KR101114446B1 - A new biomarker protein and a new detection method in the biological fluid with preeclampsia using proteomics technology - Google Patents

Abstract

The present invention is a mass spectrometer using a mass spectrometer (Insulin like growth factor binding prtein, acid labile subunit (IGFBP, ALS), Alpha2-HS-glycoprotein (AHSG) , Fetuin), Retinol binding protein 4 (RBP4), and alpha-1-microglobulin / bikunin precursor (AMBP), and the development of a novel diagnostic method for quantifying specific markers of pregnancy poisoning using the same.

Description

A new biomarker protein and a new detection method in the biological fluid with preeclampsia using proteomics technology}

The present invention relates to a method for discovering and diagnosing a pregnancy addiction-specific marker protein using a proteomic technique, and more particularly, Insulin, a diagnostic protein specific for pregnancy addiction in a biological sample (plasma/serum) using a mass spectrometer. Like growth factor binding prtein, acid labile subunit (IGFBP, ALS), Alpha2-HS-glycoprotein (AHSG, Fetuin), Retinol binding protein 4 (RBP4), and alpha-1-microglobulin/bikunin precursor (AMBP) The present invention relates to a method and the development of a new diagnostic method for quantification of a pregnancy addiction-specific label protein using the same.

The present invention is derived from a study conducted as part of the Ministry of Health and Welfare's Health and Medical Technology Research and Development Project [Task serial number: A060-769, Task name: Development of a method for diagnosing pregnancy addiction using quantitative proteomic analysis technology].

In general, pregnancy addiction is a major cause of maternal and fetal perinatal mortality, accounting for a large proportion of 5-8% of all pregnant women, and major symptoms include high blood pressure, edema, and an increase in the amount of urinary protein toxic to pregnancy. , Severe headache and sudden weight gain. Pregnancy addiction usually occurs after 20 weeks and is a major cause of premature birth. However, until now, its exact cause and treatment method have not been clearly identified. The only incentive treatment method is to get out of pregnancy through delivery. However, if pregnancy poisoning occurs in the early stages of pregnancy, early delivery can increase the mortality rate of the fetus. Therefore, early diagnosis and prolongation of the pregnancy state as much as possible may be the best method. Therefore, early diagnosis of this disease can reduce the preterm delivery of the fetus caused by pregnancy addiction and the resulting perinatal mortality of the fetus.

The discovery of new disease-specific proteins in biological fluids, such as serum, plasma, urine, and pleural effusion, has great significance for early diagnosis and treatment of diseases clinically. Because, in the case of existing disease-specific proteins, it is difficult to diagnose a low-range protein in the diagnosis of a disease, and there are not many disease-specific proteins that are recognized, so it has many limitations to represent a disease, and only marker proteins for a few diseases. Because of this, it is difficult to diagnose the entire disease. In particular, in the case of pregnancy addiction, since many marker proteins that can predict the diagnosis and prognosis of the disease in the early stage are not known, there are many vulnerabilities in early diagnosis. .

In this regard, when looking at the conventional sample processing technology, in the case of biological samples such as serum/plasma samples, about 20 components account for nearly 90% of the total components, which acts as a factor that interferes with the identification of proteins distributed in small amounts. In most cases, in the case of proteins that are considered to be disease markers (for example, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), ng/mL is present)), in most cases, trace amounts (pg. /mL~ng/mL), the removal of a large amount of protein is a particularly important factor in the discovery of a new label protein.

Currently used methods include resin based depletion, antibody based depletion, glycopeptide enrichment, and peptide enrichment. 1) Resin based depletion method includes cibacron blue and blue dye, 2) Antibody based depletion method includes MARS (Multiple Affinity Removal System, Agilent technology), which is one of the most used depletion methods for proteins to be removed from beads. By coupling antigens, the affinity between antigen and antibodies is based on the principle. The IgY-microbead method also uses an antigen-antibody reaction as a basic principle, but it is said that chicken immunoglobulins are used to reduce non-specific reactions and remove the target protein more precisely than in the case of IgG. 3) Glycosylation is one of the most important transformations, and plays an important role in cell growth, development, and communication between cells, especially in immune responses. In many cases, it is particularly important in cancer diagnosis, prognosis, and observation of disease progression. Proteins with meaning are also known to be glycoproteins. Therefore, enrichment of glycopeptide is emerging as a new enrichment technology in the discovery of marker proteins for disease diagnosis. This is to separate the peptide from the lectin beads by selectively binding only the glycopeptide through a substance that recognizes the glycopeptide, for example, a lectin, and then deglycosylation using a deglycosylase such as PNGase F. By doing so, it is a method of removing very abundant proteins and concentrating glycopeptides.

Looking at the conventional protein fractionation technology, protein fractionation technology from biological samples has been widely used up to now as the most important technology for the identification of new proteins, and the easiest method is to use SDS-PAGE and 2-DE. It is a method of fractionation. Recently, in order to obtain better results in the protein identification step, a more detailed protein classification process has been required, and new methods are being introduced for this. The methods that have recently begun to be used are non-gel based separation methods.FFE (Free Flow Electrophoresis), PF2D 2-D, MCE (multichannel electrolyte), which are technologies that fractionate according to protein size (molecular weight) and isoelectric point (pI), ZOOM (microscale solution IEF).

Diagnosis methods for biological samples that have been used up to now include serological methods, immunological methods, physicochemical methods, and pathological methods. However, the disease-specific labeling proteins used to date have many limitations in accurately and accurately predicting the stage of the disease. In many cases, it was difficult to collect samples, and thus, there were many difficulties in repeating tests for a short period of time. Therefore, the discovery of a new label protein in a biological fluid, which is non-invasive and relatively easy to collect specimens, is very important in order to diagnose the disease and determine the prognosis without pain to the patient. For this reason, there have been many attempts to discover new label proteins in biological samples until now, and due to the completion of the human genome project, a mass spectrometer capable of remarkable development of genomics and proteomics and high throughput analysis. With the development of (mass spectrometer) technology, hundreds of new marker proteins have actually been discovered. However, until now, there are few cases in which a newly discovered marker protein has been applied to the diagnosis of disease in clinical practice. The reason is that the reproducibility of the diagnostic method is difficult when using a mass spectromer, and the immunological method known to have high reproducibility, especially ELISA, takes a lot of cost and a long time to establish a diagnostic method, and targets all proteins. It has a drawback that cannot be done. Therefore, the development of new analytical methods with higher repeatability, reproducibility, sensitivity and specificity is of particular importance.

Therefore, it is necessary to develop a diagnostic method with high reproducibility, precision, and sensitivity, to discover a label protein specific to pregnancy addiction, measure a large number of samples in a short time, and have high reproducibility, precision, and sensitivity for biological samples that are relatively easy to collect.

The present invention solves the above problems and has been conceived by the necessity of the above, and an object of the present invention is to provide a label protein specific for pregnancy addiction to a biological sample.

Another object of the present invention is to measure a large number of specimens in a short time and to provide a method for diagnosing pregnancy addiction with high reproducibility, precision, and sensitivity.

In order to achieve the above object, the present invention

Insulin like growth factor binding protein, acid labile subunit (IGFBP, ALS), alpha 2-HS-glycoprotein (AHSG, fetuin), retinol binding protein 4 (RBP4), and It provides a composition for diagnosing pregnancy addiction comprising one or more proteins selected from the group consisting of alpha-1-microglobin/bikunin (AMBP) as an active ingredient .

The protein of the present invention is preferably derived from an animal, preferably a mammal, and more preferably derived from a human, but is not limited thereto.

In one embodiment of the present invention, the insulin-like growth factor binding protein acid-degradable subunit (Insulin like growth factor binding protein, acid labile subunit; IGFBP, ALS) is preferably the protein described in SEQ ID NO: 1, and the alpha2 -HS-glycoprotein (AHSG, fetuin) is preferably the protein shown in SEQ ID NO: 2, the retinol binding protein 4 (RBP4) is preferably the protein shown in SEQ ID NO: 3, and the alpha-1-microglobin/ratio Kunin (AMBP) is preferably the protein shown in SEQ ID NO: 4, but is not limited thereto.

In addition, the present invention provides a peptide for identifying and quantifying the protein of the present invention.

The peptide is preferably a peptide selected from the group consisting of SEQ ID NO: 81 in SEQ ID NO: 5, but is not limited thereto.

In addition, the present invention is a) the step of segmenting the protein in the biological sample of the normal group and the patient group: b) the total protein using a liquid chromatography/mass spectrometry (LC-MS/MS) using the fragmented peptides. To identify; And c) it provides a method for selecting a protein specific for pregnancy addiction, which is an increased or decreased protein, by comparing the total peak intensity of the protein of the normal person group and the patient group.

In one embodiment of the present invention, the biological sample is preferably selected from the group consisting of serum, plasma, urine, pleural fluid, and saliva, but is not limited thereto.

Hereinafter, the present invention will be described.

First, the present invention is a pregnancy addiction specific protein, insulin-like growth factor binding protein, acid-degradable subunit (Insulin like growth factor binding protein, acid labile subunit; IGFBP, ALS), alpha 2-HS-glycoprotein (AHSG, fetuin) , Retinol binding protein 4 (RBP4), and alpha-1-microglobin/bikunin (AMBP) were discovered.

Second, the present invention relates to a method for diagnosing a novel labeled protein using the SRM assay for the protein using a proteomics technology.

In the present invention, in order to quantify the concentration of the selected protein, a proteomics technique, SRM (selective reaction monitoring) method was used, which has reproducibility after analyzing the peptide spectrum information of a large number of proteins that can be obtained in the protein identification process. , Peptides that can be easily analyzed are selected with a mass spectrometer, and based on the selected peptide spectral information, a precursor ion and a product ion, which are the intrinsic values of the peptide, are obtained. Based on the peptide specific information obtained through this series of processes, it is analyzed in SRM mode, which is one of the quantitative analysis methods of mass spectrometers. SRM is a method of measuring the total amount of the selected protein based on the total MS/MS peak value obtained by scanning the selected peptide and the internal standard by the same method.

In the case of AHSG, the following peptide TVVQPSVGAAAGPVVPPC#PGR was selected for the peptide specific information of the peptide selected in the present invention, and the specific information is 1009.2 (mother ion) -> 683.7 (daughter ion). In the case of IGFBP and ALS, LWLEGNPWDC#GC#PLK , 923.6 -> 1132.9

For RBP4, YWGVASFLQK, 600.6 -> 849.9, AMBP, AFIQLWAFDAVK, 750.3 -> 836.9, for internal standard, DHTGFLTEYVATR 758.0 -> 1160.0.

Hereinafter, the present invention will be described in more detail.

In the present invention, using proteomics technology, in order to discover a specific marker protein for pregnancy addiction in a biological sample, an image of a change in protein between groups was analyzed for 5 patients and 5 normal pregnant women.

The same amount of the biological sample protein was digested with trypsin, peptide fragmented, and then the total protein was identified using LC-MS/MS (Figs. 1 and 2). In order to check the mass change of the protein between the normal and abnormal groups, proteins with an increase or decrease of 30% or more were first compared by comparing the total peak intensity of the protein. Among them, Insulin, which is expected to have significance as a candidate for pregnancy addiction marker protein. Some proteins such as growth factor binding prtein, acid labile subunit (IGFBP, ALS), Alpha2-HS-glycoprotein (AHSG), Retinol binding protein 4 (RBP4), and alpha-1-microglobulin/bikunin (AMBP) are unique to pregnancy. After selecting a protein candidate group, the mass change of these proteins was compared and analyzed using SRM (Selective Reaction Monitoring) method (FIG. 7), which is a proteomic quantitative method, and Western blot (FIG. 9), and ELISA (FIG. 8), which are traditional methods.

As shown in FIG. 7, it was confirmed that the candidate group selected based on the protein peak intensity was quantitatively analyzed for the labeled protein mass of the disease group and the normal group using the SRM method, and was consistent with the conventional quantitative methods Western blot and ELISA.

As can be seen from the present invention, the SRM assay, a new label protein for pregnancy addiction of the present invention and a new label protein diagnostic method using a mass spectrometer, is an excellent diagnostic method that can easily and quickly quantify several types of label proteins at once. Confirmed.

1 shows the entire process for discovering a new label protein in a biological sample (serum/plasma) using a mass spectrometer and developing a diagnostic method.
2 shows total proteins identified using mass spectrometry.
Figure 3 shows the number of proteins by group in Figure 2.
FIG. 4 shows a histogram of matching proteins and non-matching proteins by analyzing the identified proteins of FIG. 3 for each group.
5 shows the LC conditions before mass spectrometry.
6 shows analysis conditions for SRM analysis for quantitative analysis using LC-MS/MS.
7 is a graph showing the result of quantification of the selected protein using the SRM method using a mass spectrometer.
8 is a graph showing the results of ELISA of Alpha2-HS glycoprotein.
9 is a photograph showing the Western blot result of Alpha2-HS glycoprotein and a graph showing the densitometry result.
Fig. 10 is a graph showing the clarification of the biological function of the protein in which the amount of protein is increased or decreased between the pregnant woman group with pregnancy addiction and the normal pregnant woman group.
11 shows the tryptic peptide sequence of the selected protein. 11a is the tryptic peptide sequence of Insulin like growth factor binding protein, 11b is the tryptic peptide sequence of Alpha2-HS glycoprotein, 11c is the tryptic peptide sequence of alpha-1-microglobulin/bikunin, and 11d is Retinol binding protein. It shows the tryptic peptide sequence of 4.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are for explaining the present invention, but the scope of the present invention is not to be construed as being limited by the following examples.

In the present invention, after removing a large amount of proteins such as albumin, immunoprotein, transferrin, etc. using MARS, the protein is fragmented into peptide fragments with trypsin, and an internal standard is prepared, and the final quantification step using LC-MS/MS Go through.

Example 1: Preparation of sample for analysis

The analysis sample was analyzed using blood from pregnant women at the same week as the blood of patients showing signs of pregnancy addiction among pregnant women before and after 30 weeks of pregnancy. After collecting the patient's blood, the cells were aggregated at 4°C for about 4 hours, and then fractionated at 3500 rpm for about 20 minutes using a centrifuge, and the supernatant was used as a target sample.

Example 2: serum/ Plasma Albumin and Immunoprotein remove

In order to remove albumin, immunoproteins (antibodies), etc. in biological samples (serum/plasma), Agilent technology's MARS (Multiple Affinity Removal System) was used. The protein-removed serum/plasma sample was _{washed twice with 20mM NHHCO 3} , pH8 300uL using a microcon (Millipore) to change the buffer.

Example 3: of biological sample protein Peptide Fragmentation Way( In - solution digestion )

For protein identification using mass spectrometry, fragmentation of the analyzed protein into peptide fragments is required. To this end, 2.5uL of 100M DTT was added to 50uL of each total 10ug of protein, reacted at 60°C for 30 minutes, cooled at room temperature, and then 0.75uL of 1M idoacetoamide (IAA) was added and reacted in the dark for 40 minutes (Trypsin, Promega). After adding a protein-to-trypsin ratio of 20:1, the reaction mixture was reacted at 37°C for 5 to 14 hours, and the reaction solution was vaporized using SpeedVac.

Example 4: ZipTip ( C18 mini - tip column Decontamination method using)

ZipTip (C18, Millipore), which is commercially available, is used to remove salts and other impurities generated in the process of peptide fragmentation of proteins using trypsin. The test method is first, ziptip 3 times with 10uL of 80% Acetonitrile/0.1% Formic Acid. After washing, after washing three times with 10uL of 1% Formic Acid, the prepared peptide is added and reacted with C18 repeatedly 10 times.

To remove non-binding, wash 3 times with 10uL of 0.1% Formic Acid and repeat 3 times with 5uL of 80% Acetonitrile/0.1% Formic Acid to extract the peptide bound to the column.

Example 5: LC - MS / MS ( LTQ , Thermo Method for quantifying protein using)

Bioprotein identification method

The peptide fragment fragmented with trypsin was subjected to the desalination process using the ziptip described above, and then fractionated according to the LC condition (Fig. 5), and the mass spectrometry condition for the identification of the whole protein was a nano-electrospray ion source, and the electropray voltage Was set to 2.1kV, and collision energy for MS/MS fragmentation was activated at 35% of RF amplitude for 30ms. In data dependent mode to obtain the entire spectra, the MS/MS scan selects 5 high peak intensity among full mass scans and breaks the selected parent ions using helium gas. Spectrum information generated at this time After collecting the data, protein information is obtained by comparing it with the theoretical peptide spectrum using SpectroMill, a database search program. In this way, a biological protein was identified, and based on the peptide peak intensity information at this time, a protein having a difference between the disease group and the normal group was first selected (Fig. 2).

Screening process of specific protein for pregnancy addiction

Using LC-MS/MS, pregnancy poisoning-specific proteins, Alpha2-HS-glycoprotein, and alpha-1-microglobulin/bikunin (AMBP) were discovered, and parent ion information and product ion information obtained based on the peptide spectrum information obtained at this time. Based on the SRM assay (FIG. 6), which imposes selectivity during mass spectrometry, was used for quantitative analysis of the labeled protein. This was called SRM (Selective Reaction Monitoring).

Attach electronic file of sequence list

Claims (9)

A composition for internal standard material for quantitating a diagnostic protein for early diagnosis of pregnancy poisoning comprising at least one peptide selected from the group consisting of SEQ ID NO: 81 in SEQ ID NO: 5. A composition for internal standard material for quantitating a diagnostic protein for early diagnosis of pregnancy poisoning comprising a peptide consisting of SEQ ID NO: 81 in SEQ ID NO: 5. The method according to claim 1 or 2, wherein the peptide is insulin-like growth factor binding protein, acid labile subunit (IGFBP, ALS), alpha2-HS-glycoprotein (AHSG, Quantitatively diagnosing diagnostic proteins for early diagnosis of preeclampsia, characterized in that they are derived from a protein selected from the group consisting of fetuin), retinol binding protein 4 (RBP4), and alpha-1-microglobin / bikunin (AMBP). Composition for internal standard substances. According to claim 3, wherein the insulin-like growth factor binding protein acid labile subunit (IGFBP, ALS) is a protein described in SEQ ID NO: 1 diagnostic protein for early diagnosis of pregnancy Composition for internal standard for quantitative analysis. The composition of claim 3, wherein the alpha 2-HS-glycoprotein (AHSG, fetuin) is a protein of SEQ ID NO: 2. 4. The composition of claim 3, wherein the retinol binding protein 4 (RBP4) is the protein set forth in SEQ ID NO: 3. 4. The composition of claim 3, wherein the alpha-1-microglobin / bikunin (AMBP) is the protein set forth in SEQ ID NO: 4. 4. delete delete

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