WO2017039123A1 - Method for analysing n-glycans in complex biological fluids - Google Patents

Abstract

A method for analysing N-glycans in complex biological fluids may comprise the steps of: adding protein to a sample including N-glycans, the protein including first marker glycans that have a different mass from the N-glycans; denaturing the protein in the sample after the step of adding the protein; separating glycans in the sample from the protein using an enzyme after the protein has denaturised; eluting glycans from the sample in which the protein is separated; obtaining a mass spectrum of the eluted glycans; and determining the efficiency or reaction of the enzyme based on the amount of first marker glycans in the mass spectrum. Using the method, pure N-glycans can be separated and refined from various complex biological fluids including serum, and data reliability can be improved by maximising efficiency and reproducibility in a short time using a well plate.

Description

How to Analyze N-Glycans in Biological Complex Fluids

Embodiments relate to methods for analyzing n-glycans in biological complex fluids, and to techniques for reproducibly separating and purifying only pure N-glycans with high efficiency from various biological complex fluids including serum. It is about.

[Description of National Support R & D]

This study was made with the support of the Ministry of Trade, Industry and Energy, Korea Institute of Industrial Technology Evaluation and Development (New Industrial Technology Development Project, Development of Blood Glucose Marker Diagnostic Kit for Early Diagnosis of Ovarian Cancer, Task No. 10045075) under the supervision of Asa Co., Ltd.

In particular, with the development of mass spectrometry (mass spectrometry) of the analyzer, the research to make a diagnosis by incorporating a mass spectrometer for the diagnosis of various diseases are being actively conducted. Dual Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) is a method that allows vaporization and ionization of polymer materials without decomposition of the sample. It is known as a method that can be ideally applied to a biomass or a synthetic polymer that is large in mass and heat-stable.

In order to analyze the small amount of N-glycan present in the sample serum by using the advantages of the MALDI-TOF MS, only pure N-glycan must be obtained through various processing procedures on the sample. However, due to the complexity of the process, there have been many difficulties to quantitatively measure N-glycans or to implement repetitive reproducibility.

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

In addition, an aspect of the present invention, protein denaturation necessary for the isolation and concentration of N-glycans, separation of N-glycans from sugar proteins, protein precipitation and removal, and N- by Solid Phase Extraction (SPE) It is aimed at optimizing the glycan purification process to have high throughput.

In addition, an aspect of the present invention, an object of the present invention is to introduce a monitoring method that can determine the optimization of each of the above-described process using a label glycan.

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

According to one embodiment, a method for analyzing an N-glycan in a biological complex fluid includes a first labeled glycan having a different mass from the N-glycan in a sample including the N-glycan Adding a protein to make; After the step of adding the protein, denaturing the protein in the sample; Separating the glycan from the sample from the protein using an enzyme after the protein is denatured; Eluting glycan from the sample from which the protein is separated; Obtaining a mass spectrum of eluted glycans; And determining the efficiency or function of the enzyme based on the amount of the first labeled glycan in the mass spectrum.

In one embodiment, the protein comprising the first label glycan is horse radish peroxidase.

According to one embodiment, a method for analyzing N-glycans in a biological complex fluid includes a second labeled glycan having a different mass from the N-glycans in the sample from which the protein has been removed prior to eluting the glycan It further comprises the step of adding. In addition, the method for analyzing N-glycans in the biological complex fluid further includes determining an elution efficiency based on the amount of the second labeled glycan in the mass spectrum.

In one embodiment, the second labeled glycan is Malto-hexose.

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

In one embodiment, the step of denaturing the protein in the sample further comprises the step of reacting the sample mixed with the buffer solution in a bioshaker at 65 ℃ temperature.

In one embodiment, the enzyme is peptide N-glycosidase F (peptide N-glycosidase F), and the step of separating the glycan in the sample from the protein, peptide N- at a concentration of 500 units (unit) in the sample Injecting glycosidase F.

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

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

According to one embodiment, the method for analyzing N-glycans in a biological complex fluid further comprises drying the sample at a temperature of 70 ° C. after eluting glycan from the sample and before obtaining the mass spectrum. Include.

According to the method for analyzing N-glycans in a biological complex fluid according to an aspect of the present invention, only pure N-glycan can be separated and purified from various biological complex fluids including serum, and the well plate ( Well plates can be used to improve data reliability by maximizing efficiency and reproducibility in a short time. The method may be widely used for methods 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 flowchart of an N-glycan analysis method according to an embodiment.

Figure 3 shows the mass spectrum according to the pH of the ammonium bicarbonate (NH ₄ CO ₃ ) buffer solution in the course of protein denaturation.

Figure 4 shows the mass spectrum according to the concentration of dithiothreitol in the course of protein denaturation.

Figure 5 shows the mass spectrum according to the concentration of acetonitrile solution in the glycan elution process.

6A shows the mass spectrum obtained using a tube type cartridge.

6B shows a mass spectrum obtained using a 96 well type cartridge, according to one embodiment.

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

1 is a schematic diagram illustrating 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.

With reference to FIG. 1 and FIG. 2, the N-glycan analysis method which concerns on a present Example is demonstrated. The N-glycan analysis method is for reproducibly separating and purifying only pure N-glycans in the sample with high efficiency from samples which are various biological complex fluids. The sample may be blood of human or animal, or serum separated from blood. For example, the collected blood can be left at low temperature for coagulation and then only centrifuged to separate serum from the blood. Specifically, the blood can be left on ice for 1 hour to allow clot formation, and then centrifuged at an acceleration of about 3500 g at a temperature of about 4 minutes to separate serum. If storage of the separated serum is necessary, the serum may be transferred to a sterile cryogenic tube and stored at a temperature of about 80 degrees below zero.

In the N-glycan analysis method according to the present embodiment, a well plate capable of simultaneously processing a large amount of sample aliquots in a batch mode including a plurality of reaction regions (or wells) ) May be performed using In the present specification, a process for analyzing N-glycans by injecting a sample aliquot into a 96 well plate including 96 wells and processing the same by an automated device will be described. In this case, the injection may mean directly injecting a sample into each well, or when the sample is contained in another container such as a tube, it may mean that the container containing the sample is mounted in each well. Meanwhile, the 96 well plates described herein are merely exemplary, and the type of the well plates is not limited thereto.

As a specific pretreatment process, first, a protein including the first labeled glycan may be added to the sample (S1). The protein containing the first labeled glycan is added to confirm the separation efficiency in the glycan separation process by the enzyme described below, wherein the protein can be processed by the same enzyme as the protein in the sample and N in the sample. -A glycan of a different mass than the glycan (ie, the first labeled glycan).

In one embodiment, the protein comprising the first labeled glycan is Horse radish peroxidase (HRP), a glycoprotein that is not present in an animal biological fluid. For example, after dispensing 50 uL of the sample into each well of a 96 well plate, 5 uL of HRP solution prepared at a concentration of 30 mg / mL using water may be dispensed into each well. HRP is a plant-derived protein with a sugar called xylose, and has a structure and mass different from that of animal-derived glycans. Glycans isolated by digesting HRP with enzymes have a different mass than N-glycans isolated from animal serum, which may serve as markers for determining the efficiency and / or action of the enzymes. However, the type of protein containing the first labeled glycan is not limited to HRP.

Next, the sample may be mixed with dithiothreitol to denature the protein (S2). Dithiothreitol can be added to the sample by mixing in ammonium bicarbonate (NH ₄ CO ₃ ) buffer solution. For example, a mixture of ammonium bicarbonate buffer solution prepared at a concentration of 200 mM with dithiothritol at a concentration of 1 to 50 mM can be dispensed into each well of a 96 well plate by using water. Next, protein denaturation may be performed by mounting a 96 well plate on a bioshaker, followed by centrifugation and mixing, and rotating at 1500 rpm for 5 minutes at a temperature of 65 ° C. However, the specific operating conditions of the shake incubator are not limited to those described above. After denaturation, the sample can be left to cool for about 5 minutes at room temperature.

In one embodiment, the pH of the ammonium bicarbonate buffer solution in the course of protein denaturation may be adjusted in the range of 7.5 to 9, preferably to 7.5. For example, adjustment of the pH can be achieved by injecting about 10% hydrochloric acid (HCl) into the buffer solution.

Figure 3 shows the mass spectrum according to the pH of the ammonium bicarbonate buffer solution in the process of protein denaturation, Table 1 below is the pattern of N-glycan main peak (peak) by the pH of the buffer solution in the mass spectrum of Figure 3 Shows the similarity (correlation) of. The peak of FIG. 3 is ionized and extracted by matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) of N-glycans extracted through pretreatment. It is obtained by analyzing, and this process is mentioned 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 pattern was increased in the pH of the buffer solution of 7.5 to 9, in particular, the main peak pattern obtained in pH 7.5 and pH 9 conditions compared to the peak pattern obtained in pH 8 conditions Similarly, the pH8 condition is measured as the pH condition of the buffer solution most desirable for maintaining the peak pattern. As a result of measuring the pH change according to the storage condition of the buffer solution, the pH was increased according to the temperature and duration. It can be seen that.

Also in one embodiment, the concentration of dithiothreitol in the course of protein denaturation may be adjusted to 1mM to 2mM, preferably 1mM. Figure 4 shows the mass spectrum according to the concentration of dithiothreitol in the process of protein denaturation, Table 2 below shows the intensity of the main peak in the mass spectrum of FIG. In addition, n in the tables of the present specification means the number of samples to obtain data.

Dithiothreitol Concentration		0.5mM	1 mM	2mM	10 mM
Peak intensity (n = 4)	10% ACN use elution	1.8 × 10 4	6.6 × 10 4	6.6 × 10 4	5.4 × 10 4
	Elution with 20% ACN	1.2 × 10 4	7.2 × 10 4	6.7 × 10 4	5.9 × 10 4

Referring to Table 2, when the concentration of dithiothreitol is 1mM to 2mM, the intensity of the N-glycan main peak is large, especially when the concentration of dithiothreitol is 1mM, the highest peak is obtained. Could. As shown in Table 2, the peak intensity is also affected by the concentration of acetonitrile (ACN) solution used for the elution, which will be described later in detail.

In one embodiment, protein denaturation is performed using a shaker while appropriately controlling the temperature and operating time of the shaker. In the past, protein denaturation was performed in a high temperature water bath, but due to the high temperature and low temperature crossing in the water bath, the tube containing the sample expands and contracts, and in this process, the tube stoppers are opened and foreign substances are introduced. There was a problem that occurred. In addition, it is difficult to control the temperature in the water tank method, and the tube plug is likely to be opened at a high temperature of 80 ° C. or higher, the aggregation of proteins is severe, and there are variables caused by the experimenter's manual work to improve accuracy and efficiency. It is difficult. In this embodiment using a shake incubator in place of the bath, the temperature of the shake incubator is maintained at 65 ° C., and the time for performing protein denaturation in the shake incubator is adjusted to 5 minutes.

Table 3 below shows the similarity of the peak pattern according to the operating temperature and time of the shaker as compared with the peak pattern obtained by performing protein denaturation in a 95 ° C water tank as in the prior art.

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

As shown, when protein denaturation was performed using a shake incubator at a temperature of 65 ° C. for 5 minutes, a peak pattern virtually similar to a method using a conventional water tank was obtained.

Table 4 below compares the case where protein denaturation was performed in a 95 ° C. water tank as in the prior art and when the protein denaturation was performed at 65 ° C. for 5 minutes using a shake incubator according to the present embodiment.

	Conventionally (water tank)	Example
Temperature (n = 3)	95 / 20.5 ℃	65 ℃
time	3 minutes	5 minutes
Main peak intensity	4.5 × 10 4	4.8 × 10 4
Relative standard deviation (RSD)	2.8	1.64

As shown in Table 4, it can be seen that by using the present example, the peak intensity was increased and the relative standard deviation (RSD) of the peaks was reduced as compared with the prior art, thereby improving the reproducibility and efficiency of the analysis.

Referring back to FIGS. 1 and 2, the glycan may be separated from the protein by mixing a sample in which the protein is denatured with an enzyme (S3). In one embodiment, a peptide N-glycosidase F (PNGase F) enzyme may be injected into a sample to cut glycans by enzymatic mixing, and the ammonium bicarbonate solution described above may be used as a buffer. Can be. For example, 2 uL of PNGase F enzyme solution at a concentration of 250 to 1000 units and 2 uL of ammonium bicarbonate buffer at 200 mM and pH 7.5 were mixed and stored at 4 until used, and then stored in each well of a 96 well plate. The mixed solution may be added by 4 uL and reacted by centrifugation and mixing.

Table 5 below shows the main peak intensities of the mass spectrum according to the concentration of PNGase F during the enzymatic reaction. As shown in the table, the highest intensity peak was obtained when the concentration of PNGase F was 500 units.

PNGase F concentration	1000unit (n = 4)	500unit (n = 4)	250unit (n = 4)
Main peak intensity	7.5 × 10 4	7.8 × 10 4	7.6 × 10 4

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

Microwave intensity and temperature	350 W, 37		400 W, 37
Microwave irradiation 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 × 10 4	3.6 × 10 4	3.7 × 10 4

Table 7 below compares the case where the enzyme reaction was performed overnight in a water tank as compared with the case where the microwave of 400W intensity was irradiated for 8 minutes according to the present embodiment.

	Conventionally (water tank)	Example
Temperature	37	37 ℃
Century / time	Overnight (16 hours)	400 W / 8 minutes
Major peak intensity (n = 8)	6.9 × 10 4	7.4 × 10 4

As shown in Table 7, by using this example, it is possible to obtain a higher intensity peak while performing the enzymatic reaction for a short time significantly compared to the conventional.

Referring back to Figures 1 and 2, after the enzyme reaction using ethanol to precipitate the protein and the precipitated protein 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 enzyme reaction is completed. After vortexing was repeated several times (for example, three times) for about 1 to 2 seconds after completion of ethanol addition, centrifugation may be performed for 50 minutes at 4 ° C and 3,700 rpm. If the protein is precipitated by ethanol and centrifugation to separate the sample into two layers, only 400 uL of supernatant in each well can be taken and transferred to another 96 well plate. As a result, the protein in the lower layer solution is removed from the sample. In one embodiment, the protein-depleted sample may be dried for about an hour in a concentrator under a nitrogen stream and the dried 96 well plate may be stored at minus 20 ° C. for subsequent processing.

In one embodiment, the second labeled glycan is added to the sample from which the protein is removed (S5). The second labeled glycan, like the first labeled glycan, has a different mass from the N-glycans in the sample and is added to the sample for the purpose of measuring the efficiency of the Solid Phase Extraction (SPE) process described below. The second labeled glycan added to the sample before SPE can be analyzed with the serum derived glycan after SPE to determine the change in SPE efficiency by tracking the change in the relative amount of serum glycans. In one embodiment, the second labeled glycan may be Malto-hexose. For example, maltohexose prepared at a concentration of 100 ug / mL with water may be dispensed 5 uL into each well of a 96 well plate. However, the kind of the second label glycan is not limited to maltohexose.

Next, the glycan may be eluted from the sample by the SPE process (S6). For the SPE process, conditioning can be achieved by mounting a 96 well type cartridge in a vacuum manifold, and then sequentially flowing the following solutions into a 96 well type cartridge.

end. 3 times with 1 mL of deionized water

I. 3x 1 mL of acetonitrile (ACN) solution at 80% concentration with 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. Prior to loading, 500 uL of deionized water may be added to the sample, followed by agitation and centrifugation for 10 seconds. In addition, 1 mL of deionized water may be flushed three times to a 96-well cartridge that has been loaded for washing.

Next, using a vacuum manifold, an ACN solution for glycan elution can be injected into the sample. The eluted sample can then be completely dried, then deionized and added to the dried sample again to add 15 uL to redissolve and transfer to the plate for analysis. In one embodiment, the concentration of ACN solution for elution is 20%. For example, a 20% concentration of ACN solution may be injected into the sample twice, 1 mL.

Figure 5 shows the mass spectrum according to the concentration of the ACN solution in the glycan elution process, Figure 5 (a) shows the mass spectrum of the glycan eluted using a 10% concentration of ACN solution, 5 (b) shows the mass spectrum of the glycan eluted using the 20% concentration of ACN solution. The relative amount of glycan in the mass spectrum varies with the concentration of the ACN solution used for the elution, as shown by the red circle, and the amount of glycan actually present in the sample when a 20% concentration of ACN solution is used. It appeared to reflect more accurately.

Table 8 below shows the number and intensity of M + Na (M: any atom) peaks appearing in the mass spectrum according to the concentration and composition of the ACN solution used for elution, and a large number when using a 20% concentration of ACN solution. It can be seen that the peak intensity is also large while detecting the peak of.

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 intensity	5.9 × 10 4	8.4 × 10 4	5.0 × 10 4	6.1 × 10 4	6.5 × 10 4

In one embodiment, the drying of the sample after the SPE process is at a temperature of 70 ° C. Table 9 shows the characteristics of the peaks of the mass spectrum according to the drying temperature of the sample. When the sample is dried at a temperature of 70 ° C., the relative standard deviation of the peaks is small, indicating that the analysis is reproducible and the drying time is short. Can be.

Drying temperature	37 ℃	60 ℃	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 mean	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 deviation and intensity of the N-glycan peaks according to the column characteristics used.

Bed Mass (mg) / Volume (ml)	Graphitized Carbon (Particle Size 38-125μm) 150mg / 4ml	Ultra-pure Graphitized Carbon 250mg / 6ml	100% porous graphitic carbon (PGC) (particle size 30-40μm, pore size 250mm) 50mg / 1ml	100% porous graphitic carbon (PGC) (particle size 30-40μm, pore size 250mm) 100mg / 1ml
Relative standard deviation (RSD,%) (n = 4)	4.2	4.32	1.81	2.65
Peak intensity	3.0 × 10 4	1.0 × 10 4	6.3 × 10 4	5.5 × 10 4

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

Analysis of serum-derived N-glycans and first and second labeled glycans is by obtaining a mass spectrum of the sample by MALDI-TOF MS. Specifically, first, the glycan eluted from the sample is mixed with a predetermined matrix material. The matrix material is a material that easily absorbs energy from the laser and ionizes, and the MALDI-TOF MS is configured to indirectly ionize the sample by an 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 substances may be used as the matrix material in order to dissolve the organic compound well and to dissolve the sample well.

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

Meanwhile, in one embodiment of the present invention, the elution process is different from the conventional glycan elution process using the tube type cartridge in that the 96 well type cartridge is used. FIG. 6A shows a mass spectrum obtained using a tube type cartridge, and FIG. 6B shows a mass spectrum obtained using a 96 well type cartridge according to this embodiment. As shown, no difference in mass spectral peak pattern due to the type of cartridge was observed.

Table 11 below shows the relative standard deviations of the mass spectra of FIGS. 6A and 6B for verification of the assay, showing a reproducibility of less than 10% in both a tube type cartridge and a 96 well type cartridge. It is confirmed that a slightly better result can be obtained numerically using a 96 well type cartridge.

	Relative standard deviation (%)
	Tube type cartridge	Use of 96-well cartridges
Day 1	9.81 (n = 48)	5.39 (n = 39)
Day two	8.30 (n = 48)	5.41 (n = 39)
Each primary mean	9.06	5.40

In the present specification, the N-glycan analysis method according to the embodiments has been described with reference to the flowchart shown in the drawings. Although the method is shown and described in a series of blocks for the sake of simplicity, the invention is not limited to the order of the blocks, and some blocks may occur in different order or simultaneously with other blocks than those shown and described herein. Various other branches, flow paths, and blocks may be implemented in order to achieve the same or similar results. In addition, not all illustrated blocks may be required for the implementation of the methods described herein.

In addition, the present invention described above has been described with reference to the embodiments shown in the drawings, but this is only exemplary and those skilled in the art will understand that various modifications and variations are possible from this. will be. However, such modifications should be considered to be within the technical protection scope of the present invention. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Embodiments relate to methods for analyzing n-glycans in biological complex fluids, and to techniques for reproducibly separating and purifying only pure N-glycans with high efficiency from various biological complex fluids including serum. It is about.

Claims (12)

1. Adding to the sample comprising N-glycans a protein comprising a first labeled glycan having a different mass than the N-glycans;

   After the step of adding the protein, denaturing the protein in the sample;

   Separating the glycan from the sample from the protein using an enzyme after the protein is denatured;

   Eluting glycan from the sample from which the protein is separated;

   Obtaining a mass spectrum of eluted glycans; And

   Determining the efficiency or function of the enzyme based on the amount of the first labeled glycan in the mass spectrum.
2. The method of claim 1,

   Wherein the protein comprising the first labeled glycan is horseradish peroxidase, the method of analyzing N-glycans in a biological complex fluid.
3. The method of claim 1,

   Before eluting the glycan, adding a second labeled glycan having a different mass from the N-glycan to the sample from which the protein has been removed;

   Determining the elution efficiency based on the amount of the second labeled glycan in the mass spectrum.
4. The method of claim 3, wherein

   Wherein said second labeled glycan is maltohexose.
5. The method of claim 1,

   Denaturing the protein in the sample comprises mixing the sample with a buffer solution comprising dithiothreitol and ammonium bicarbonate.
6. The method of claim 5,

   PH of the buffer solution is 7.5 to 9, method for analyzing N-glycans in a biological complex fluid.
7. The method of claim 5,

   The concentration of dithiothreitol in the buffer solution is 1mM to 2mM, method for analyzing N-glycans in a biological complex fluid.
8. The method of claim 5,

   Denaturing the protein in the sample further comprises reacting the sample mixed with the buffer solution in a shaker at 65 ° C. temperature.
9. The method of claim 1,

   The enzyme is peptide N-glycosidase F,

   Separating glycan in the sample from the protein comprises injecting 500 units of peptide N-glycosidase F into the sample. .
10. The method of claim 9,

    Separating the glycan from the protein from the protein further comprises irradiating the sample mixed with the enzyme with microwave for 8 minutes at 400 W intensity, analyzing the N-glycans in the biological complex fluid.
11. The method of claim 1,

    Eluting glycan from the sample comprises mixing the sample with a 20% concentration of acetonitrile solution.
12. The method of claim 1,

    And drying the sample at a temperature of 70 ° C. after eluting glycan from the sample, before obtaining the mass spectrum.

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