KR101648926B1 - Method for analyzing n-glycans in complex biological fluids - Google Patents

Abstract

A method for analyzing N-glycan in a biological complex fluid comprises: a step of adding, to a sample including N-glycan, protein including first marker glycan having a mass different from that of the N-glycan; a step of denaturation of the protein in the sample after the step of adding the protein; a step of separating glycan in the sample from protein by using enzyme after the protein is denatured; a step of eluting glycan from the sample from which the protein is separated; a step of acquiring mass spectrum of the eluted glycan; and a step of determining the efficiency of the enzyme or operation on the basis of the amount of first marker glycan in the mass spectrum. According to the method, it is possible to separate and refine only pure N-glycan from various biological complex fluids including serum, and it is possible to improve reliability of data by maximizing efficiency and reproducibility for a short time by using a well plate.

Description

[0001] METHOD FOR ANALYZING N-GLYCANS IN COMPLEX BIOLOGICAL FLUIDS [0002]

Embodiments relate to methods for analyzing N-glycans in biological complex fluids, and techniques for separating and purifying only pure N-glycans from a variety of biological complex fluids including serum in a reproducible manner with high efficiency It is about.

Especially, mass spectrometry of analytical instruments has been actively developed for the diagnosis of various diseases by combining mass spectrometry. MALDI-TOF MS (MALDI-TOF MS) is a method capable of vaporizing and ionizing a polymeric material without decomposition of the sample. In general, the method of mass spectrometry It is known as a method that can be ideally applied to biopolymers or synthetic polymers that are large in mass and unstable to heat.

In order to analyze a small amount of N-glycan present in the serum of a sample using the merits of such MALDI-TOF MS, pure N-glycans should be obtained through various processes for the sample. However, due to the complexity of the process, there are many difficulties to quantitatively measure N-glycans or to realize repetitive reproducibility.

Japanese Patent Application Laid-Open No. 10-2013-0066481

One aspect of the present invention is to provide an optimized process for efficiently separating and concentrating N-glycan, which is bound to a glycoprotein present in serum, which is one of the most complex samples among biological fluids The purpose.

Another aspect of the present invention relates to a method for producing a protein, comprising the steps of: denaturing a protein required for separation and concentration of N-glycans; separating N-glycans from glycoproteins; precipitating and removing proteins; and solid phase extraction The goal is to optimize the purification process of glycans to have high throughput.

Another aspect of the present invention is to introduce a monitoring method capable of determining the optimization of each process described above using labeled glycans.

Further, one aspect of the present invention is to introduce a method for shortening the time required for the N-glycan separation and concentration process.

According to one embodiment, an N-glycan analysis method in a biological composite fluid comprises adding a first labeled glycan having a different mass to the N-glycan to a sample containing N-glycans Adding a protein to the protein; Denaturing the protein in the sample after the step of adding the protein; Separating the glycans in the sample from the protein using an enzyme after the protein is denatured; Eluting glycans from the sample from which the protein is isolated; Obtaining a mass spectrum of the eluted glycans; And determining the efficiency or the function of the enzyme based on the amount of the first labeled glycane in the mass spectrum.

In one embodiment, the protein comprising said first labeled glycan is Horse radish peroxidase.

According to one embodiment, the method for N-glycan analysis in a biological composite fluid comprises, prior to eluting the glycans, a second labeled glycan having a mass different from the N-glycan, Lt; / RTI > In addition, the method for N-glycan analysis in the biological complex fluid further comprises determining elution efficiency based on the amount of the second labeled glycans in the mass spectrum.

In one embodiment, the second labeled glycane is malto-hexose.

In one embodiment, the step of denaturing the protein in the sample comprises mixing the sample with a buffer solution comprising dithiothreitol and ammonium bicarbonate (NH ₄ CO ₃ ) .

In one embodiment, the pH of the buffer solution is from 7.5 to 9. Also in one embodiment, the concentration of dithiothreitol in the buffer solution is from 1 mM to 2 mM.

In one embodiment, denaturing the protein in the sample further comprises reacting the sample mixed with the buffer solution in a bioshaker at a temperature of 65 < 0 > C.

In one embodiment, the enzyme is a peptide N-glycosidase F, and the step of separating the glycans in the sample from the protein comprises contacting the sample with a 500 unit concentration of peptide N- 0.0 > F < / RTI >

In one embodiment, separating the glycans in the sample from the protein further comprises irradiating the sample mixed with the enzyme with microwave for 8 minutes at 400 W intensity.

In one embodiment, the step of eluting glycans from the sample comprises mixing the sample with a 20% concentration of acetonitrile solution.

According to one embodiment, the method for N-glycan analysis in a biological composite fluid further comprises the step of drying the sample at a temperature of 70 DEG C prior to obtaining the mass spectrum after eluting the glycan from the sample .

According to one aspect of the present invention, the N-glycan analysis method in a biological composite fluid can separate and purify pure N-glycan from a variety of biological complex fluids including serum, well plates can be used to maximize efficiency and reproducibility in a short time to improve data reliability. The above method can be widely used for diagnosis, such as cancer diagnosis.

1 is a schematic diagram showing a pretreatment process of a sample for N-glycan analysis according to an embodiment.
2 is a flow chart of a method for N-glycan analysis according to one embodiment.
FIG. 3 shows the mass spectrum according to the pH of an ammonium bicarbonate (NH ₄ CO ₃ ) buffer solution during protein denaturation.
Figure 4 shows the mass spectrum according to the concentration of dithiothreitol in the protein denaturation process.
Figure 5 shows the mass spectrum according to the concentration of acetonitrile solution in the course of glycan elution.
Figure 6a shows the mass spectrum obtained using a tube type cartridge.
6B shows a mass spectrum obtained using a 96 well type cartridge in accordance with one embodiment.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

FIG. 1 is a schematic diagram showing a pretreatment process of a sample for N-glycan analysis according to an embodiment, and FIG. 2 is a flowchart of an N-glycan analysis method according to an embodiment.

The N-glycan analysis method according to this embodiment will be described with reference to FIGS. 1 and 2. FIG. The N-glycan analysis method is intended to reproducibly separate and purify only pure N-glycans in the sample from samples of various biological complex fluids with high efficiency. The sample may be blood of a person or animal, or serum separated from blood. For example, the collected blood can be left at low temperature for a certain period of time for coagulation, and only the serum can be separated from the blood by centrifugation. Specifically, the blood is allowed to stand for 1 hour on ice to form a clot, and the serum can be separated by centrifugation at a temperature of about 4 for about 10 minutes at an acceleration of about 3500 g. If separate serum storage is required, the serum may be transferred to a sterile cryogenic tube and stored at a temperature of about -80 ° C.

The N-glycan analysis method according to the present embodiment is a method for analyzing a large number of sample fractions including a plurality of reaction regions (or wells) in a well plate ). ≪ / RTI > In this specification, the process of analyzing N-glycans by injecting a sample fraction into a 96-well plate containing 96 wells and treating it with an automated instrument will be described. Injection may mean injecting the sample directly into each well, or it may mean mounting a container containing the sample in each well when the sample is contained in another container such as a tube. On the other hand, the 96-well plate described herein is merely exemplary, and the type of well plate is not limited thereto.

As a specific pretreatment process, a protein containing the first labeled glycan can be added to the sample (S1). The first labeled glycan-containing protein is added in order to confirm the separation efficiency in the glycan separation process by an enzyme described later. The protein can be treated by the same enzyme as the protein in the sample, - a glycan of a different mass than the glycan (i.e., the first labeled glycan).

In one embodiment, the protein comprising the first marker glycan is horse radish peroxidase (HRP), a glycoprotein not present in mammalian biological fluids. For example, the samples can be dispensed in 50 uL aliquots into each well of a 96-well plate, and then 5 uL of HRP solution prepared at a concentration of 30 mg / mL can be dispensed into each well using water. HRP is a plant-derived protein and has a sugar called xylose, which has a different structure and mass from an animal-derived glycan. Since the glycans separated from the HRP by the enzymatic decomposition have different masses from the N-glycans isolated from the animal serum, they can serve as markers for determining the efficiency and / or the function of the enzyme. However, the kind of protein containing the first labeled glycan is not limited to HRP.

Next, the protein can be denatured by mixing the sample with dithiothreitol (S2). Dithiothreitol may be added to the sample by mixing with an ammonium bicarbonate (NH ₄ CO ₃ ) buffer solution. For example, a mixture of ammonium bicarbonate buffer solution prepared at a concentration of 200 mM and dithiothreitol at a concentration of 1 to 50 mM using water can be dispensed into each well of a 96-well plate in an amount of 50 uL. Next, the 96-well plate is mounted on a bioshaker, subjected to centrifugation and mixing, and protein denaturation can be carried out by rotating at a temperature of 65 ° C for 5 minutes at 1500 rpm. However, the specific operating conditions of the shaking incubator are not limited to those described above. After denaturation, the sample can be allowed to cool for about 5 minutes at room temperature.

In one embodiment, the pH of the ammonium bicarbonate buffer solution in the protein denaturation process can be adjusted in the range of 7.5 to 9, preferably 7.5. For example, the pH can be adjusted by injecting hydrochloric acid (HCl) at a concentration of about 10% in the buffer solution.

FIG. 3 shows the mass spectra of the ammonium peat ammonium buffer solution according to the pH in the protein denaturation process, and Table 1 below shows the mass spectra of the peaks of N-glycan main peaks And the correlation between them. 3, the N-glycans extracted through the pretreatment process are ionized by a matrix assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) This process will be described later in detail.

pH 6 pH 7 pH 7.5 pH 8 pH 9 pH 6 ㅡ 0.965 0.822 0.807 0.743 pH 7 0.965 ㅡ 0.936 0.931 0.891 pH 7.5 0.822 0.936 ㅡ 0.994 0.983 pH 8 0.807 0.931 0.994 ㅡ 0.993 pH 9 0.743 0.891 0.983 0.993 ㅡ

Referring to Table 1, the similarity of the main peak patterns was increased under the pH of the buffer solution of 7.5 to 9, and in particular, the main peak patterns obtained at the pH 7.5 and pH 9 conditions Similarly, a pH 8 condition is measured as the pH condition of the buffer solution most favorable for maintaining the peak pattern. As a result of measuring the pH change according to the storage state of the buffer solution, the pH was increased according to the temperature and the period. From the above results, it was found that when the pH of the ammonium bicarbonate buffer solution was 7.5, Respectively.

Also, in one embodiment, the concentration of dithiothreitol in the protein denaturation process can be adjusted from 1 mM to 2 mM, preferably 1 mM. FIG. 4 shows the mass spectrum according to the concentration of dithiothreitol in the protein denaturation process, and Table 2 shows the intensity of the main peak in the mass spectrum of FIG. In the tables of the present specification, n means the number of samples for obtaining data.

Dithiothreitol concentration 0.5 mM 1 mM 2 mM 10 mM Peak strength
(n = 4) 10% ACN
Use elution 1.8 x 10 ⁴ 6.6 x 10 ⁴ 6.6 x 10 ⁴ 5.4 × 10 ⁴ 20% ACN
Use elution 1.2 x 10 ⁴ 7.2 x 10 ⁴ 6.7 x 10 ⁴ 5.9 x 10 ⁴

Referring to Table 2, when the concentration of dithiothreitol was 1 mM to 2 mM, the intensity of the N-glycan main peak was large. In particular, when the concentration of dithiothreitol was 1 mM, I could. As shown in Table 2, the peak intensity is also affected by the concentration of acetonitrile (ACN) solution used for elution, which will be described in detail below.

In one embodiment, protein denaturation is performed using a shaking incubator while appropriately controlling the temperature and operating time of the shaking incubator. Conventionally, protein denaturation is performed in a high-temperature water bath. However, the tube containing the sample is expanded and contracted due to the high temperature and low temperature crossing in the water tank. In this process, . In addition, it is difficult to control the temperature in the method using the water tank. At a high temperature of 80 ° C or higher, the possibility of opening the tube cap is high and protein aggregation phenomenon is severe. it's difficult. In this example using a shaker incubator in place of the aquarium, the temperature of the shaker incubator is maintained at 65 DEG C and the time to perform protein denaturation in the shaker incubator is adjusted to 5 minutes.

The following Table 3 shows the similarity of the peak pattern with the operating temperature and time of the shaking incubator when compared with the peak pattern obtained by performing protein denaturation in a water bath at 95 캜 as in the prior art.

Time / Temperature 65 75 85 5min 0.992 0.975 0.925 10 min 0.989 0.950 0.910 20min 0.987 0.939 0.852

As shown in the figure, when protein denaturation was carried out using a shaking incubator at a temperature of 65 ° C for 5 minutes, it was possible to obtain a virtually similar peak pattern as in the conventional method using a water bath.

Table 4 below compares the case of performing protein denaturation in a water bath at 95 ° C as in the conventional method and the case of performing protein denaturation at 65 ° C for 5 minutes using a shaking incubator according to the present embodiment.

Conventional (aquarium) example Example Temperature (n = 3) 95 / 20.5 DEG C 65 ℃ time 3 minutes 5 minutes Major peak intensity 4.5 x 10 ⁴ 4.8 × 10 ⁴ Relative standard deviation ( RSD ) 2.8 1.64

As shown in Table 4, by using the present embodiment, the peak intensity is increased and the relative standard deviation (RSD) of the peaks are reduced compared to the prior art, and the reproducibility and efficiency of the analysis are improved.

Referring again to FIGS. 1 and 2, glycans can be separated from proteins by mixing the protein-denatured sample with an enzyme (S3). In one embodiment, a peptide N-glycosidase F (PNGase F) enzyme is injected into a sample and glycans can be cleaved by enzyme blending. As the buffer, the above-described ammonium peatate solution is used . For example, 2 uL of a PNGase F enzyme solution at a concentration of 250 to 1000 units and 2 uL of ammonium citrate buffer at a concentration of 200 mM and pH 7.5 were mixed and stored at 4 until use. After adding 4 uL of the mixed solution, the mixture can be reacted by centrifugation and mixing.

Table 5 shows the main peak intensities of the mass spectra according to the concentration of PNGase F in the enzyme reaction. As shown in the table, when the concentration of PNGase F is 500 units, peak having the strongest intensity can be obtained.

PNGase F concentration 1000 units (n = 4) 500 units (n = 4) 250 units (n = 4) Major peak intensity 7.5 x 10 ⁴ 7.8 x 10 ⁴ 7.6 × 10 ⁴

In one embodiment, an enzymatic reaction is performed while irradiating a 96 well plate with microwaves. Table 6 below shows the major peaks of the mass spectra according to temperature, microwave intensity and irradiation time during the enzyme reaction. As shown in Table 6, in order to obtain the optimum efficiency, the microwave can be irradiated at a temperature of 37 DEG C at 400 W for 8 minutes.

Microwave intensity
And temperature 350W, 37 400W, 37 microwave
Investigation time 9 minutes 10 minutes 8 minutes 9 minutes 10 minutes Major peak intensity
(n = 8) 3.7 × 10 ⁴ 3.6 × 10 ⁴ 4.1 x 10 ⁴ 3.6 × 10 ⁴ 3.7 × 10 ⁴

Table 7 below compares the case where the enzymatic reaction was performed overnight in the water bath and the case where the microwave of 400 W intensity was irradiated for 8 minutes according to this embodiment.

Conventional (aquarium) example Example Temperature 37 37 ℃ Century / Hour Overnight (16 hours) 400W / 8 min Major peak intensity
(n = 8) 6.9 × 10 ⁴ 7.4 × 10 ⁴

As shown in Table 7, by using this embodiment, it is possible to obtain a peak of a larger intensity while performing an enzymatic reaction for a remarkably short time compared with the conventional method.

Referring again to FIGS. 1 and 2, proteins can be precipitated using ethanol after the enzymatic reaction, and the precipitated proteins can be removed from the sample (S4). Specifically, 450 uL of ethanol may be added to each well of the 96 well plate in which the enzymatic reaction has been completed. The ethanol-added 96-well plate may be repeatedly vortexed several times (for example, three times) for about 1 to 2 seconds, followed by centrifugation at 4 ° C and 3,700 rpm for 50 minutes. After the proteins are precipitated by ethanol and centrifugation and the sample is separated into two layers, only 400 μL of the supernatant from each well can be transferred to another 96-well plate. As a result, the protein in the submerged solution is removed from the sample. In one embodiment, the protein-free sample may be dried in a concentrator for about one hour in a nitrogen stream, and the dried 96-well plate may be stored at -20 ° C for subsequent processing.

In one embodiment, a second labeled glycan is added to the sample from which the protein has been removed (S5). The second labeled glycans, like the first labeled glycans, have a mass different from that of the N-glycans in the sample and are added to the sample for the purpose of measuring the efficiency of the solid phase extraction (SPE) process described below. The second labeled glycane added to the sample before SPE can be analyzed with serum-derived glycans after SPE to track changes in the relative amounts of serum glycans to determine the change in SPE efficiency. In one embodiment, the second labeled glycane may be malto-hexose. For example, maltose sources prepared at a concentration of 100 ug / mL using water can be dispensed into each well of a 96-well plate in 5 uL. However, the type of the second labeled glycans is not limited to maltose.

Next, glycans can be eluted from the sample by the SPE process (S6). For the SPE process, conditioning can be accomplished by mounting a 96 well type cartridge in a vacuum manifold and serially flushing the following solutions into a 96 well type cartridge.

end. Deionized water 3 times in 1 mL increments

I. 1 mL of 80% acetonitrile (ACN) solution containing 0.1% Trifluoacetic acid (TFA)

All. 3 times with 1 mL of deionized water

Thereafter, the sample contained in the 96 well plate is loaded into the 96 well type cartridge. Before loading, 500 μL of deionized water is added to the sample, stirred, and centrifuged for 10 seconds. In addition, deionized water may be poured into the 96-well type cartridge that has been loaded for 3 times in 1 mL increments for cleaning.

Next, an ACN solution for glycan elution can be injected into the sample using a vacuum manifold. After the eluted sample is completely dried, it can be re-dissolved by adding 15 μL to the dried sample and transferred to a plate for analysis. In one embodiment, the concentration of the ACN solution for elution is 20%. For example, 20% ACN solution can be injected into the sample twice in 1 mL increments.

5 shows a mass spectrum according to the concentration of the ACN solution in the course of glycan elution. FIG. 5 (a) shows the mass spectrum of the glycans eluted using a 10% ACN solution, 5 (b) shows the mass spectrum of the glycans eluted using a 20% ACN solution. As shown by the red circle, there is a change in the relative amount of glycans in the mass spectrum depending on the concentration of the ACN solution used in the elution. When using 20% ACN solution, the amount of glycans actually present in the sample Of the respondents.

Table 8 shows the number and intensity of M + Na (M: Ar atom) peaks in the mass spectrum depending on the concentration and composition of the ACN solution used for elution. When using 20% ACN solution, It is possible to confirm that the peak intensity is also large.

The solution used for elution 10% CAN 20% ACN after 10% ACN 10% ACN
: 20ACN = 1: 1 15% ACN 20% ACN M + Na peak number 50 0 40 to 50 45 to 50 45 to 50 Peak strength 5.9 x 10 ⁴ 8.4 × 10 ⁴ 5.0 × 10 ⁴ 6.1 × 10 ⁴ 6.5 x 10 ⁴

In one embodiment, drying of the sample after the SPE process is at a temperature of 70 ° C. Table 9 shows the characteristics of peaks of the mass spectrum according to the drying temperature of the sample. It is known that when the sample is dried at a temperature of 70 캜, the relative standard deviation of the peaks is small and thus the reproducibility of the analysis is high and the drying time is low .

Drying temperature 37 ℃ 60 ° C 70 ℃ Relative standard deviation
(RSD,%) (n = 4) 13.5 8.0 7.0 Relative standard deviation
(RSD,%) (n = 9) 4.5 2.4 2.9 Relative standard deviation average 9.00 5.20 4.95 Drying time 5 hours 3.5 hours 3 hours

Also, in one embodiment, the properties of the column used in the SPE procedure can be adjusted to optimize for the analysis of N-glycans. Table 10 below shows the relative standard deviations and intensities of N-glycan peaks according to the column characteristics used.

Bed mass (mg) / volume (ml) Graphitized carbon
(Particle size 38-125 [mu] m) 150 mg / 4 ml Ultra-pure graphitized carbon 250mg / 6ml 100% porous graphitic carbon (PGC)
(Particle size 30-40 mu m, pore size 250 ANGSTROM) 50 mg / 1 ml 100% porous graphitic carbon (PGC)
(Particle size 30-40 m, pore size 250 Å) 100 mg / 1 ml Relative standard deviation
(RSD,%) (n = 4) 4.2 4.32 1.81 2.65 Peak strength 3.0 x 10 ⁴ 1.0 x 10 ⁴ 6.3 x 10 ⁴ 5.5 × 10 ⁴

Next, the glycans eluted by mass spectrometry can be analyzed (S7). At this time, the enzyme activity and elution efficiency can be evaluated in the N-glycan analysis method according to the present embodiment based on the amounts of the first and second labeled glycans analyzed (S8). Since the first and second labeled glycans have masses different from those of the N-glycans contained in human or animal serum, the first and second labeled glycans can be easily identified in the mass spectrum. The enzyme activity efficiency can be evaluated in the N-glycan analysis method according to the present embodiment based on the peak of the first labeled glycan in the mass spectrum. In addition, the efficiency of the SPE process can be evaluated in the N-glycan analysis method according to this embodiment based on the peak of the second labeled glycan in the mass spectrum. This efficiency evaluation is used as information for optimizing conditions such as reaction material, temperature and time involved in each process.

Analysis of serum-derived N-glycans and first and second labeled glycans is accomplished by obtaining the mass spectrum of the sample by MALDI-TOF MS. Specifically, first, the glycans eluted from the sample are mixed with a predetermined matrix material. The matrix material is an easily ionized material that absorbs energy from the laser, and MALDI-TOF MS is configured to indirectly ionize the sample by ion transfer process using a matrix. For example, the matrix material may be an organic compound having a structure that is easily excited by a laser, or may be an aromatic organic compound. In addition, a mixture of two or more materials may be used as the matrix material so that the sample can be dissolved well while dissolving the organic compound well.

Next, the ionized sample fragments are moved by the electric field using the matrix material, and the molecular weight distribution and the relative intensities of the fragments having the mass (m / z) relative to the specific charge amount are obtained as mass spectra have. However, in the embodiments of the present invention, the method of obtaining the mass spectrum of the eluted glycans is not limited to MALDI-TOF MS, and can be performed by, for example, Fourier Transform Ion Cyclotron Resonance (FT-ICR) Other mass spectrometry methods not described herein may be used to derive mass spectra.

In an embodiment of the present invention, the elution process is different from the conventional glycan elution process using a tube type cartridge in that a 96-well type cartridge is used. Figure 6a shows the mass spectrum obtained using a tube type cartridge and Figure 6b shows the mass spectrum obtained using a 96 well type cartridge according to this embodiment. No difference in mass spectral peak pattern due to the type of cartridge was observed as shown.

The following Table 11 shows the relative standard deviations of the mass spectra of FIGS. 6A and 6B for verification of the assay, showing reproducibility of less than 10% for both tube type and 96 well type cartridges , And it is confirmed that a case of using a 96 well type cartridge is numerically better than that of a 96 well type cartridge.

Relative standard deviation (%) Using tube type cartridges Using 96 well type cartridges Day 1 9.81 (n = 48) 5.39 (n = 39) Day 2 8.30 (n = 48) 5.41 (n = 39) Each primary average 9.06 5.40

In the present specification, the N-glycan analysis method according to the embodiments has been described with reference to the flowcharts shown in the drawings. While the above method has been shown and described as a series of blocks for purposes of simplicity, it is to be understood that the invention is not limited to the order of the blocks, and that some blocks may be present in different orders and in different orders from that shown and described herein And various other branches, flow paths, and sequences of blocks that achieve the same or similar results may be implemented. Also, not all illustrated blocks may be required for implementation of the methods described herein.

While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the scope of the invention as defined by the appended claims. will be. However, it should be understood that such modifications are within the technical scope of the present invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (12)

Adding to the sample containing N-glycan a protein comprising a first labeled glycan not present in the sample and having a mass different from the N-glycan;
Denaturing the protein in the sample after the step of adding the protein;
Separating the N-glycan and the first labeled glycans in the sample from the protein using an enzyme after the protein is denatured;
Eluting the N-glycan and the first labeled glycan from the sample from which the protein is separated;
Obtaining a mass spectrum of the eluted N-glycan and the first labeled glycan;
Determining the efficiency or the function of the enzyme based on the amount of the first labeled glycane in the mass spectrum; And
A method for analyzing N-glycans in a biological composite fluid, comprising the step of controlling the reaction material, temperature or time of separation from the protein based on the determined efficiency or the function of the enzyme.
The method according to claim 1,
Wherein the protein comprising the first labeled glycan is a horseradish peroxidase.
The method according to claim 1,
Further comprising adding to the sample from which the protein has been removed a second labeled glycan not present in the sample and having a different mass than the N-glycan, prior to the eluting step,
Determining elution efficiency based on the amount of the second labeled glycane in the mass spectrum; And
Further comprising the step of adjusting the reaction material, temperature or time of the eluting step based on the determined elution efficiency.
The method of claim 3,
Wherein said second labeled glycan is a maltoxhose.
The method according to claim 1,
Wherein denaturing the protein in the sample comprises mixing the sample with a buffer solution comprising dithiothreitol and ammonium di-carbonate.
6. The method of claim 5,
Wherein the pH of the buffer solution is between 7.5 and 9.
6. The method of claim 5,
Wherein the concentration of dithiothreitol in the buffer solution is 1 mM to 2 mM.
6. The method of claim 5,
Wherein the step of denaturing the protein in the sample further comprises the step of reacting the sample mixed with the buffer solution in a shaking incubator at a temperature of 65 ° C.
The method according to claim 1,
The enzyme is the peptide N-glycosidase F,
Wherein said step of separating from said protein comprises the step of injecting a 500 unit concentration of peptide N-glycosidase F into said sample.
10. The method of claim 9,
Wherein separating from the protein further comprises irradiating the sample mixed with the enzyme with microwave for 8 minutes at 400 W intensity.
The method according to claim 1,
Wherein the eluting step comprises mixing the sample with an acetonitrile solution containing 20% by weight of acetonitrile.
The method according to claim 1,
Further comprising the step of drying the sample at a temperature of 70 DEG C before the step of obtaining the mass spectrum after the eluting step.

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