KR20200038449A - A method of isolation of protein and lipid using zwitterionic detergents and the use thereof - Google Patents

Abstract

The present invention relates to a method for analyzing proteins and lipids, and to a method for isolation and mass spectrometry of proteins and lipids using an amphoteric surfactant. As such, the present invention provides a method for efficiently identifying proteins and lipids by extracting and mass-analyzing proteins and lipids specifically present in cells and exosomes using an amphoteric surfactant. The present invention also provides a method for diagnosing diseases using the activity of the identified enzymatic proteins.

Description

{A method of isolation of protein and lipid using zwitterionic detergents and the use thereof}

The present invention relates to methods for the separation, purification and / or analysis of proteins and lipids using amphoteric or zwitterionic surfactants. Specifically, using an amphoteric surfactant, lipids of exosomes including cytoplasmic proteins, membrane proteins, and proteins present in the lipid layer, and blood derivatives including biological tissues, blood, and blood exosomes are extracted and mass spectroscopically analyzed. Therefore, it relates to a method of efficiently separating / identifying proteins and lipids and using the result. It also relates to a method for diagnosing dementia using enzyme activity of lactate dehydrogenase and pyruvate kinase obtained by analyzing blood exosomes of dementia model animals with amphoteric surfactants.

Proteins and lipids are important substances responsible for signaling in vivo and are associated with various diseases. It is essential to observe changes in protein and lipids in studying the causes of disease and developing treatments. Proteins have a variety of structures and are characterized by having a variety of hydrophilicity or hydrophobicity depending on the biochemical nature of the amino acids that are composed to perform their own functions. In order to study the mechanism of disease and develop disease treatment, accurate and sufficient identification and study of proteins must be performed. However, it is difficult to identify proteins because they cannot be amplified, have low stability, and have different molecular properties. In the case of a membrane protein, a protein that is an important target of a disease treatment agent such as an ion transporter or a receptor, or a protein that is still unidentified due to strong hydrophobicity, and even if identified, is identified and excluded from the hydrophobic portion, It is difficult to predict the complete function. Even in the case of cytoplasmic proteins, there are proteins that are difficult to identify due to properties such as hydrophobicity and strong ionicity. The typical one is ubiquitin. For these highly hydrophobic proteins, research on effective separation and identification methods has been ongoing for decades, in addition to Molly's method using surfactants such as urea, thiourea, and CHAPS, as well as centrifugation, chromatography, and glass beads. Methods of separation using a lamp have been reported. However, these methods have disadvantages such as requiring an additional verification process and taking a long time. Therefore, the prior arts do not present satisfactory results in separating and purifying membrane proteins.

On the other hand, because of its strong hydrophobicity, lipids have limitations in their analysis with chromatography or mass spectrometry equipment. In general, a method of separating lipids from cells or biological tissues with a mixture of chloroform / methanol / water and a method using metyl tert-butyl ether / methanol / water are well known (Trends Biochem Sci. 2016 Nov; 41 (11 ): 954-969). Lipids extracted by the above method are analyzed using MALDI mass spectrometry equipment and liquid chromatography-based electrospray mass spectrometry equipment. However, only lipids soluble in the mixture can be separated or recovered, and it is difficult to identify lipids present in various biological derivatives by mass spectrometry due to various ionic states of lipids. Recently, it has been proposed that lipids present in exosomes present in blood have intrinsic properties and can be used as biomarkers for diagnosing diseases (Prog Lipid Res. 2017 Apr; 66: 30-41). . However, there is no optimized method for exosome lipid analysis.

Thus, the present inventors have conducted studies in various ways to overcome the limitations and problems of the prior art in separating and purifying proteins and lipids. As a result, by using amphoteric surfactants, proteins and lipids of various origins are obtained. It was confirmed that it can be effectively dissolved and extracted, and the present invention was completed. The present invention includes separating proteins and lipids, and then performing mass spectrometry for identification and sequence analysis.

Journal of Biomedicine and Biotechnology, 2003: 4 (2003) pp249-255) Trends Biochem Sci. 2016 Nov; 41 (11): 954-969 Prog Lipid Res. 2017 Apr; 66: 30-41

The present invention aims to provide a method for the separation and purification of proteins and lipids.

In addition, an object of the present invention is to provide a method for isolating and purifying all kinds of proteins, including membrane proteins of biological tissues and cells, and a method for isolating and purifying lipids from blood exosomes.

In addition, an object of the present invention is to provide a mass spectrometry method for the identification and sequencing of proteins and lipids.

In order to achieve the above object, the present invention is a protein analysis method using an amphoteric surfactant, i) separating cell and tissue fractions from biological samples isolated ex vivo, and ii) dissolving the fraction in amphoteric surfactant. , Iii) performing SDS-PAGE of the lysed protein fraction, and iii) in-gel digestion or in-solution digestion of the SDS-PAGE product in iii). Thus, a method for isolating proteins or peptides is provided.

In one embodiment of the invention in this regard, the amphoteric surfactant is myristyltrimethylammonium bromide, N-dodecyl-N, N'-dimethyl-3-amino-1-propanesulfonate (N- Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent, 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent, 3- (N, N-dimethylmyristylammonio) propanesulfonate (3- (N, N-dimethylmyristylammonio) propansulfonate), Thesit (trademark), Triton X-144 (trademark), n-dodecyl β-D-malto And one or more amphoteric surfactants selected from the group consisting of seeds (n-Dodecyl β-D-maltoside), and octyl β-D-glucopyranoside.

In another embodiment of the invention in this regard, the protein includes a cytoplasmic protein, a receptor cell membrane protein, an exosome protein, or both.

In another embodiment of the present invention related to this, for the separation of the membrane protein, after performing the SDS-PAGE of iii), or in-gel digestion or in-solution of iv) (in-solution) It may further comprise sonicating the lysed protein prior to digestion).

In another embodiment of the present invention related to this, further comprising the step of iv) further comprising in-gel digestion or in-solution digestion (in-solution digestion), to obtain a transcript Provides a method.

In addition, the present invention is a lipid analysis method using an amphoteric surfactant, i) separating cell and tissue fractions from biological samples isolated ex vivo, ii) dissolving the separated fraction in amphoteric surfactant, and ⅲ ) Provides a method for separating and analyzing lipids, including performing mass spectrometry.

In one embodiment of the invention in this regard, the amphoteric surfactant is myristyltrimethylammonium bromide, N-dodecyl-N, N'-dimethyl-3-amino-1-propanesulfonate (N- Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent, 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent, 3- (N, N-dimethylmyristylammonio) propanesulfonate (3- (N, N-dimethylmyristylammonio) propansulfonate), Thesit (trademark), Triton X-144 (trademark), n-dodecyl β-D-malto And one or more amphoteric surfactants selected from the group consisting of seeds (n-Dodecyl β-D-maltoside), and octyl β-D-glucopyranoside.

In addition, the present invention, i) isolating blood exosomes from biological samples isolated in vitro, ii) dissolving the separated blood exosomes in amphoteric surfactants, iv) SDS-PAGE the dissolved protein fraction And iii) the SDS-PAGE result in iii) is treated with in-gel digestion or in-solution digestion, lactate dedydrogenase and pyruvate It provides a method for providing information for predicting brain disease, including isolating kinase (pyruvate kinase), and v) measuring the relative activity ratios of the lactate dehydrogenase and pyruvate kinase.

In addition, the present invention, i) a blood exosome is separated from a biological sample separated from a normal control group and a subject, ii) the isolated blood exosome is dissolved in an amphoteric surfactant, and iv) lipid is analyzed by mass spectrometry. A method for providing information for diagnosing Alzheimer's disease patients, including iv) confirming that there is a lipid having a mass to charge ratio (m / z) different from a normal control from a subject's blood exosome do.

In addition, the present invention, i) a blood exosome is separated from a biological sample separated from a normal control group and a subject, ii) the isolated blood exosome is dissolved in an amphoteric surfactant, and iv) lipid is analyzed by mass spectrometry. And iv) screening markers in blood exosomes for diagnosing Alzheimer's disease patients, including identifying lipids whose mass-to-charge ratio (m / z) from the subject's blood exosomes is different from the normal control. Provides a method.

The amphoteric surfactant in the present invention, as shown in Figure 1, myristyltrimethylammonium bromide (myristyltrimethylammonium bromide), N-dodecyl-N, N'-dimethyl-3-amino-1-propanesulfonate (N- Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent, 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent, 3- (N, N-dimethylmyristylammonio) propanesulfonate (3- (N, N-dimethylmyristylammonio) propansulfonate), Thesit (trademark), Triton X-144 (trademark), n-dodecyl β-D-malto Can be selected from seeds (n-Dodecyl β-D-maltoside), and octyl β-D-glucopyranoside, 2-10% (w / v) for protein and lipid separation ), Preferably 1% is used.

In the present invention, the protein includes a cytoplasmic protein, a receptor (receptor), a transporter (transporter), etc., or a membrane protein, and these proteins can be concentrated.

In the present invention, the sonication is further included, and the sonication may be repeatedly performed 1 to 5 times for 2 to 10 seconds with a voltage of 10 to 20 V. In addition, in the present invention, it may further include centrifugation after the sonication, the centrifugation may be repeatedly performed 1 to 5 times for 40 to 80 minutes at 12,000 to 16,000 x g.

The method for separating proteins and lipids using the amphoteric surfactant of the present invention is very effective in selectively concentrating, separating and purifying proteins and lipids present in cells and biological samples, respectively. In addition, various diseases such as Alzheimer's dementia can be diagnosed by using the enzyme activity of the identified protein.

1 is a table showing amphoteric surfactants used in the present invention.
Figure 2 is a graph showing the total number of proteins identified in U118 MG glioblastoma cells (glioblastoma cells) according to the amphoteric surfactant treatment according to the method of the present invention.
Figure 3 is a graph showing the total number of proteins identified in the astrocytes according to the treatment of the present invention according to the amphoteric surfactant (Astrocyte).
Figure 4 shows the number of membrane proteins of U118 MG glioblastoma cells (glioblastoma cells) according to the treatment of the present invention in amphoteric surfactant in%.
Figure 5 shows the number of membrane proteins of astrocytes according to amphoteric surfactant treatment according to the method of the present invention in%.
6 is a graph showing the number of proteins identified in the mouse brain according to amphoteric surfactant treatment according to the method of the present invention.
Figure 7 shows the mass-to-charge ratio (m / z) of lipids identified in the blood exosomes of Alzheimer's mouse model (TG6799) and normal mice according to surfactant # 2 treatment according to the method of the present invention.
Figure 8 shows the mass-to-charge ratio (m / z) of lipids identified in the blood exosomes of Alzheimer's dementia patients and normal people according to the surfactant # 2 treatment according to the method of the present invention.
9 is a graph showing that the activity of lactate dehydrogenase identified in human blood exosomes identified in the protein analysis according to the treatment of surfactant # 7 according to the method of the present invention is increased compared to normal in Alzheimer's dementia patients. It is shown.
10 is a graph expressing the relative enzyme activity ratio as pyruvate kinase enzyme activity of the same sample by measuring the enzyme activity in human blood exosomes of lactate dehydrogenase identified in protein analysis according to the treatment of surfactant # 7 according to the method of the present invention It was confirmed that the activity is increased in Alzheimer's dementia patients compared to normal people.

Hereinafter, the present invention will be described based on Examples. The following examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples. It is needless to say that those skilled in the art to which the present technology pertains can be easily modified or substituted from the contents described in the detailed description including the drawings and claims of the present invention are included in the scope of the present invention.

Manufacturing example

* (1) Preparation of cell fraction from U118MG and astrocyte

U118 MG cells and astrocyte cells (from: ATCC® HTB15 ™) were subconfluent in DMEM-F12 medium (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS) And cultured in a 5% CO ₂ humidified atmosphere at 37 ° C. U118MG cells were collected in 80% confluence state, centrifuged at 500 xg for 10 minutes at 4 ° C, and the supernatant was removed and a cell fraction was used as a precipitate.

(2) Peptide preparation from cell fraction

Cell fractions were each lysed with 4% (w / v) amphoteric surfactant in 1x TBS (Tris-buffered saline, Sigma-Aldrich). The mixture was vortexed and incubated at room temperature for 12 hours. Next, the mixture was sonicated 5 times for 5 seconds at 15 V. The mixture was mixed with 4x SDS sample buffer to which 10% beta-mercaptoethanol was added. After boiling for 10 minutes, the sample was subjected to SDS-PAGE. The gel plug was washed with 30% ACN (acrylonitrile), and then treated with 10 mM DTT (1,4-dithiothreitol, Sigma-Aldrich) at 56 ° C for 30 minutes. Again, the gel plug was washed with 100% ACN, and then treated with 100 mM IAA (Iodoacetamide, Sigma-Aldrich) in the dark at room temperature for 30 minutes. After washing the gel plug with 100% ACN, the gel plug was dried with a lyophilizer, and trypsin (1:50 ratio) was treated at 37 ° C for 16 hours.

(3) Preparation of lipid samples from exosome fractions

Mice (Purchased from: Korea Institute of Brain Research, Animal Research Center; 3 month old male) and human plasma (Purchased from: Korean Human Resource Bank; men and women over 60 years old; normal, early dementia, maturity dementia 5 plasma each) (Use) was diluted in 10 times the volume of HBS (HEPES buffered saline, Sigma-Aldrich), and centrifuged at 12,000 rpm at 4 ° C for 20 minutes. The supernatant was removed and 1 ml of HBS was added, followed by centrifugation at 120,000 x g for 90 minutes at 4 ° C, the supernatant was discarded, and the precipitate was dissolved in 200 μl of HBS.

For lipid analysis, 4% of surfactant # 2 was added to blood exosomes and incubated at 37 ° C for 10 minutes. For mass spectrometry with MALDI-TOF, 2,5-Dihydroxybenzoic acid (2,5-DHB, Bruker Doltonics) was added at a concentration of 10 mg / ml, 0.1% formic acid, and a TA80 solution made of 30:70 ACN and distilled water. Dissolved in. After mixing the lipid solution and 2,5-DHB solution 1: 1, MALDI-TOF mass spectrometry was performed.

Peptide mass spectrometry and database search

The trypsin degradant obtained from each peptide preparation method was dissolved in 12 µl of 0.1% formic acid in ddH ₂ O (double-distilled water). Trypsin digests were analyzed using a Q-exactive plus hybrid quadrupole orbit-trap mass spectrometer (Thermo Fisher scientific, San Jose, CA, USA) connected to a nano-electrospray ion source. Acclaim PepMap ^TM 100 (75 um x 2 cm, Thermo Fisher scientific, San Jose, CA, USA) and Acclaim PepMap ^TM RSLC (75 um x 15 cm, 100 A resin, Thermo Fisher scientific, San Jose, as a loading column) (USA, CA), and separated by chromatography on an Ultimate 3000 RSLCnano system (Thermo Fisher scientific, San Jose, CA, USA). The peptide was loaded with RS auto-sampler and separated using a linear gradient ACN / water containing 0.1% formic acid at a flow rate of 300 nl / min. The LC eluent was electrosprayed directly from the analytical column and a voltage of 2.0 kV was applied via the liquid phase crossing of the nanospray source. The peptide mixture was separated for 40 minutes with a 5% to 40% gradient ACN. The analytical method consisted of full MS (Mass Spectrometry) scanning ranging from 350 to 2,000 m / z, and data-dependent MS / MS (MS2) for the ten most intense ions from the total MS scanning results. The mass spectrometer was programmed to obtain results in a data-dependent mode. The mass spectrometer was calibrated using the proposed calibration solution according to the manufacturer's instructions. For database search, a tandem mass spectra was performed with Thermo Fisher Scientific Proteome Discoverer software version 2.1.1.21. Spectrum data was searched against the human Uniprot database. The analysis workflow used included 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). Did. All identified proteins showed a false discovery rate (FDR) of ≤1% calculated at the peptide level. It was evaluated based on the q-value. The search parameters allowed up to two uncut trypsin specificities, with methylthio-modification of cysteine as a fixed modification, and oxidation of methionine as a dynamic modification. Mass search parameters for +1, +2, and +3 ions included 20 ppm for precursor ions and 0.6 Da allowable mass error for fragment ions.

Lipid mass spectrometry (MALDI-TOF)

Blood exosome samples from each of the lipid sample preparation methods were dispensed 2 µl into ground steel plates for analysis of Rapiflex MALDI-TOF (Bruker Daltonics) and dried in air at room temperature. The plate was inserted into the MALDI-TOF, and the spectra range was set to 500-1200 m / z. The smart-beam 3D laser was set to single mode, and the number of shots was set to 500 shots. The laser was injected into the sample under the conditions, and the generated results were collected to obtain mass spectrometry results expressed as intensity values for the mass-to-charge ratio (m / z).

Bioinformatics analysis

DAVID software was used for functional enrichment analysis for Gene Ontology biological process (GOBP) and Gene Ontology cellular component (GOCC). Each identified protein was provided and analyzed using fixed value parameters.

Analysis of lactate dehydrogenase and pyruvate kinase activity

To analyze the activity of lactate dehydrogenase in blood exosomes, 20 µl of blood exosomes was measured using an enzyme activity kit (ab102526, abcam). In addition, to analyze pyruvate kinase activity, it was measured using an enzyme activity kit (MAK072, Sigma-aldrich) with 20 μl of blood exosomes. Both measurement methods were measured by a colorimetric method.

Example

Example 1: Preparation of U118 MG glioblastoma cells and astrocyte cell fraction

The inventors prepared a cell fraction containing protein from U118 MG glioblastoma and astrocytes. When each cultured cell was at 80% confluence, it was collected and centrifuged to obtain a cell fraction. In this example, a method of treating eight types of amphoteric surfactants was designed to identify the optimal protein separation and analysis method (see FIG. 1).

(1-1) Peptide preparation from cell fraction

For cell lysis, amphoteric surfactants were each used. Cell fractions were dissolved in 4% (w / v) surfactant and subjected to incubation and sonication. Next, after SDS-PAGE treatment, in-gel digestion was performed. Subsequently, LC-MS / MS mass spectrometry was performed and the database was searched to identify proteins. In U118 MG cells, 448 in PBS, 611 in surfactant # 1, 787 in surfactant # 2, 550 in surfactant # 3, 29 in surfactant # 4, 87 in surfactant # 5, 138 in surfactant # 6, 707 in surfactant # 7, and 374 in surfactant # 8 were identified (see FIG. 2). In astrocyte cells, 1027 in PBS, 1112 in surfactant # 2, 970 in surfactant # 3, 935 in surfactant # 4, 395 in surfactant # 5, 1020 in surfactant # 6, surfactant 427 to active agents # 7 and 234 to surfactant # 8 were identified (see FIG. 3). Protein was not identified in surfactant # 1 of Astrocyte cells. From the above results, it was confirmed that there is an amphoteric surfactant that can identify proteins better than the PBS condition as a control group, and it was confirmed that the number of proteins to be identified varies according to the type of cell line.

Example 2: Comparative analysis

Subsequently, screening with Gene Ontology selected membrane proteins with hydrophobic properties. Expressed as a percentage of membrane protein relative to the total identified protein, 53% in PBS conditions, 62% in surfactant # 1, 57% in surfactant # 2, 63% in surfactant # 3 in 118 MG cells, surfactant 62% in active agent # 4, 68% in surfactant # 5, 64% in surfactant # 6, 61% in surfactant # 7, and 54% in surfactant # 8 were identified (see FIG. 4). In astrocyte cells, 59% in PBS, 60% in surfactant # 2, 65% in surfactant # 3, 64% in surfactant # 4, 62% in surfactant # 5, 57% in surfactant # 6, surfactant 66% in active agent # 7 and 55% in surfactant # 8 were identified (see FIG. 5). In the above results, the percentage of the membrane protein identified does not show a large difference between the conditions, but considering the total number of proteins identified, when the amphoteric surfactant was treated, the number identified was increased, and therefore analysis was not easy. It was confirmed that the identification efficiency of the membrane protein was increased.

Example 3: Comparative analysis

The method of Example 2 was applied to the brain tissues of mice, and brain proteins according to amphoteric surfactant treatment were analyzed. Using the same preparation method for brain tissue instead of cells, peptides were prepared and LC-MS / MS mass spectrometry and database search were performed.

Brain proteins according to amphoteric surfactants are surfactant # 1 to 677, surfactant # 2 to 821, surfactant # 3 to 1213, surfactant # 4 to 1043, surfactant # 5 to 371, and surfactant 142 from surfactant # 6, 260 from surfactant # 7, and 229 from surfactant # 8 were identified (see FIG. 6). From the above results, it was confirmed that surfactants # 3 and # 4 were relatively high in the number of proteins identified when analyzing proteins for more complex biological tissues than cell lines.

Example 4: Geological analysis

For lipid analysis, blood exosomes were separated from mouse and human plasma, and then surfactant # 2 was treated and incubated to analyze MALDI-TOF. By comparing blood exosomes of normal and TG6799 Alzheimer's dementia model mice, the mass to charge ratio of Alzheimer specific lipids compared to normal mice including mass to charge ratio (m / z) 888.550 in the blood exosomes of Alzheimer's dementia model (m / z) It was confirmed that the peak was present (see Fig. 7). By comparing blood exosomes of normal people with Alzheimer's patients, it was confirmed that there was a peak mass-to-charge ratio (m / z) of lipids specific to Alzheimer's patients in the blood exosomes of Alzheimer's patients (see FIG. 8).

Example 5: Analysis of lactate dehydrogenase enzyme activity

To confirm the possibility of disease diagnosis utilizing the activity of the identified protein, the activity of the identified protein, lactate dehydrogenase enzyme, was measured by treating surfactant # 7 with mouse blood exosome. After dividing the normal and Alzheimer's patient groups into early and late stages, blood exosomes were obtained to confirm the activity of the enzyme of lactate dehydrogenase, and it was confirmed that the enzyme activity of lactate dehydrogenase was increased in Alzheimer's patients (see FIG. 9). In addition, by measuring the activity of pyruvate kinase, which was regularly identified in blood exosomes, the activity of lactate dehydrogenase against pyruvate kinase activity was confirmed, and it was confirmed that the enzyme activity ratio was increased in Alzheimer's patients compared to normal people (see FIG. 10). This result shows the possibility of diagnosing Alzheimer's dementia of the lactate dehydrogenase, a blood exosome protein discovered by the technology of this patent.

The present invention relates to a method for isolating and identifying proteins and lipids. According to the present invention, it is possible to effectively separate and analyze the membrane protein, which has been difficult in conventional separation and analysis. New lipids can also be analyzed. Therefore, the present invention can be widely used for research on proteins and lipids and drug development targeting proteins and lipids.

Claims (11)

As a protein analysis method using an amphoteric surfactant,
i) separating cell and tissue fractions from biological samples isolated ex vivo,
Ii) the fraction is dissolved in an amphoteric surfactant;
Iv) SDS-PAGE was performed on the lysed protein fraction; And
Iii) In the step iii), the SDS-PAGE product is processed by in-gel digestion or in-solution digestion.
Methods for isolating proteins or peptides. The method of claim 1, wherein the amphoteric surfactant
Myristyltrimethylammonium bromide, N-dodecyl-N, N'-dimethyl-3-amino-1-propanesulfonate (N-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate) , 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent (3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent), 3- (N, N-dimethylmyristylammonio) propanesulfonate (3 -(N, N-dimethylmyristylammonio) propansulfonate), Thesit (trademark), Triton X-144 (trademark), n-dodecyl β-D-maltoside (n-Dodecyl β-D-maltoside), and octyl β- Characterized in that it is at least one amphoteric surfactant selected from the group consisting of D-glucopyranoside (Octyl β-D-glucopyranoside),
Methods for isolating proteins or peptides. According to claim 1 or claim 2, wherein the protein is characterized in that it comprises a cytoplasmic protein, receptor cell membrane protein, exosome protein, or both,
Methods for isolating proteins or peptides. The lysed protein according to claim 1 or 2, after the SDS-PAGE of iv), or before in-gel digestion or in-solution digestion of iv). It includes additional sonication,
Methods for isolating proteins or peptides. The method of claim 4, wherein the protein is a membrane protein,
Methods for isolating proteins or peptides. As a lipid analysis method using an amphoteric surfactant,
i) separating cell and tissue fractions from biological samples isolated ex vivo;
Ii) the isolated fraction is dissolved in an amphoteric surfactant; And
Iv) involving performing mass spectrometry,
Methods for isolating and analyzing lipids. The method of claim 6, wherein the amphoteric surfactant
Myristyltrimethylammonium bromide, N-dodecyl-N, N'-dimethyl-3-amino-1-propanesulfonate (N-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate) , 3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent (3- (decyldimethylammonio) propanesulfonate inner salt zwitterionic detergent), 3- (N, N-dimethylmyristylammonio) propanesulfonate (3 -(N, N-dimethylmyristylammonio) propansulfonate), Thesit (trademark), Triton X-144 (trademark), n-dodecyl β-D-maltoside (n-Dodecyl β-D-maltoside), and octyl β- Characterized in that it is at least one amphoteric surfactant selected from the group consisting of D-glucopyranoside (Octyl β-D-glucopyranoside),
Methods for isolating and analyzing lipids. i) separating blood exosomes from biological samples isolated ex vivo,
ii) dissolving the isolated blood exosomes in amphoteric surfactants;
Iv) SDS-PAGE was performed on the lysed protein fraction; And
Iv) The SDS-PAGE product in ⅲ) is treated with in-gel digestion or in-solution digestion, and lactate dedydrogenase and pyruvate kinase ),
v) comprising measuring the relative activity ratios of the lactate dehydrogenase and pyruvate kinase,
How to provide information to predict brain disease. i) Isolating blood exosomes from biological samples isolated from normal controls and subjects,
Ii) dissolving the isolated blood exosomes in amphoteric surfactants; And
Iv) to separate lipids by mass spectrometry,
iv) including confirming that the mass to charge ratio (m / z) from the blood exosomes of the subject is different from the normal control,
How to provide information to diagnose Alzheimer's disease patients. i) Isolating blood exosomes from biological samples isolated from normal controls and subjects,
Ii) dissolving the isolated blood exosomes in amphoteric surfactants; And
Iv) to separate lipids by mass spectrometry,
iv) from the subject's blood exosomes, including identifying lipids with a mass to charge ratio (m / z) different from the normal control,
A method for screening markers in blood exosomes to diagnose Alzheimer's disease patients. The method of claim 1 or 2, wherein the isolated protein is further treated with in-gel digestion or in-solution digestion to obtain transcripts.

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