KR102100158B1 - Method for disease screening by relative quantification of N-Glycan chromatogram peak - Google Patents

Abstract

Task: The method of confirming the presence or absence of a disease using a difference in the composition of the conventional sugar chain does not have many specific sugar chain compositions for each disease, and has a problem in that it takes a long time to analyze.
Problem solving method: The present invention revealed that even if the oligosaccharide composition is the same, there exist oligomeric isomers showing a difference in quantitative values, and it can be used as a marker for a specific disease. The present invention selectively separates and concentrates oligosaccharides containing sialic acid (NeuAc) in N-glycosaccharides in liberated body fluids, and then performs mass analysis using a triple quadruple LC / MS analyzer and multiple reaction monitoring analysis method. It is related to a screening method to confirm and compare the ratio of isomer quantitative values to determine whether a specific disease is present.

Description

Method for disease screening by relative quantification of N-Glycan chromatogram peak}

The present invention selectively isolates isomers of N-glycosyls containing sialic acid NeuAc among N-glycans in free plasma, and each isomer obtained by multiple reaction monitoring (MRM) mass spectrometry It relates to a screening method to identify a specific disease by comparing the ratio of the quantitative value of.

More than 60% of proteins in the blood are glycoproteins with oligosaccharides on the surface, most of which are associated with immune and inflammatory reactions, such as immunoglobulin G, haptoglobin, alpha-1-acid glycoprotein, serum amyloid A, etc. The protein of is also a glycoprotein. Since the location and structural diversity of protein glycosylation is sensitive to the environment inside and outside the body, disease-related research and disease diagnosis system development using the sugar chain are currently actively underway.

After the protein is synthesized, the glycoprotein is synthesized from the endoplasmic reticulum to the Golgi Apparatus and then added with sugar by various glycosylases (glycosyltransferase). However, depending on the state of biological change, such as disease, a specific glycosylation is abnormally expressed or abnormal glycosylation occurs due to a catalyst or a transition material, resulting in cell mutation and loss of function. In addition, in the process of biosynthesis of oligosaccharides, numerous oligosaccharides and isomers may exist due to competitive reactions between enzymes, and the characteristics of each oligosaccharide are very diverse. Therefore, qualitative and quantitative analysis including isomer information is essential to reveal the relationship between the disease and the company. However, up to now, disease-based disease-related studies have been conducted to compare the characteristics of human chains (blood, urine, tears, saliva, etc.) and tissues by comparing the entire profiling analysis of the chains, which excludes isomer information by liberating N- or O-glycosylase. I have been focusing. This method is not only time-consuming, but it is very difficult to find disease-specific sugar chains among numerous sugar chains. Therefore, in order to confirm the association between the oligosaccharide and the disease, a new approach that reflects the quantitative characteristics and qualitative information considering the oligomeric isomer is required.

Porous Graphitized Carbon column-based liquid chromatography is pre-treated without additional derivatization step compared to reverse phase (C18) or hydrophilic interaction column (HILIC), which is commonly used in conventional chain analysis. Is simple and excellent for isomer separation.

In addition, multiple reaction monitoring (MRM) mass spectrometry is one of the mass spectrometry techniques that can be quantified through a triple quadrupole mass spectrometry (QQQ), and has excellent selectivity and sensitivity. The mass spectrometer filters only the ion (m / z) of the target material in the first quadrupole (Q1) part, and then in the second quadrupole (Q2) a collision with inert gas and electrical energy is generated (Fragmentation, Tandem, MS / MS) Produces product ions (m / z). It consists of selecting and scanning the characteristic ions of the target material among the fragment ions generated in the last quadrupole (Q3) part. This analysis method can minimize the influence of interference materials such as the matrix, so that the target material can be analyzed with higher sensitivity, and absolute quantitative analysis is possible through the area derived through ion monitoring.

Behcet's disease is an unexplained autoimmune disease in which the prevalence of 10 to 300 cases per 100,000 people is reported mainly in the Middle East and Asia, which causes inflammation of the oral cavity, genitals, eyes, and skin, leading to death through complications. To date, the exact cause or pathophysiology of the disease has not been established, no established diagnostic guidelines have been established, and there is no specific treatment. Therefore, there is a need for a diagnosis method that can be used to specifically diagnose Behcet's disease specifically, to measure disease activity, and to develop specific therapeutic agents.

The present invention is to describe a diagnostic method from the perspective of oligosaccharide isomers that can specifically identify Behcet's disease.

Korean Registered Patent Publication No. 10-1452730 Korean Patent Publication No. 10-2016-0146102

De Leoz, Maria Lorna A., et al. "High-mannose glycans are elevated during breast cancer progression." Molecular & Cellular Proteomics (2010): mcp-M110. Trbojevic Akmacic, Irena, et al. "Inflammatory bowel disease associates with proinflammatory potential of the immunoglobulin G glycome." Inflammatory bowel diseases 21.6 (2015): 1237-1247. Miyamoto, Suzanne, et al. "Multiple Reaction Monitoring for the quantitation of serum protein glycosylation profiles: Application to Ovarian Cancer." Journal of proteome research 17.1 (2017): 222-233.

In order to solve the limitations and limitations of the indirect guidelines that standardize the in vitro symptoms used for the diagnosis of Behcet's disease, the present invention extracts N-glycosyl from a plasma sample and then confirms the quantitative ratio between N-glycomeric isomers. It aims to provide an enemy diagnostic method.

In addition, the present invention extracts N-glycosyl of all glycoproteins rather than specific glycoproteins from a bodily fluid sample, and then uses N-glycosyl as a triple quadruple LC / MS analyzer and multiple reaction monitoring analysis It is intended to provide a method for analyzing the presence or absence of a disease in a subject by separating the N-glycoside isomer by mass spectrometry and using the peak as a biomarker when there is a significant difference between the normal control group and the patient group between the isomer peaks.

In order to solve the above problems, the present inventors separated and analyzed N-glycosylated chains containing acidic monosaccharides (sialic acid, sialic acid) in about 90 plasmas and compared the specific ratio of isomeric isomers between normal and patient. Markers for bottle identification were selected.

The present invention

A) preparing a body fluid sample from a specific disease patient group and a normal control group;

B) separating the N-saccharide chain by treating the sample with an enzyme;

C) purifying or concentrating the separated N-saccharide chain;

D) mass spectrometry of purified or concentrated N-glycosyl with a triple quadruple LC / MS analyzer and multiple reaction monitoring analysis method;

E) comparing the ratio of quantitative values of each of the N- sugar chain isomers obtained in step d); And

F) selecting a specific N-glycomeric isomer as a marker of the specific disease when there is a significant difference in the ratio of specific N-glycomeric isomers between the patient group and the normal control group; and disease screening using the N-glycomeric isomer Provides a method.

The present invention provides a disease screening method using an N-glycoside isomer, characterized in that the step of denaturing the protein of the sample before step a) and step b) is added.

The present invention is after the step f) above

G) Taking a test sample of body fluid of the test subject, separating the N-sugar chain from the test sample, purifying or concentrating the separated N-sugar chain, and liquid chromatography of the purified or concentrated N-sugar chain-multiple reaction monitoring mass spectrometry Then, when the ratio of the quantitative value of the N-glycomeric isomer obtained by mass spectrometry is compared with the ratio of the quantitative value of the normal control group and the patient group obtained in step E), if there is a significant difference from the ratio of the normal control group, the subject It provides a disease screening method using the N- sugar chain isomer, characterized in that it further comprises the step of confirming that the jam.

In the present invention, the above step b)

a) treating the body fluid sample with a protein denaturing reagent to denature the protein;

b) separating the N- sugar chain by treating the sample with PNGase F enzyme for 16 to 24 hours;

c) b) precipitating the protein in the sample that has been subjected to step; And

d) centrifuging the sample from which the protein has been precipitated and obtaining a supernatant; a disease screening method using an N-glycoside isomer is provided.

In addition, the present invention is the step c)

a) washing the porous graphitized carbon column with a purification solvent;

b) washing the column of a) with an acetonitrile solution containing trifluoroacetic acid;

c) washing the column subjected to step b) with a purification solvent;

d) c) loading the sample to the column that has been subjected to step;

e) washing the column loaded with the sample with a purification solvent; And

f) e) flowing a concentration gradient solution to a column that has undergone the step of eluting the sample; provides a disease screening method using N-glycosyl isomers, which comprises sequentially.

In addition, the present invention has the step of mass-analyzing the purified or concentrated N- sugar chain by a triple quadruple LC / MS analyzer and a multiple reaction monitoring analysis method.

a) separating the N-glycomeric isomer containing sialic acid with a porous graphitized carbon packed column using a gradient elution;

b) Single sialic acid complex N- sugar chain with Hex ₅ HexNAc ₄ NeuAc ₁ in the first quadrupole of the triple quadruple LC / MS analyzer, single sial with Hex ₅ HexNAc ₄ Fuc ₁ NeuAc ₁ Oxidizing single fucosylated complex N-sugar chain, oligosaccharide composition Hex ₅ HexNAc ₄ NeuAc ₂ Double sialic acid complex N-sugar chain ions selected and passed through;

c) generating a fragment ion by fragmenting the ions that have passed through the first quadrupole by applying specific energy in a collision cell that is the second quadrupole;

d) detecting only fragment ions specific to the target substance among the fragment ions generated in the collision cell by passing through a third quadrupole;

e) comparing the chromatogram area ratio between isomers of the fragment ions detected in step d); provides a disease screening method using N-glycosyl isomers.

In addition, the present invention provides a disease screening method using the N- sugar chain isomer, characterized in that the specific disease is an autoimmune disease.

In addition, the present invention provides a disease screening method using the N- sugar chain isomer, characterized in that the autoimmune disease is Behcet's disease.

In addition, the present invention is a fragment ion specific to the target material of the step d) has a theoretical molecular weight of 203.0794 Da, a fragment ion having a oligosaccharide composition of HexNAc ₁ and a theoretical ion molecular weight of 365.1322 Da, and at least one selected from a fragment ion of a oligosaccharide composition of Hex ₁ HexNAc ₁ It provides a disease screening method using the N- sugar chain isomer, characterized in that.

In addition, in the present invention, step e) includes 6 isomer peaks corresponding to Hex ₅ HexNAc ₄ NeuAc ₁ , 3 isomer peaks corresponding to Hex ₅ HexNAc ₄ Fuc ₁ NeuAc ₁ , or ₂ corresponding to Hex ₅ HexNAc ₄ NeuAc 2 Deriving the ratio of the chromatogram area of each of the four isomer peaks; And Comparing the ratio of the quantitative value of each oligomeric peak derived in the above step compared to the normal control group, when the difference in expression level more than 2 times in the patient group is confirmed to be diseased; disease using the N-glycan isomer comprising a Provides a screening method.

Furthermore, in the present invention, when the specific disease is Behcet's disease, the marker of step f) is linked to (α 2, 3) binding of one of the terminal galactoses to the terminal sialic acid in the N- sugar chain composition Hex ₅ HexNAc ₄ NeuAc ₁ Among the isomers, the two anomer isomers resulting from the formation of the hemiacetal ring structure of monosaccharides and the oligosaccharide composition Hex ₅ HexNAc ₄ NeuAc ₂ is a double sialic acid complex N-glycan (Di-sialylated complex-type N-glycan) isomer Provided is a disease screening method using N-glycosyl isomers, characterized in that the two terminal sialic acids are at least one selected from isomers linked by two (α 2, 3) and (α 2, 6) bonds with two terminal galactose do.

When explaining the configuration of the present invention in more detail, the present invention

A) separating the N-glycoline in the sample by treating the enzyme with PNGase F in 50 μl of body fluid, preferably plasma or serum;

B) desalting the sample subjected to step a) by porous graphitized carbon solid phase extraction, separating, purifying and concentrating the N-sugar chain containing sialic acid (NeuAc, NeuGc);

C) Quantitative analysis of concentrated sialic acid-containing N-saccharide chain by porous graphitized carbon-based liquid chromatography-multiple reaction monitoring mass spectrometry (PGC-LC / MRM-MS);

D) comparing the ratio of the quantitative values of each of the sialic acid-containing N-sugar chain isomers obtained by mass spectrometry; relates to a method for determining whether or not Behcet's disease.

In addition, the present invention is the method of processing the enzyme of step a)

a) taking a sample of 50 μl of body fluid;

b) adding a protein denaturing reagent to the sample and treating it at 90 to 110 ° C. for 1-3 minutes to denature the protein;

c) separating the N- sugar chain by treating 2 μl of PNGase F enzyme with the sample for 16 to 24 hours;

d) precipitating the protein in the sample; And

e) centrifuging the sample from which the protein has been precipitated and obtaining a supernatant;

In addition, the present invention is the porous graphitized carbon solid phase extraction of step b) above

a) washing the porous graphitized carbon column with purified water 2.5 to 3.5 ml twice or more;

b) washing the column of a) twice or more with 3 ml of 80% acetonitrile solution with 0.1% trifluoroacetic acid;

c) washing the column subjected to step b) with purified water 2.5 to 3.5 ml twice or more;

d) loading the sample;

e) washing two or more times with 2.5 to 3.5 ml of purified water;

f) washing with 10% acetonitrile solution 2.5 to 3.5 ml twice or more; And

g) washing two or more times with 2.5-3.5 ml of 20% acetonitrile solution; And

h) eluting the sample two or more times with 2.5-3.5 ml of a 40% acetonitrile solution containing 0.05% trifluoroacetic acid; a method for separating and purifying N- sugar chains containing sialic acid sequentially will be.

In addition, the present invention is a porous graphitized carbon-based liquid chromatography in step c)-multiple reaction monitoring mass spectrometry

a) Using a gradient gradient elution of solvent A (3% acetonitrile with 0.1% formic acid) and solvent B (90% acetonitrile with 0.1% formic acid) Separating the acid-containing N- sugar chain isomer by liquid chromatography;

b) Single sialic acid complex N- sugar chain (theoretical molecular weight 1931.6876 Da, oligosaccharide composition Hex ₅ HexNAc ₄ NeuAc ₁ ), single sialoxidation single in the first quadrupole (Q1) of the quadrupole mass spectrometer (QqQ-MS) Corresponds to fucosylated complex N- sugar chains (theoretical molecular weight 2077.7455 Da, oligosaccharide composition Hex ₅ HexNAc ₄ Fuc ₁ NeuAc ₁ ), dual sialic acid complex N-glycosyl (theoretical molecular weight 2222.7830 Da, oligosaccharide composition Hex ₅ HexNAc ₄ NeuAc ₂ ) Selecting and passing ions to pass;

c) generating a fragment ion by fragmenting by applying specific energy to a molecule selected from a collision cell (ie, a second quadrupole; q2);

d) In the third quadrupole (Q3), only fragment ions specific to the target material (theoretical molecular weight 203.0794 Da, oligosaccharide composition HexNAc ₁ / theoretical molecular weight 365.1322 Da, oligosaccharide composition Hex ₁ HexNAc ₁ ) among the fragment ions generated in the collision cell pass through To detect;

e) comparing the area ratio of chromatograms between isomers of the fragment ions obtained in step d); and relates to a method for quantitative analysis of N-saccharides containing sialic acid.

In addition, in the present invention, the step of comparing the ratio of the chromatogram area between the isomers of the fragment ions obtained in step e) in step d) is a peak of six isomers corresponding to Hex ₅ HexNAc ₄ NeuAc ₁ , Hex ₅ HexNAc ₄ Fuc ₁ NeuAc two isomers for the three isomers peak or Hex ₅ HexNAc ₄ NeuAc ₂ corresponding to the _first peak deriving the ratio of each area of the chromatogram; And at least _one of the first isomer of Hex ₅ HexNAc ₄ NeuAc ₁ , the second isomer, and the first isomer of Hex ₅ HexNAc ₄ NeuAc ₂ , which is a marker of Behcet's disease patients in the area ratio of each target oligomeric peak derived A result interpretation step of comparing and comparing the difference between the patient group sample and the normal control sample other than Behcet's disease;

f) when compared to the normal control group, when the patient group shows a difference of 5 times or more of the expression level, identifying with Behcet's disease; relates to a specific Behcet's disease screening analysis method.

For reference, the " first " isomer and the " second " isomer are expressions based on the order in which they are eluted from the column, and are used in the same meaning in the following description and claims.

In the conventional Behcet's disease diagnosis method, it is possible to draw an inaccurate result using a standardized classification table that standardizes the symptoms and prognosis results of existing clinical patients. The present invention specifically diagnoses Behcet's disease by using a relatively small amount of body fluid, preferably 50 microliters (μl) of plasma, to determine the ratio of quantitative values of N-glycomeric isomers containing sialic acid. As a method, it can be used to measure disease activity and develop specific therapeutic agents.

In the present invention, since only N-sugar chains containing sialic acid as a target in a plasma sample are separated and subjected to purification and concentration steps, it can be accurately quantitatively analyzed with high sensitivity without other interfering substances.

In addition, since the present invention separates structural isomers of oligosaccharides through porous graphitized carbon-based liquid chromatography, it is quantitatively analyzed based on multiple reaction monitoring mass spectrometry, so that each oligomeric isomer can be specifically quantified.

In addition, the present invention is an analysis method for comparing the frequency and expression level between isomers by converting the area values of the isomers to ratios rather than the absolute quantification method through the quantitative curve of the standard product. ) It is a new data interpretation method that can overcome the limitations of the field.

The specific diagnostic method of the present invention can not only solve the cost problem of purchasing a standard product necessary for quantification, but also can be used by anyone in hospitals and medical institutions to diagnose Behcet's disease in the future because it is a method to convert at an accurate rate. This is a useful method.

The present invention relates to a method for diagnosing Behcet's disease using a small amount of serum, and is very useful in a situation in which there is currently no method for specifically diagnosing Behcet's disease.

The method of the present invention separates the total N-sugar chain of the specific protein from the extract, and separates the N-sugar chain isomer peak using a triple quadruple LC / MS analyzer and a multiple reaction monitoring analysis method and compares the ratio between peaks. Therefore, it is an analysis of the presence or absence of a disease, and it is a new method that cannot be found in disease diagnosis methods to date. In addition, the method of the present invention is a convenient method that does not have any inconvenience such as biopsy tests for collecting organs and tissues, and does not have the inconvenience and burden of preparing a standard that is absolutely necessary in quantitative analysis.

1 is a flow chart schematically illustrating an embodiment of a method for diagnosing Behcet's disease using a relative quantitation of the N-glycomeric isomer of the present invention.
2 is a representative chromatogram result of isolating and analyzing oligomeric isomers containing sialic acid using the plasma of normal subjects and Behcet's disease patients as a sample by the method of the present invention.
Figure 3 is a result of comparing the ratio of quantitative values of oligomeric isomers containing sialic acid in normal and Behcet's disease patients with the method of the present invention.
Figure 4 is a single sialic acid complex N-glycan (Mono-sialylated complex-type N-glycan) Hex ₅ HexNAc ₄ NeuAc ₁ of the first and second isomers, double sial oxidation confirmed in the sample of 90 people with normal and Behcet's disease Box whiskers plot for the first isomer ratio of a di-sialylated complex-type N-glycan Hex ₅ HexNAc ₄ NeuAc ₂ .
5 is a chromatogram result confirming the isomer structure of the specific sugar chain of Behcet's disease.
Figure 6 is the first isomer of Mono-sialylated complex-type N-glycan (Hex ₅ HexNAc ₄ NeuAc ₁ ), an indicator of Behcet's disease patients, Di-sialylated complex-type N-glycan (Hex ₅ HexNAc ₄ NeuAc ₂ ) Is the result of Receiver Operating Characteristic (ROC) Curve analysis showing the comparison of the difference between the plasma of the first isomer of Behcet's disease patient and the person who is not Behcet's disease.
7 schematically illustrates the screening method of the present invention.
Figure 8 is a schematic diagram showing the three N- sugar chain isomers selected as markers of Behcet's disease through the method of the present invention.
2,4,5,7,8, etc., the green circle represents mannose (Man), the yellow circle represents galactose (Gal), and "OAc" means O-acetylation, and the blue square is N-acetyl Glucosamine (GlcNAc), the red triangle represents fucose (Fuc), and the purple rhombus represents N-acetylneuraminic acid (NeuAc) (also called sialic acid).

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific examples. However, the examples are merely illustrative of the present invention, it is obvious to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited only to the description of the examples.

1. Sample preparation

1) Protein denaturation reagent was added to 50 μl of human plasma, and the protein was denatured by treatment at 90 to 110 ° C. for 1 to 3 minutes.

2) 2 μl (1000 units) of PNGase F was added to the sample subjected to step 1), and the enzyme reaction was performed at 37 ° C. for 16 hours in a constant temperature water bath.

3) 800 µl of cold ethanol (-42 ° C) was added to the sample that was subjected to step 2), and then left at -20 to -80 ° C for 1 hour, followed by centrifugation (14,000 rpm, 4 ° C, 10 to 30 minutes), The isolated protein was removed.

4) Sialic acid by eluting the sample more than 2 times with 2.5 ~ 3.5ml of 40% acetonitrile solution containing 0.05% trifluoroacetic acid through a solid phase extraction method using a porous graphitic carbon ("PGC") cartridge The sugar chain containing was separated and concentrated.

2. Analysis method

1) Quantitative analysis was performed using a multiple reaction monitoring (MRM) of LC / QqQ MS (Triple Quadrupole MS) for a oligosaccharide sample containing sialic acid obtained in the above pretreatment. More specifically, oligosaccharides containing diluted sialic acid were analyzed using an UPLC-MS / MS system consisting of an autosampler (maintained at 4 ° C.), an ultra high pressure pump and a column oven. A PGC column (100 × 2.1 mm, 3 μm) was used, and separation by chromatography was performed according to optimized separation conditions. Briefly, after loading the sample, 0.3 ml per minute of a gradient gradient solution of oligosaccharides containing sialic acid was flowed. For the concentration gradient solution, (A) 3.0% acetonitrile solvent containing 0.1% formic acid (v / v) and (B) 90% acetonitrile solvent containing 0.1% formic acid (v / v) were used. Used in concentration: 0 to 0.25 min; 7% B, 0.25-10 min; 7-15% B, 25-30 min; 15-40% B, 30-35 min; 40-100% B. Finally, the analytical column was re-equilibrated with 7% B for 10 minutes. The column temperature was maintained as follows: 30-60 ° C. Table 1 shows an example of the concentration gradient.

Time (min) Solvent B (%) Flow rate (ml / min) 0 7 0.3 0.25 7 0.3 10 15 0.3 25 40 0.3 30 100 0.3 35 100 0.3 35.1 7 0.3 45 7 0.3

2) Multi-reaction monitoring (MRM) is one of the selective methods available in QqQ MS. In the first quadrupole, only molecules of the substance to be analyzed are passed through, resulting in collision cells (ie, second 4). Medium electrode) and applying specific energy in the collision cell to fragment selected molecules to generate fragment ions. In the third quadrupole, only fragment ions specific to the target substance are detected from the fragment ions generated in the collision cell. When using multi-reaction monitoring, it is possible to minimize the influence on the interfering substances in the matrix, so that the target substances can be analyzed with higher sensitivity, and when monitoring a plurality of fragment ions, it can also be used to quantify substances through their area ratio. Table 2 shows the MRM transition conditions for quantitative analysis of the present isomers.

Target (Hex_HexNAc_Fuc_NeuAc) Quantitative ion Qualitative ion 5_4_0_1 644.9-> 366.1 644.9-> 274.1 5_4_1_1 1039.9-> 366.1 1039.9-> 274.1 5_4_0_2 741.9-> 366.1 741.9-> 274.1

3) The analyzed data were processed using Masshunter software version 7.

4) To investigate the difference and intensity between the control group and the patient group, it was performed using the Student's t-test analysis method. In addition, it was determined that it is meaningful when the P-value <10 ^-20 .

Hereinafter, FIGS. 2 to 3 showing the results obtained through the above sample preparation and analysis method will be described.

Figure 1: Acid N- in serum Our own  Purification and analysis steps

1 is a flow chart of the entire process corresponding to the acidic N- sugar chain separation and analysis of the present invention. In this method, the step of isolating the N-glycoside of the glycoprotein contained in human serum by an enzyme called N-glycosidase (PNGase F) is primarily carried out. N- sugar chains separated from proteins by enzymes are fractionated by the difference in size and polarity of oligosaccharides by solvents of different polarity based on cartridges filled with PGC, and in one preferred embodiment, 40% aceto containing 0.05% TFA It is analyzed by selectively eluting only acidic sugar chains with nitrile aqueous solution. The isomers of the separated and concentrated acidic sugar chains are separated by PGC-LC and subjected to relative quantification through the area value of the target sugar chains by MRM mass spectrometry.

Figure 2: serum acid N- of Behcet's disease patients and normal people Our chain  Profiling

Serum from 47 patients with Behcet's disease (26 males; 21 females) and 47 healthy control serums (26 males; 21 females) were used for our chain-based Behcet's disease diagnosis study.

A representative chromatogram of PGC-LC / MRM-MS analysis of acidic sugar chains isolated from serum of normal and Behcet's disease patients according to an embodiment of the present invention is shown in FIG. 2.

Mono-sialylated complex-type N-glycan Hex ₅ HexNAc ₄ NeuAc ₁ , a single sialoxidation single fucosylated complex N-glycosyl ( Mono-sialylated mono-fucosylated complex-type N-glycan) Hex ₅ HexNAc ₄ Fuc ₁ NeuAc ₁ , double sialicated complex-type N-glycan Hex ₅ HexNAc ₄ NeuAc ₂ isomers Eleven chromatographic peaks (Peaks) were identified. The number of oligomeric isomers in the patient group and the normal control group was similar, but the ionic strength, i.e., the ionic strength at the three peaks corresponding to the first and second isomers of Hex ₅ HexNAc ₄ NeuAc ₁ and the first isomer of Hex ₅ HexNAc ₄ NeuAc ₂ , The difference between the area values was confirmed to confirm the possibility as a marker to distinguish the patient from the normal.

Figure 3: Comparison between Behcet's disease patients and normal through ratio of each isomer area

For direct comparison between Behcet's disease patients and normal controls, the area values of each isomer observed were normalized by the total area value of each structural isomer. No statistically significant difference occurred when comparing patients and controls for each N-glycan structure excluding isomer information, and statistical analysis confirming the difference between patients and controls when the area values of each structural isomer were directly compared (t-test ), The result value was lower than the data interpretation of the present invention. However, the result obtained through the method of the present invention can be confirmed that the P-value of the isomer showing a difference between normal and patient is 10 ^-20 or less.

Figure 4: In Behcet's disease patients Marker  Isomer changes

Figure 5 is between the patient and the control group with the first and second isomers, Box Lewis coarse plots (box whiskers) for the first isomer ratio of Hex ₅ HexNAc ₄ NeuAc ₂ of Hex ₅ HexNAc ₄ NeuAc ₁ make a total of 94 people Sample It shows a dramatic difference. These are isomers of oligosaccharides composed of Hex ₅ HexNAc ₄ NeuAc _{1 ~ 2} , which increased 4-5 times in patients than in normal cases. Interestingly, this difference was more apparent in the male group than in the female group. The P-values representing the difference between the three isomers of Behcet's disease and the control group were found to be 10 ^-20 or more, and these three isomers were determined as diagnostic markers of Behcet's disease.

Figure 5: Behcet's disease specific Our chain  Structure check

The sialic acid of the main N-glycoside present in serum has α-2,3 or α-2,6 binding to the terminal galactose. In order to reveal the structure of the specific chain of Behcet's disease identified in the present invention, a sialic acid degrading enzyme (Neuraminidase or Sialidase) treatment was performed on an N- sugar chain sample containing sialic acid. 5 is a chromatogram comparing before and after treatment with α-2,3 structure-specific degrading enzyme. Enzyme hayeoteum-treated (upper) enzyme treatment after (bottom) Hex ₅ HexNAc ₄ 1 peak reduction times has a structure of NeuAc structure 1 and 2 times the peak, and Hex ₅ HexNAc with a _{1 4} NeuAc ₂ as compared to can see. From these results, it can be seen that the oligomeric isomer of the Behcet's disease diagnostic marker contains sialic acid bound to α-2,3.

Figure 6: Evaluation of Behcet's disease diagnosis method

A tool for evaluating the performance of biomarkers for disease detection is the ROC (Receiver Operating Characteristic) curve. The ROC curve with the derived AUC result value of 0.6 or more is judged to have validity to distinguish between normal and patient. The first and second isomers of the single sialic acid complex N- sugar chain Hex ₅ HexNAc ₄ NeuAc ₁ selected as the biomarker of Behcet's disease patients in the present invention, the _first of the double sialic acid complex N- sugar chain Hex ₅ HexNAc ₄ NeuAc ₂ The AUC was calculated through the ROC curve for the ratio of the second isomer. As a result, all three types of oligosaccharide markers selected from the present invention showed excellent results of 0.98 or more. These results indicate that the method and marker for diagnosis of Behcet's disease of the present invention are useful in determining Behcet's disease.

Figure 7: Our chain  Behcet's disease diagnosis method by isomer profiling

In the method of the present invention, first, a small amount of human body fluid, preferably plasma or serum, is concentrated by N-glycosylation by enzymatic treatment, etc., and then the isomer isomers are separated and MRM mass spectrometry is performed on the separated isomers. By quantifying the ratio of isomers, it is to diagnose whether a person has a disease.

Figure 8: Behcet's disease With marker  Selected N- Our chain  3 isomers

Through the mass values of the isomers confirmed by mass spectrometry, it is possible to confirm the constituents of sugars constituting the sugar chain. In addition, as a result of further confirming through an enzyme treatment for separating sialic acid having a specific structure, referring to an existing paper in which N- sugar chains were separated and analyzed using a PGC column, FIG. 8 It can be described as At this time, 5401-1 and 5401-2 are anomer isomer relations due to the formation of hemiacetal ring structures of monosaccharides.

That is, as a specific example in the disease screening method using the N-glycomeric isomer of the present invention, the N-glycomeric isomer marker that can be used for Behcet's disease screening, as shown in FIG. 8, the terminal sialic acid in the N-glycomeric composition Hex ₅ HexNAc ₄ NeuAc ₁ Of the isomers linked by (α 2, 3) bonds with the terminal galactose, two anomer isomers resulting from the formation of a hemiacetal ring structure of monosaccharides and oligosaccharide composition Hex ₅ HexNAc ₄ NeuAc ₂ double sialic acid complex N- Of the di-sialylated complex-type N-glycan isomers, two or more terminal sialic acids are selected from isomers linked by two (α 2, 3) and (α 2, 6) bonds with two terminal galactose. It is characterized by.

Claims (9)

A) obtaining a body fluid from a specific disease patient group and a normal control group, and denaturing the protein in the body fluid to prepare a sample;
B) separating the N-saccharide chain from the sample by treating the sample with an enzyme;
C) purifying or concentrating the separated N-saccharide chain;
D) mass spectrometry of the purified or concentrated N-glycosyl through the steps of a) to d) below with a triple quadruple LC / MS analyzer and multiple reaction monitoring analysis method;
a) separating sialic acid-containing N-sugar chains into sialic acid-containing N-sugar chains having the same sugar composition and the same molecular weight with a porous graphitized carbon packed column using a concentration gradient elution;
b) selecting and passing one of the ions corresponding to each sialic acid-containing N- sugar chain separated in step a) in the first quadrupole of the triple quadruple LC / MS analyzer;
c) generating a fragment ion by fragmenting ions corresponding to the sialic acid-containing N- sugar chain that has passed through the first quadrupole by applying specific energy in a collision cell that is a second quadrupole; And
d) obtaining a mass spectrometry chromatogram of sialic acid-containing N-glycosyl by detecting only fragment ions specific to the target substance among the fragment ions generated in the collision cell by passing through a third quadrupole;
E) comparing the peak area ratios of isomers showing differences between the patient group and the normal control group among the isomers which are peaks in the sialic acid-containing N- sugar chain mass spectrometry chromatogram obtained in step d) above; And
F) comparing the patient group and the normal control group with a specific N-glycan chain chromatogram peak quantitative value, selecting the specific N-glycan chain chromatogram peak as a marker of the specific disease.
Wherein b) a sialic acid-containing sugar chain of N- phase, the composition of the oligosaccharide Hex ₅ HexNAc ₄ NeuAc ₁ is a single sialic oxide complex type N- linked sugar chains, the sugar chain composition of Hex ₅ HexNAc ₄ Fuc ₁ NeuAc ₁ is a single sialic oxide single pyuko acylated compound Type N- sugar chain and sugar chain composition Hex ₅ HexNAc ₄ NeuAc ₂ Double sialic acid complex N- sugar chain containing sialic acid N- sugar chain chromatogram peak characterized in that the disease screening method.
delete The method according to claim 1,
After step f) above
G) Performing steps a) to d) by taking a test sample of the body fluid of the test subject, and normal to the area ratio of the chromatogram peak selected as a marker for a specific disease in step f) among the chromatogram peaks obtained by mass spectrometry. Disease screening method using a sialic acid-containing N- sugar chain chromatogram peak further comprising the step of confirming that the test subject has a specific disease when there is a significant difference compared to the control value.
The method according to claim 1,
Step b) above
a) treating the sample with the enzyme PNGase F for 16 to 24 hours to separate the N-saccharide chain;
b) a) precipitating the protein in the sample that has been subjected to step a); And
c) centrifuging the sample in which the protein is precipitated and obtaining a supernatant; disease screening method using a sialic acid-containing N- sugar chain chromatogram peak, which comprises sequentially.
The method according to claim 1,
The specific disease is an autoimmune disease, the disease screening method using a sialic acid-containing N- sugar chain chromatogram peak.
The method according to claim 5,
The autoimmune disease is a disease screening method using a sialic acid-containing N- sugar chain chromatogram peak, characterized in that Behcet's disease.
delete The method according to claim 1,
The isomers appearing as peaks in the sialic acid-containing N- sugar chain mass spectrometry chromatogram of step E) are six isomers corresponding to Hex ₅ HexNAc ₄ NeuAc ₁ , three isomers corresponding to Hex ₅ HexNAc ₄ Fuc ₁ NeuAc ₁ and Hex ₅ HexNAc ₄ NeuAc _{2 A} screening method for disease using a sialic acid-containing N- sugar chain chromatogram peak, characterized in that it is at least one isomer selected from two isomers.
The method according to claim 1,
When the specific disease is Behcet's disease,
The marker of step f) is an anomaly due to the formation of a hemiacetal type ring structure of a monosaccharide among isomers in which terminal sialic acid is linked to (α 2, 3) by terminal sialic acid in the N- sugar chain composition Hex ₅ HexNAc ₄ NeuAc ₁ Two isomers and two oligosaccharides Hex ₅ HexNAc ₄ NeuAc ₂ of the two sialic acid complex N-glycan isomers of two terminal sialic acids with two terminal galactose and (α 2, 3) A disease screening method using a sialic acid-containing N- sugar chain chromatogram peak, characterized in that it is at least one selected from isomers linked by (α 2, 6) bonds.

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