, and/or surfactants remaining in biological samples from which lipids are removed by treatment with anionic surfactants Thus, the present invention was completed.

US8507291B1 Detergent removal from protein samples prior to mass spectrometry analysis US7662411B2 Process for removal of solvent and detergent from plasma US4801691A Method for removing sodium dodecyl sulfate from sodium dodecyl sulfate solubilized protein solutions

A.BlankR.H., Sugiyama, Charles A.Dekker, Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels, Analytical Biochemistry, 1982;120:2:267-275 Zhou JY, Dann GP, Shi T, Wang L, et al. Simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics applications. Anal Chem. 2012;84:2862-8. Soundharrajan Ilavenil, Naif Abdullah Al-Dhabi et al., Removal of SDS from biological protein digests for proteomic analysis by mass spectrometry, Proteome Science 2016;14:11

As such, it is an object of the present invention to provide a composition for removing an anionic surfactant from a biological sample, including a tertiary amine as an active ingredient.

Another object of the present invention is to provide a method for removing an anionic surfactant from a biological sample, comprising treating the biological sample containing the anionic surfactant with a composition comprising a tertiary amine.

In order to achieve the above object, the present invention provides a composition for removing anionic surfactants in living tissues, including a solution in which a tertiary amine is added as a main component.

In one embodiment of the present invention, the biological sample may be a biological sample including a tissue, an organ, or a part thereof separated out of the body.

In one embodiment of the present invention, the tertiary amine may be represented by Formula 1.

Formula 1

Formula 1

In the above formula,

R ¹ , R ² and R ³ are each independently an alkyl group, an aryl group or a cycloalkyl group having 1 to 10 carbon atoms; The alkyl group, aryl group, or cycloalkyl group may include -O-, -SO ₂ -, -CO-, -S-, -CF ₂ -, or -OH.

In another embodiment of the present invention related thereto, the tertiary amine is trimethylamine, triethylamine, tripropylamine, 1-methylpiperidine, N, N-dimethylethylamine (N,N-dimethylethylamine), N,N-diethylmethylamine (N,N-diethylmethylamine), N,N-dimethylethanolamine (N,N-dimethylethanolamine), triethanolamine, 2,2',2"-nitrilotriethanol (2,2',2"-nitrilotriethanol), 2,2'-ethyliminodiethanol (2,2'-ethyliminodiethanol), 2-diethylaminoethanol (2- diethylamino ethanol), 2-diisopropylaminoethanol (2-diisopropylaminoethanol), N,N,N',N'-tetrakis (2-hydroxypropyl)ethylenediamine (N,N,N'N'-tetrakis ( 2-hydroxypropyl) ethylenediamine), N,N,N'N'-tetrakis(2-hydroxyethyl)ethylenediamine (N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine), N,N ,N',N',N''-pentakis(2-hydroxypropyl)diethylenetriamine (N,N,N',N',N''-pentakis(2-hydroxypropyl) diethylenetriamine) and their It may be selected from the group consisting of mixtures, but is not limited thereto, and a buffered saline may be provided.

In another embodiment of the present invention, the anionic surfactant is, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate (ALS), sodium lauryl ethylene sulfate (Sodium Lauryl Ethylene Sulfate) ;SLES), Linear Alkylbenzene Sulfonate (LAS), Alpha-olefin Sulfonate (α-OlefinSulfonate; AOS) Alkyl Sulfate (AS), Alkyl Ether Sulfate (AES), and It may be one or more selected from the group consisting of sodium alkane sulfonate (SAS), but is not limited thereto.

In another embodiment of the present invention, the tertiary amine may be included in a concentration of 0.01 to 50% (wt/vol%) in the composition, and a concentration of 1 to 10 w/v% is preferred. In this case, the solution for indicating the concentration may be a simulated body fluid used in a conventional field, and more specifically, water (distilled water), phosphate buffered saline, etc. may be used. However, the present invention is not limited thereto. When the concentration of the compound containing the tertiary amine is less than 1 w/v%, the removal rate of the anionic surfactant and its by-products present in the biological sample may be significantly slowed down, and if it exceeds 10 w/v%, If included, the antigenicity of the biological sample may be lost.

In another embodiment of the present invention, in order to remove the anionic surfactant into the tissue of the biological sample, the compound containing the tertiary amine may be treated to the biological sample, preferably for 1 hour to 24 hours. , but is not limited thereto. In another related embodiment, the processing of the biological sample may be placing on a bench at a low temperature without agitation.

In another embodiment of the present invention, the treatment of the biological sample may be treatment at a temperature below the freezing point of the tertiary amine (25° C.) to the protein modification (60° C.), but is not limited thereto. In addition, the temperature may be carried out in a temperature range of 25 ° C to 60 ° C, may be carried out in a temperature range of 25 to 37 ° C, may be carried out in a range of 37 to 42 ° C, may be carried out in a range of 42 to 60 ° C, and , may be carried out in the range of 32 to 42 ° C, may be carried out in the range of 20 to 40 ° C, may be carried out in the range of 24 to 39 ° C, may be carried out in the range of 28 ° C to 38 ° C, and may be carried out in the range of 30 ° C to 37 ° C It can be carried out in, it can be carried out at 33 to 34 ℃

In another embodiment of the present invention, treating the biological sample with a solution in which a tertiary amine is added as a main component may be immersing the biological sample in a solution in which the tertiary amine is added as a main component.

The method of removing the anionic surfactant from a living tissue of the present invention can quickly and uniformly remove the anionic surfactant and by-products made therefrom, particularly in the deep part of a tissue or organ having a thickness of 0.1 mm or more.

In the present invention, the "biological sample" includes biological tissues, organs, and cells separated in vitro, preferably biological tissues having a thickness of 0.1 mm or more, but in particular, brain, It is preferable to apply to blood vessels, liver, lung, kidney, pancreas, intestine, etc., but is not limited thereto.

The present invention provides a method for detecting important information in the biological sample, ie, DNA, RNA, protein, fluorescent signal, and the like.

A composition related to a washing buffer solution for a biological sample, comprising the tertiary amine of the present invention as an active ingredient, and after lipid removal, anionic surfactant can be removed from a biological sample that is not removed by conventional methods. In particular, it is possible to remove the anionic surfactant present in the biological tissue having a thick thickness from the deep part of the tissue.

In one aspect of the present invention, it may be used as a process after clearing a biological sample including biological tissue or as a pre-stage process before immunochemical staining. Through the present invention, since the three-dimensional distribution of cells and molecules in living tissue can be imaged and observed while maintaining antigenicity, it can be effectively used to obtain various information within the tissue as well as to identify the causes of various diseases related to living tissue. there is.

In another aspect of the present invention, in the protein identification process for mass spectrometry, after removing lipids, more anionic surfactants are removed to remove artifacts from mass spectrometry after peptide treatment.

1 shows a conceptual diagram of a method for removing anionic surfactants remaining in a biological sample by using a washing buffer solution for a biological sample containing the tertiary amine of the present invention as an active ingredient.
Figure 2 shows the whole brain tissue image after washing with a phosphate buffer solution containing 1% tetrakis (2-hydroxypropyl) ethylenediamine and a buffer solution containing 1% Tween 20 in the brain of a mouse that has been tissue cleared; is the result of
3 shows brain tissue of different sizes after tissue transparency is washed using a phosphate buffer solution containing 1% tetrakis (2-hydroxypropyl) ethylenediamine and a phosphate buffer solution containing 1% Tween 20. It is a graph measuring the amount of SDS in one solution.
Figure 4 shows when the cleared biological tissue is washed using a buffer solution containing 0.1%, 1%, 10% tetrakis (2-hydroxypropyl) ethylenediamine and a buffer solution containing 1% triethanolamine. It is a graph measuring the amount of SDS in one solution.
Figure 5 shows that exosomes isolated from the blood of genetically modified mice were treated with 5% SDS and then removed by a general method, including 1%, 5%, and 10% tetrakis (2-hydroxypropyl) ethylenediamine. These are the results regarding the types of proteins found when performing liquid chromatography mass spectrometry after treatment with the buffer solution.

In one aspect of the present invention, there is provided a cleaning solution composition for removing residual anionic surfactants in a biological sample, including a tertiary amine or a hydrate thereof.

The method of removing the anionic surfactant remaining in the biological sample includes washing the biological sample by contacting it with the composition.

Specifically, the anionic surfactant washing method according to the present invention is an anionic surfactant such as sodium dodecyl sulfate remaining in the living tissue by contacting the biological tissue with a composition containing the tertiary amine or its hydrate as an active ingredient, and micelles. It serves to wash so that anionic surfactant by-products such as anionic surfactants can escape from the biological sample.

The method for removing the residual anionic surfactant and byproducts thereof according to the present invention causes a normal antigen-antibody immunochemical reaction in a living tissue. If an anionic surfactant such as sodium dodecyl sulfate (SDS) remains in the biological sample, the remaining anionic surfactant interferes with the antigen-antibody reaction and prevents binding, so it is easy to immunize using the present invention. It can be observed with a fluorescence microscope using a chemical tissue staining method. In addition, since sodium dodecyl sulfate micelles remaining in living tissue create a fluorescence background signal, the fluorescence signal cannot be normally observed unless washed. Through the method of the present invention, it is possible to observe a sample through a normal fluorescence microscope by removing the anionic surfactant and its micelles to the inside even in thick living tissue.

Since the method of removing the residual anionic surfactant and its by-products according to the present invention can preserve most of the protein, the protein information in the sample is not lost or distorted, and various fluorophores are used in the tissue. Detection can be useful.

Hereinafter, the present invention will be described in detail with reference to Preparation Examples, Examples, Comparative Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

production example

Preparation Example 1. Preparation of cleared brain tissue

The transparent brain tissue is prepared as follows. However, all animal experiments in this specification were performed according to the guidelines of the Korea Brain Research Institute's Laboratory Animal Committee. Experimental mice were allowed to freely ingest water under a 12-hour light/dark cycle condition.

First, experimental mice (Korea Brain Research Institute or The Jackson Laboratory) were anesthetized using an inhalation anesthetic, Isoflurane (Isoflurane, Sigma-Aldrich, USA), and a phosphate-buffered saline containing 4% paraformaldehyde (phosphate-buffered saline). Transcardial perfusion fixation was performed using pH 7.4, Thermo Fisher Scientific, USA). The brains of mice were enucleated and then placed at 4°C for 12 hours. Then, the immobilized brain tissue was washed several times using a phosphate buffer solution, and then placed again at 4°C for 24 hours.

Brain immobilized in a solution of 0.5 mL and 50 mL of 25% X-CLARITY™ Polymerization Initiator (#13104, Logos Bios Systems, Korea) and X-CLARITY™ Hydrogel Solution (#13103, Logos Bios Systems, Korea), respectively The tissue was placed and incubated at 4°C for 24 hours. The brain tissue and solution thus treated were put back into the X-CLARITY™ Polymerization System (Logos Biosystems, Korea) and run under the following conditions.

Vacuum (kPa) -90 Temperature (℃) 37 hour (hour) 3

After the execution was completed, the brain tissue in the solution was mixed well for 1 minute, and then the brain tissue was washed using a phosphate buffer solution. The washed brain was again placed in Electrophoretic Tissue Clearing Solution (Cat# C13001, Logos Bios Systems, Korea) and cleared for 6-8 hours using the following protocol.

Current (A) 1.0 to 1.2 Temperature (℃) 37 Pump speed (rpm) 30

Preparation Example 2. Preparation of blood-derived exosomes

To separate plasma from blood collected from experimental mice (Korea Brain Research Institute or The Jackson Laboratory), centrifuge the blood at 400 g for 10 minutes, and then centrifuge the supernatant at 2,000 g for 10 minutes sequentially to prevent cell destruction. prevented. The separated plasma was centrifuged for 15 minutes at 3000 g at room temperature to remove cells remaining in the plasma. To separate exosomes from plasma from which cells and impurities have been removed through centrifugation, 67 µl of exosome separation solution (EXOTC20A-1, System Biosciences, USA) was mixed with 250 µl of plasma and reacted at 4°C for 12 hours. In order to precipitate exosomes from the reaction solution, the reaction solution was centrifuged at 3,000 g for 30 minutes at 4°C, the supernatant was removed, and the supernatant was completely removed by centrifugation once more at 3,000 g for 5 minutes. At this time, the precipitate was mixed with 100 μl of HEPES buffered saline prepared by exosomes and stored at -80°C.

Preparation Example 3. Preparation of liquid chromatography mass spectrometry sample

The prepared sample was reacted in 5% SDS solution at room temperature for 2 hours. Commonly treated with Detergent Removal Spin Column (87777, Pierce, USA). The sample was put into the column included in the kit and allowed to react at room temperature for 2 minutes. After centrifugation at 1,500 g for 2 minutes, 75 μl of 40 mM ammonium bicarbonate and 500 mM DL-Dithiothreitol (DL-Dithiothreitol; D0632, Sigma, USA) were added to a solution obtained through a flow-through treatment process. ) was added and reacted at 56°C for 30 minutes. 8 μl of 500 mM iodineacetamide (Iodoacetamide; I1149, Sigma, USA) was added, and after blocking the light at room temperature for 20 minutes, 4 μl of 500 mM DL-dithiothreitol was added once more. Then, 0.5 μg/μl Trypsin Protease (90058, Pierce, USA) was added to the sample by 2 μl and reacted at 37° C. for one day.

Example

Example 1

Drd-1/Cre;Ai6 transgenic adult mice (8 weeks old, B6;129-Tg(Drd1-cre)120Mxu/Mmjax and B6.Cg-Gt(ROSA)26Sor ^{tm6(CAG-ZsGreen1)Hze} /J cross, The The brain tissue of Jackson Laboratory, USA) was transparently processed as in Preparation Example 1. After clearing, the brain tissue was placed in a phosphate buffer containing 1% tetrakis(2-hydroxypropyl)ethylenediamine and washed for 8 hours. In order to efficiently wash, it was performed using a rocker having a tilt motion of 30° and a speed of 100 rpm. The brain tissue after washing was placed in X-CLARITY™ Mounting Solution (Cat# C13101, Logos Bios Systems, Korea) at room temperature for 1 hour. For brain tissue, a three-dimensional whole brain tissue image was acquired using a planar laser fluorescence microscope (Lightsheet Z.1, Carl Zeiss, Germany). Brain tissue was placed in the prepared frame and a 1% low melting agarose (Ararose Low Melt 100g, Carl Roth GmbH, Germany) solution set at 37°C was poured. Before gelation, air bubbles around the brain tissue were removed using a brush. After gelation, the brain tissue embedded in the gel was removed from the mold and placed in a pre-prepared mixed solution of urea and sucrose at 4° C. for 12 hours. The brain tissue contained in the gel was fixed in a plane laser fluorescence microscope frame, and a three-dimensional whole brain tissue image was acquired.

Example 2

Brain tissues of normal adult mice (C57BL/6, Korea Brain Research Institute) having various age groups (1 to 12 months) were transparently treated as in Preparation Example 1. After clearing, the brain tissue was placed in a phosphate buffer containing 1% tetrakis(2-hydroxypropyl)ethylenediamine and washed for 8 hours. For the washed solution, the amount of SDS present in the solution was measured using the SDS Detection & Estimation Reagent Kit (786-129, G-Biosciences, USA). Put 2 ml blue dye (included in the kit, G-Biosciences, USA) and 1 ml dye extraction buffer (included in the kit, G-Biosciences, USA) into a 15 ml centrifuge tube, and 5 μl of the washed solution was placed in a 15 ml centrifuge tube and thoroughly mixed using votex for 30 seconds. To this mixed solution, 2 ml of chloroform was added and left at room temperature for 5 minutes. After processing the solution in which 6 brain tissues were washed, it was transferred to a 96-well dish. The optical density of the chloroform layer was measured at 600 nm using a spectrometer (Flex Station3, Molecular Devices, USA). For normalization of the measured values, 1% tetrakis (2-hyd Only the phosphate buffer solution containing hydroxypropyl)ethylenediamine was measured in the same way using the SDS Detection & Estimation Reagent Kit, and the average value (n=3) was calculated and subtracted from the measured value.

Example 3

The brain tissue of a normal adult mouse (8 weeks old, C57BL/6, Korea Brain Research Institute) was transparently treated as in Preparation Example 1. The experiment was carried out by combining two brain tissues extracted from mice of the same number of weeks each. After clearing, the brain tissue was placed in a phosphate buffer solution containing tetrakis (2-hydroxypropyl) ethylenediamine and triethanolamine (90279, Sigma-Aldrich, USA) and washed for 8 hours. Tetrakis(2-hydroxypropyl)ethylenediamine was treated with 0.1%, 1%, and 10%. Triethanolamine was treated with 1%. The method of measuring the amount of SDS present in the washed solution is the same as in Example 2.

Example 4

The extraction process of exosomes from the blood of mice (8 weeks old, C57BL/6, Korea Brain Research Institute) was the same as in Preparation Example 2. The extracted exosomes were reacted in 5% SDS solution at room temperature for 2 hours. In the exosomes of the experimental group, the final concentrations of 40 mM ammonium bicarbonate (A6141, Sigma-Aldrich, USA) and tetrakis (2-hydroxypropyl) ethylenediamine were 5% and 10%, and The reaction was carried out for minutes. In the control exosomes, only 40 mM ammonium bicarbonate was added and reacted at 37° C. for 30 minutes. Thereafter, the pretreatment process for liquid chromatography mass spectrometry was the same as in Preparation Example 3. After pre-treatment was completed, the samples of the experimental group and control group were evaporated using a high-speed vacuum concentrator (SpeedVac; SPD1010, Thermo, USA), and then desalted of proteins/peptides present in the sample using C18 Spin Columns (898709, Pierce, USA). (protein/peptide desalting) was performed.

The re-extracted experimental and control samples were evaporated using a high-speed vacuum concentrator and then re-dissolved in 0.1% formic acid. Q-exactive plus hybrid quadrupole orbit-trap mass spectrometer combined with nano-electrospray ion source for the trypsin digest of each sample liquid chromatography mass spectrometer (Ulimate 300 / Q Exactive plus, Thermo, USA) was analyzed with Peptides were loaded from an RS auto-sampler, and a 30% aqueous solution of acetonitrile containing 0.1% formic acid was separated by a linear gradient at a flow rate of 300 nl/min. The LC eluent was electrosprayed from the analytical column and a voltage of 2.0 kV was applied through the liquid contact of the nanospray source. The peptide mixture was separated using acetonitrile with a gradient of 5% to 40% for 40 minutes. The assay consisted of a full MS scan ranging from 350 to 2000 m/z, and data-dependent MS/MS (MS2) on the 10 strongest ions from the full MS scan. The mass spectrometer was programmed to acquire in a data-dependent mode. Basic calibration of the mass spectrometer's instrument was performed with the proposed calibration solution according to the manufacturer's instructions. The generated data were analyzed with Proteome Discoverer v2.4 (Thermo, USA). The spectral data were retrieved against the mouse Uniprot database (release 2019_11). The analysis flow used at this time has four nodes: Spectrum Files (data input), Spectrum Selector (spectrum and feature retrieval), Sequest HT (sequence database search), and Percolator (peptide spectral match or PSM Validation and FDR analysis) ) were included. All identified proteins had a false discovery rate (FDR) of < 1% calculated at the peptide level. The evaluation was based on the q-value. The search parameters were methionine oxidation as dynamic modification and methylthio-modification of cysteine as fixed modification together with tryptic specificity up to a maximum of 2 missed cleavages. . The mass search parameters for +1, +2, and +3 ions included mass error tolerances of 10 ppm for precursor ions and 0.02 Da for fragment ions.

Proteome Discoverer Database UniProt Fasta file (Mus musculus) Mass tolerance Precursor (10 ppm), Fragment (0.02 Da)

comparative example

Comparative Example 1

For the brain tissue of the same genetically modified adult mouse of Example 1, the clearing process was the same as in Preparation Example 1. After clearing, the brain tissue was placed in a phosphate buffer solution containing 1% Tween 20 and washed for 8 hours. The brain tissue after washing was observed using a flat-panel laser fluorescence microscope in the same manner as in Example 1.

Comparative Example 2

The brain tissue of a normal adult mouse (8 weeks old, C57BL/6, Korea Brain Research Institute) having the same number of weeks as in Example 2 was transparently treated as in Preparation Example 1. After clearing, the brain tissue was placed in a phosphate buffer solution containing 1% Tween 20 and washed for 8 hours. A method for measuring the amount of SDS present in the washed solution was the same as in Example 2. For normalization of the measured values, only the phosphate buffer solution containing 1% Tween 20 was measured in the same way using the SDS Detection & Estimation Reagent Kit, and the average value (n=3) was calculated and subtracted from the measured value.

Experimental example

Experimental Example 1

A fluorescence microscope is a microscope for observing a fluorescence signal emitted from a sample when a light source is applied to a sample having a fluorescent dye. If the sample itself has inherent fluorescence or if there is a material that can absorb the fluorescence property, the sample generates a stronger fluorescence signal than its surroundings, so it is possible to determine the state or location of a specific material in the sample. However, when sodium dodecyl sulfate micelles (SDS micelles) are present in the sample, a background signal is created in the fluorescence microscope, making it impossible to observe fluorescence in the sample. Since the size of a general SDS micelle is formed with a diameter of 3.5 to 4 nm, when short-wavelength light of 400 to 700 nm, the light source of a general fluorescence microscope, reaches the fluorescence in the sample, it interferes to create a background signal, and normal fluorescence It prevents the image from being created.

Example 1 and Comparative Example 1 are Drd-1/Cre;Ai6 transgenic adult mice having a green fluorescence source (8 weeks old, B6;129-Tg(Drd1-cre)120Mxu/Mmjax and B6.Cg-Gt(ROSA) ) 26Sor ^{tm6(CAG-ZsGreen1)Hze} /J cross, The Jackson Laboratory, USA) was used. As can be seen from the results of Comparative Example 1, when a general washing method was used, while a green fluorescent signal was seen in other brain tissues, the signal disappeared in the deep brain tissue and appeared black. On the contrary, in the result of Example 1, a green fluorescence signal was seen in all regions including the deep part of the brain tissue. As shown in Figure 2, it shows that the hydrate containing tertiary amine as a main component can wash SDS and its by-products deep into the brain tissue.

Experimental Example 2

When the SDS remaining in the living tissue is removed, the removed SDS remains in the washing solution. By comparing the amount of SDS present in the washing solution, it can be confirmed how much SDS is removed by the solution containing the tertiary amine as a main component compared to the general washing method. Since mice of the same age (C57BL/6, Korea Brain Research Institute) also have a similar volume of brain tissue, two brain tissues of the same age were bundled and divided into A, B, C, D, E, F, and compared with Example 2 A comparison was made as in Example 2. As can be seen from the results of Figure 3, it can be seen that the amount of SDS of Example 2 is greater than that of Comparative Example 2 in brain tissues of all weeks. It can be seen that when a composition containing a tertiary amine as a main component is treated, the amount of SDS remaining in the living tissue is washed more.

Experimental Example 3

When a solution containing other tertiary amines as a major component was treated on a biological sample, it was tested whether the SDS remaining in the biological tissue could be treated. It can be seen that when brain tissue with a similar volume was washed with a solution containing 1% triethanolamine as a main component, it was removed higher than 0.1% tetrakis(2-hydroxypropyl)ethylenediamine solution and lower than 1% solution. there is.

From the results of FIG. 4 , it was confirmed that the anionic surfactant remaining in the living tissue could be sufficiently removed even by using other tertiary amines. In addition, it was possible to confirm the concentration of triethanolamine. The 1% solution removed more SDS compared to the 0.1% solution, but the 10% solution removed less than the 1% solution. From this, it can be seen that washing should be performed using a buffer solution containing an appropriate concentration of tertiary amine.

Experimental Example 4

Liquid mass spectrometry was performed to determine whether antigenicity was preserved when a biological sample was treated with a solution containing a tertiary amine as a main component. When treated with different concentrations of tetrakis(2-hydroxypropyl)ethylenediamine, relative to the total protein type, 8.39% for 1% solution, 6.96% for 5% solution, and 17.83 for 10% solution It can be seen that only % of the protein types are lost. It can be seen that more than 90% of the total protein is preserved even if the tetrakis (2-hydroxypropyl) ethylenediamine solution is treated within 5%. In addition, it can be seen that 0.73% of the total protein in a 1% solution, 11.89% in a 5% solution, and 8.39% in a 10% solution are newly discovered, which could not be found through the existing method of removing SDS. From the results of Figure 5, it can be seen that the solution containing tertiary amine as a main component presented by the present invention preserves most antigenicity when anionic surfactants such as SDS present in biological samples are removed. .

With respect to the present invention, the above embodiments have been mainly examined. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

The present invention relates to a composition for removing anionic surfactants, byproducts of surfactants, and/or micelles remaining in a biological sample, and anionic surfactants and surfactants remaining in a biological sample from which lipids are removed by treatment with anionic surfactants It can be used to remove micelles made of proteins or biopolymers bound to and/or surfactants.

Claims (11)

A composition for removing an anionic surfactant from a biological sample, comprising a tertiary amine compound of Formula 1 below:

Formula 1
In the above formula,
R ¹ , R ² and R ³ are each independently an alkyl group, an aryl group or a cycloalkyl group having 1 to 10 carbon atoms; The alkyl group, aryl group or cycloalkyl group may include -O-, -SO ₂ -, -CO-, -S-, -CF ₂ - or -OH (provided that among R ¹ , R ² and R ³ ) if either two are CH ₃ -, the other R ¹ , R ² or R ³ is not CH ₂ OH). According to claim 1, wherein the tertiary amine of Formula 1,
Trimethylamine, triethylamine, tripropylamine, 1-methylpiperidine, N,N-dimethylethylamine, N,N -Diethylmethylamine (N,N-diethylmethylamine), triethanolamine (triethanolamine), 2,2',2"-nitrilotriethanol (2,2',2"-nitrilotriethanol), 2,2'-ethyliminodi Ethanol (2,2'-ethyliminodiethanol), 2-diethylaminoethanol (2-diethylamino ethanol), 2-diisopropylaminoethanol (2-diisopropylaminoethanol), N,N,N',N'-tetrakis (2 -Hydroxypropyl) ethylenediamine (N,N,N'N'-tetrakis(2-hydroxypropyl) ethylenediamine), N,N,N'N'-tetrakis(2-hydroxyethyl)ethylenediamine (N,N ,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine), N,N,N',N',N''-pentakis(2-hydroxypropyl)diethylenetriamine (N,N,N',N',N''-pentakis(2-hydroxypropyl) diethylenetriamine) and mixtures thereof,
A composition for removing anionic surfactants from biological samples. The method according to claim 1 or 2, wherein the anionic surfactant is sodium dodecyl sulfate (SDS), ammonium lauryl sulfate (ALS), sodium lauryl ethylene sulfate (Sodium Lauryl Ethylene Sulfate). ;SLES), Linear Alkylbenzene Sulfonate (LAS), Alpha-olefin Sulfonate (α-OlefinSulfonate; AOS) Alkyl Sulfate (AS), Alkyl Ether Sulfate (AES), and One or more selected from the group consisting of sodium alkane sulfonate (SAS),
A composition for removing anionic surfactants from biological samples. The method according to claim 1 or 2, wherein the tertiary amine is included in an amount of 1 to 10 w/v% with respect to the composition,
A composition for removing anionic surfactants from biological samples. A method for removing an anionic surfactant from a biological sample, comprising treating the biological sample comprising the anionic surfactant with a composition comprising a tertiary amine of Formula 1 below:
Formula 1

In the above formula,
R ¹ , R ² and R ³ are each independently an alkyl group, an aryl group or a cycloalkyl group having 1 to 10 carbon atoms; The alkyl group, aryl group or cycloalkyl group may include -O-, -SO ₂ -, -CO-, -S-, -CF ₂ - or -OH (provided that among R ¹ , R ² and R ³ ) if either two are CH ₃ -, the other R ¹ , R ² or R ³ is not CH ₂ OH). The method of claim 5, wherein the biological sample includes a biological tissue, an organ, or a cell separated in vitro, and has a thickness of 0.1 mm or more,
A method for removing anionic surfactants from biological samples. The method according to claim 5 or 6, wherein the tertiary amine is included in an amount of 1 to 10 w/v% with respect to the composition,
A method for removing anionic surfactants from biological samples. The method of claim 5, wherein the biological sample is treated with the composition comprising the tertiary amine for 10 minutes to 24 hours.
A method for removing anionic surfactants from biological samples. The method according to claim 5 or 6, wherein the composition comprising the tertiary amine is treated at a temperature of 25°C to 60°C,
A method for removing anionic surfactants from biological samples. The method of claim 5 or 6, wherein the treatment of the biological sample with the composition comprising the tertiary amine is performed for 1 to 24 hours,
A method for removing anionic surfactants from biological samples. 7. The method according to claim 5 or 6,
The application of the composition comprising the tertiary amine to the biological sample is performed simultaneously with or after the treatment of the anionic surfactant to the biological sample,
A method for removing anionic surfactants from biological samples.

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