KR101544591B1 - Development of high-throughput quantitative glycomics platform using mass spectrometry and chromatography system - Google Patents

Abstract

The present invention relates to a method for mass spectrometry analysis using a mass spectrometer and a chromatography system. The ultrafast glucose analysis method according to the present invention is characterized in that sugar extraction, purification and labeling are sequentially carried out on a 96-well plate, and specific sugar labeled with isotopes is mass analyzed using MALDI-TOF MS and UPLC, The cost is remarkably reduced, and there is an effect that the relative quantitative determination and the absolute quantitative determination of the sugar in the glycoprotein are enabled.

Description

TECHNICAL FIELD The present invention relates to a mass spectrometry apparatus and a mass spectrometry system for mass spectrometry,

The present invention relates to a high speed saccharification and quantitative analysis method using a mass spectrometer and a chromatography system.

Glycoprotein is a complex protein made by covalent bonding of protein and carbohydrate of single structure. The distribution of the glycoprotein is variously present in the cell membrane or in the cell surface layer, and as an extracellular protein. Most of the commercial therapeutic proteins are glycoproteins. Also, since glycosylation, a component of carbohydrates, affects the stability, efficacy and consistency of the product, proper glycosylation of recombinant therapeutic glycoproteins has been emphasized in the biopharmaceutical field. In particular, the glycation level of the glycoprotein is very important because glycoproteins with desialylated glycans are rapidly removed by blood purification.

The oligosaccharides in the glycoprotein are responsible for maintaining the steric structure of the protein and obtaining resistance to prevent degradation from the protease. Recently, it has become clear that oligosaccharides in glycoproteins are involved in life phenomena such as fertilization, differentiation, signal transduction, encryption, intracellular transport of proteins, and regulation of physiological activity.

At present, the center of glyceral function research is mainly concerned with the glycosyltransferase (oligosaccharide gene) responsible for the biosynthesis, but considering that glycosyltransferase also participates in the function of life by cooperating with other proteins , It is necessary to perform function analysis of oligosaccharides by structural analysis technique which grasps and analyzes the whole phase of oligosaccharide expressed in cell tissue.

At present, as a structural analysis method for oligosaccharides of glycoproteins, a method of fluorescently labeling oligosaccharides cut out from glycoproteins and then analyzing them using ultra high performance liquid chromatography (UPLC) or mass spectrometry (MS) is a promising means, and sialylation Only anion exchange column chromatography has been used for separation of the oligosaccharide. However, analytical methods for sialylated N-glycans should provide a quantitative information on the glycan structure and should be fast, robust, and reliable.

Matrix-Assisted Laser Desorption Ionixation Time of Flight Mass Spectrometry (MALDI-TOF-MS) was developed in the 1980s, and unlike conventional mass spectrometry, extreme conditions (eg pyrolysis) It is relatively easy to handle because it is not needed and does not use a column. Recently, it has been widely used in biology, especially in the field of proteomics and bioinformatics. It has also been applied as a method of analyzing the specificity of biomaterials such as antigen-antibody reaction, DNA-DNA hybridization and protein adsorption for biocompatibility experiments .

The inventors proceeded in sequence with extraction, purification and labeling of sugars on a 96-well plate while searching for methods to reduce sugar analysis time and cost, and the specific sugar labeled with isotopes was analyzed by MALDI-TOF MS and UPLC Mass spectrometry was carried out, and the present invention was completed.

Accordingly, the present invention is intended to provide a high-throughput sugar analysis method using a mass spectrometer and a chromatography system.

The present invention provides a method for mass spectrometry analysis using a mass spectrometer and a chromatography system.

The ultrafast glucose analysis method using the mass spectrometer and the chromatography system according to the present invention has the effect of drastically reducing the analysis time and cost of sugar and permitting the relative quantitation and absolute quantitation of sugar in the glycoprotein.

≪ RTI ID = 0.0 > 1 & Figure 2 is a schematic representation of the analysis of N-glycans from glycoproteins using amidation and 2-AA labeling.
Figure 2 is a diagram showing the acetylhydrazide amidation of sialylated N-glycans.
Figure 3 shows the 2-AA (2-aminobenzoic acid) labeling of N-glycans.
Fig. 4 is a diagram showing a relative quantitative method of sugar; Fig.
5 is a diagram showing a method for absolute determination of sugar.
6 is a diagram showing MALDI-TOF spectra of N-glycans labeled with (a) ¹² [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA in positive mode, respectively.
7 is a diagram showing MALDI-TOF spectra of N-glycans labeled with (a) ¹² [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA in negative mode, respectively.
FIG. 8 is a graph showing the relationship between the concentration of glycoprotein in the positive mode (a) ¹² shows the intensity of the MALDI-TOF spectra of N-glycans labeled with [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA, respectively.
9 is a graph showing the relationship between the concentration of glycoprotein in the negative mode (a) ¹² shows the intensity of the MALDI-TOF spectra of N-glycans labeled with [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA, respectively.

The present invention

1) reducing the glycoprotein by adding dithiothreitol (DTT) to the glycoprotein;

2) alkylating the reduced glycoprotein with iodoacetamide;

3) adding amido to the reduced and alkylated glycoprotein by adding acetohydrazide;

4) adding the PNGase F to the amidated glycoprotein to separate the amidated N-glycans and extracting them using a 96-well 10K MWCO plate;

5) Purifying the extracted amidated N-glycans using a porous graphite carbon (PGC) plate on a 96-well plate;

6) adding 2-aminobenzoic acid (2-AA) to the purified amidated N-glycan to form amidated N-glycans labeled with 2-aminobenzoic acid; And

7) Amidated N-glycans labeled with 2-aminobenzoic acid were quantified and quantified using Matrix-Assisted Laser Desorption Ionixation Time of Flight Mass Spectrometry (MALDI-TOF-MS) And a qualitative analysis step.

Hereinafter, the present invention will be described in detail.

Mass analysis using MALDI-TOF-MS according to the present invention can analyze a large number of samples at the same time by introducing a 96-well plate. By neutralizing the glycoprotein by acetohydrazide amidation, the same time the analysis is to be further N- glycan to ¹² [C _6] respectively in 2-aminobenzoic acid (2-AA) and ¹³ [C _6] -2-AA-substituted carbon of the benzene ring isotopically Whereby the relative quantification and the absolute quantification are enabled.

The "glycoprotein" refers to a complex protein produced by covalent bonding of a protein and a carbohydrate having a single structure. It is widely distributed in all cells and becomes the subject of viscous mucin. It is also included in saliva, gastric juice, blood secretion fluid, similar organs such as digestive tract mucosa, submandibular gland and pituitary gland, other connective tissues, plant seeds, have. The main sugar that constitutes the glycoprotein is glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (NeuAc, NeuGc), and the like.

The glycoprotein preferably comprises but is not limited to sialylated N-glycans. Glycans refer to the sugar chain structure, and are also referred to as oligosaccharides. It is an important element of cell wall elements and tissue supporting the skin. The mode of binding between the oligosaccharide and the protein includes N-glycoside bond between N-acetyl-D-glucosamine and aspartic acid, O-glycoside bond between N-acetyl-D-galactosamine and serine or threonine, D-galactose and hydroxy And the O-glycosidic bond of lysine.

In order to efficiently obtain the glycan from the glycoprotein obtained from the sample of the subject, a sample pretreatment process such as a reduction and an alkylation process which can denature the tertiary structure of the protein may be performed. In addition, the denaturation process of the glycoprotein can be performed by heating the glycoprotein at 90 DEG C for 3 minutes.

Quantitative analysis of sialylated glycans by mass spectrometry is not reliable due to the different ionisation efficiencies of neuronal and acidic glycans, so the acidic N-glycans attached to the protein are amidated and neutralized . The resulting amidated N-glycan can be extracted from the glycoprotein by adding PNGase F. [

The amidation is preferably performed by adding acetohydrazide (Ah), HCl, and EDC (N- (3-dimethylaminopropyl) N'thylcarbodiimidehydrochloride) to the reduced and alkylated glycoprotein.

The "N-glycans" are used interchangeably with N-linked glycans and are N-linked oligosaccharides, such as N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of the asparagine residue of the protein, or N-acetylglucosamine As shown in FIG. The major N-glycan is a fully sialylated glycan represented by GlcNAc ₂ Man ₃ GlcNAc ₂ Gal ₂ NeuAc ₂ . Other embodiments may be partially sialylated glycans, sialic acid-depleted glycans, fully galactosylated glycans, partially galactosylated glycans, or mangalactosylated glycans.

The N-glycan is selected from the group consisting of GlcNAc ₂ Man ₃ GlcNAc ₂ Gal ₂ NeuAc ₂ , GlcNAc ₂ Man ₃ GlcNAc ₂ Gal ₂ , GlcNAc ₂ Man ₃ GlcNAc ₂ , GlcNAc ₂ Man ₅ GlcNAcGalNeuAc, GlcNAc ₂ Man ₅ GlcNAcGal, GlcNAc ₂ Man ₅ GlcNAc , GlcNAc ₂ Man ₃ and GlcNAc ₂ Man ₅ , but is not limited thereto.

As used herein, the term " well plate " refers to a substrate that is divided into specific sites that prevent migration from specific sites to other sites. Here, the specific region is a well. Each well in the well plate is physically separated from the other well. Typical well plates typically have 48, 96, 384, or 1356 wells and are made of polystyrene, polypropylene, polycarbonate, glass, or equivalent materials.

The extraction, purification and labeling of the N-glycan is preferably carried out in a single-pot method using a 96-well plate.

In the method, the N- amination glycans was substituted with ^{13 12} [C _6] -2- amino acid (2-AA) and of the benzene ring carbon ¹² [C _6] the isotope ¹³ [C _6] [C ₆ ] -2-AA, respectively, followed by mass spectrometry, whereby relative quantitation and absolute quantification are possible.

A mass spectrometer is an analytical instrument that measures the mass of a compound. After the compound is charged and ionized, the mass to charge amount is measured to determine the molecular weight of the compound. Examples of the method of ionizing a compound include an electron ionization method using an electron beam, a method of colliding atoms at high speed, and a method using a laser. Among these methods, a method of using a laser is characterized in that a matrix for assisting ionization of a compound is mixed with a compound (sample) to be analyzed and then placed on a target of the analyzer, and then the laser is irradiated onto the sample, Thereby ionizing the sample.

The above-mentioned "matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF MS)" is a process for producing a low molecular weight (100 to 300 Da) organic or inorganic matrix and a cationic To make uniform crystals, and then to irradiate a strong pulse N ₂ laser of ~ 337 nm to accurately perform mass spectrometry with a TOF-MS (Time-of-Flight Mass Spectrometer). The above method is advantageous in that the molecular weight of the polymer substance of 300 Da or more can be measured, the sensitivity of the polymer substance is high, the sample of the peptomol level can be analyzed, and the phenomenon of fragmentation of the compound to be analyzed at the time of ionization can be greatly reduced. Therefore, it is effective for mass analysis of large molecular weight biochemicals such as proteins and nucleic acids.

In the above method, it is preferable to use MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, and UPLC having higher resolution than HPLC is introduced, It is more preferable to use it for mass analysis.

In the mass spectrometry, the peak area is proportional to the concentration of the constituent material, so that the concentration of the constituent material can be quantitatively analyzed by measuring the area of the peak.

In the method, MALDI-TOF-MS using a quantitative analysis of different cells the following steps: 1) to 5), the amidation of N- glycans obtained respectively by a step ¹² from the (e. G., Normal cells and specific cell) C ₆ ] -2-AA and ¹³ [C ₆ ] -2-AA, respectively. The mass spectra were then measured using MALDI-TOF-MS and the relative quantities were calculated by calculating the peak area . In addition, the N-glycans of the mixed structure obtained from the one cell (for example, normal cells) obtained in the above 1) to 5) may be added to the N-glycans of the specific structure according to the above 1) Glycans were labeled with ¹² [C ₆ ] -2-AA, N-glycans of a specific structure were labeled with ¹³ [C ₆ ] -2-AA By measuring the mass spectrum using MALDI-TOF-MS and calculating the peak area, an absolute amount of N-glycan having a specific structure can be determined.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1 Isolation of N-Glycans (Saccharides) from Glycoproteins

(10K MWCO plates), tablets (PGC plates) and labeling ( ¹² [C ₆ ] -2-AA / ¹³ [C ₆ ] -2-AA) on 96-well plates for separation, purification and analysis of sugars -2-AA plate). Acetohydrazide A summary of the analysis of glycans from glycoproteins using amidation and 2-AA labeling is shown in Fig.

As shown in FIG. 1, the sialylated glycans were reacted with acetohydrazide Amidation neutralizes and allows simultaneous analysis with other sugars. It can be labeled with ¹² [C ₆ ] -2-AA and ¹³ [C ₆ ] -2-AA to determine whether the protein is glycosylated. .

1-1. Reduction and alkylation of glycoproteins

5 uL of glycoprotein was added to a 96-well plate (Harvard Apparatus, Holliston, MA) with a molecular weight cutoff (MWCO) of 10 K and then spun down to dryness. Then 5 uL of the glycoprotein was mixed with 9 uL of deionized water and 4 uL of buffer. To the mixture was added 2 μL of 0.5 M dithiothreitol (DTT), followed by incubation at 70 ° C. for 10 minutes to reduce the glycoprotein. To this, 2 uL of 100 mM iodoacetamide (IAA) was added and the glycoprotein was alkylated by incubation in the dark room for 30 minutes at room temperature. Then, 28 uL of water was added to make a 50 uL mixture.

1-2. Acetohydrazide amidation of glycoprotein

The acetohydrazide amidation of the glycoprotein is shown in Fig.

As shown in Fig. 2, the denatured glycoprotein was desalt by centrifugation and washing 5 times at 5000 rpm on a 96-well 10K MWCO plate with 200 uL of water. The demineralized glycoprotein was reconstituted with 100 uL of 1 M acetohydhydrazide, 20 uL of 1 M HCl and 20 uL of 2 M EDC. The mixture was incubated at room temperature for 4 hours. The aminated glycoprotein was desalt by centrifugation and washing 5 times at 5000 rpm with 200 uL of water.

1-3. Preparation of 96-well based N-glycans (PNGase F treatment and N-glycan purification)

A 96-well 10K MWCO plate was prewetted with 100 uL of 70% ethanol and washed twice with 200 uL of water. The above reduced, alkylated and amidated mixture was added to the filter and allowed to stand for 5 minutes, and then the remaining solution was removed by centrifugation. Then, the mixture was washed three times with 20 mM of NaHCO ₃ 200 uL. 50 uL of a 200 μU / μL PNGase F solution (45 μL of 20 mM NaHCO ₃ + 5 μL of stock) was added thereto, followed by incubation at 37 ° C. overnight. The plate was placed on a new deep well plate washed with Milli Q and distilled water, and 50 uL of water was added thereto, followed by centrifugation to remove the residual enzyme reaction medium. Then, 200 μL of water was added to the filter, and the solution was allowed to stand for 5 minutes, dissolved, and then separated and collected three times. The released N-glycans were extracted from 96-well 10 K MWCO plates with 200 uL of water and immediately added to 96-well plates containing PGC (porous graphite carbon).

The PGC wells were pre-washed with 200 uL of 30% acetic acid / water (v / v) solution and eluted with 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile (ACN) (v / v) solution. The wells were then pre-equilibrated with 200 uL of 0.1% TFA (v / v) in a 5% ACN / water (v / v) solution. And N-glycans were loaded into the wells. Wells were washed successively with 200 uL of water and 200 uL of 0.1% TFA (v / v) in 5% ACN / water (v / v) solution.

N-glycans were extracted into a collection plate with 200 uL of 0.1% TFA (v / v) in 50% ACN / water (v / v) The extracted N-glycans were then dried under vacuum.

1-4. 2-AA labeling

The 2-AA (2-aminobenzoic acid) labeling of N-glycans is shown in FIG.

As shown in Figure 3, 10 uL of N-glycans in a 50/50 (v / v) methanol / water solution were mixed with 100 uL of 2-aminobenzoic acid (2-AA) solution. The 2-aminobenzoic acid (2-AA) solution was prepared by dissolving 30 mg of 2-AA, 2% boric acid in methanol (w / v) and 1% sodium cyanoborate in 1 mL of 4% sodium acetate trihydrate And 20 mg of sodium cyanoborohydride.

The mixture was heated at 80 < 0 > C for 40 minutes. After cooling at room temperature, the mixture was then diluted with 1 mL of 80% acetonitrile (ACN). The excess reagent was removed using a C18 cartridge. The cartridge was purged twice with 1 mL of 95% acetonitrile (ACN) in water. After loading the N-glycans, the excess reagent was washed twice with 1 mL of 95% acetonitrile (ACN) in water. The N-glycans were then extracted twice with 0.5 mL of 20% acetonitrile (ACN) in water.

1-5. Quantitative analysis of sugars using MALDI-TOF MS

The relative quantitation method of sugars obtained from different cells or tissues is shown in Fig.

As shown in Fig. 4, N-glycans obtained from different cells (normal cells and specific cells) were each acetohydrazide amidated according to Example 1, and then the resulting amidated N- The cells were labeled with ¹² [C ₆ ] -2-AA and the amidated N-glycans from specific cells were labeled with ¹³ [C ₆ ] -2-AA. MS spectra of N-glycans labeled with ¹² [C ₆ ] -2-AA and ¹³ [C ₆ ] -2-AA respectively were then measured and analyzed using a MALDI-TOF mass spectrometer.

A method for absolute determination of a specific sugar structure in a mixed sugar structure is shown in Fig.

As shown in Figure 5, the structure obtained (N- glycans) per mixed from normal cells in Example 1, after each of acetonitrile Hydra Zaid amide in accordance with the screen, the mixed N- glycan ¹² [C _6] - 2-AA, and double specific N-glycans were labeled with ¹³ [C ₆ ] -2-AA. MS spectra of N-glycans labeled with ¹² [C ₆ ] -2-AA and ¹³ [C ₆ ] -2-AA respectively were then measured and analyzed using a MALDI-TOF mass spectrometer.

Experimental Example 1. Quantitative analysis of N-glycans

1-1. Measurement method of MALDI-TOF MS

1 μL of N-glycan sample (10 to 50 pmol) was mixed with 1 μL of 2,5-dihydroxybenzoic acid (DHB) solution (30 mg / mL in 70/30% (v / v) acetonitrile / water) Respectively. Thereafter, 0.5 μL of the mixture was spotted three times on a stainless steel MALDI plate and dried at room temperature. MS spectra were obtained using a MALDI-TOF mass spectrometer (Autolflex MALDI-TOF / TOF mass spectrometry (Bruker, Leipzig, Germany). Relative% is the ratio of the intensity of each ion to the summed intensity of the total sensed ions.

1-2. UPLC measurement method

The UPLC profiling for 2-AA labeled N-glycans was obtained with a 1.7 μm HILIC stationary phase column (BEH glycan, 1.7 μm, 2.1 mm id × 50 mm L, Waters, Milford, Mass.). The peak area of the UPLC profile was obtained using Empower software (Waters, Milford, Mass.).

1-3. Quantitative analysis using MALDI-TOF MS

Sialylated N-glycans obtained from bovine fetuin were amidated, N-glycans were extracted using PNGase F, and purified using PGC (porous graphite carbon). After dividing the purified N-glycans into two batches, one batch of N-glycan was labeled with ¹² [C ₆ ] -2-AA and the other batch of N-glycans was labeled with ¹³ [ ₆ ] -2-AA. The MALDI-TOF spectra of the N-glycans labeled with (a) ¹² [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA, respectively, measured in the positive mode, The MALDI-TOF spectra of the N-glycans labeled with (a) ¹² [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA measured in the negative mode are shown in FIG.

As shown in FIGS. 6 and 7, the N-glycans labeled with ¹³ [C ₆ ] -2-AA had the same intensity as the N-glycans labeled with ¹² [C ₆ ] -2-AA (m / z = 6) while increasing the mass (intensity).

(A) the concentration of the glycoprotein measured in the positive mode, The intensity of the MALDI-TOF spectrum of the N-glycans labeled with ¹² [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA is shown in FIG. 8, Based on the measured concentration of glycoprotein (a) The intensity of the MALDI-TOF spectra of N-glycans labeled with ¹² [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA is shown in FIG.

As shown in Figs. 8 and 9, quantitative results obtained by labeling with ¹² [C ₆ ] -2-AA and (b) ¹³ [C ₆ ] -2-AA using a MALDI-TOF mass spectrometer . That is, the above-mentioned results were obtained by labeling the sample to be quantified with ¹² [C ₆ ] -2-AA and identifying the specific double sugar with ¹³ [C ₆ ] -2-AA and measuring the MALDI-TOF spectrum together By measuring, absolute quantification is possible.

Claims (6)

1) reducing the glycoprotein by adding dithiothreitol (DTT) to the glycoprotein;
2) alkylating the reduced glycoprotein with iodoacetamide;
3) adding amido to the reduced and alkylated glycoprotein by adding acetohydrazide;
4) adding the PNGase F to the amidated glycoprotein to separate the amidated N-glycans and extracting them using a 96-well 10K MWCO plate;
5) Purifying the extracted amidated N-glycans using a porous graphite carbon (PGC) plate on a 96-well plate;
6) adding 2-aminobenzoic acid (2-AA) to the purified amidated N-glycan to form amidated N-glycans labeled with 2-aminobenzoic acid; And
7) Amidated N-glycans labeled with 2-aminobenzoic acid were quantified and quantified using Matrix-Assisted Laser Desorption Ionixation Time of Flight Mass Spectrometry (MALDI-TOF-MS) Qualitative analysis,
The 7) from the quantitative analysis of the phase, different cells the following steps: 1) to 5) according to the amidation step with N- glycans obtained, respectively ¹² [C _6] -2-AA and ¹³ [C _6] -2- AA, and mass spectrometry is measured using MALDI-TOF-MS, and then relative quantification is performed by calculating a peak area. The method according to claim 1, wherein the sugar constituting the glycoprotein in step 1) is at least one selected from the group consisting of glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine N-acetylglucosamine (GlcNAc) and sialic acid (NeuAc, NeuGc). 2. The method according to claim 1, wherein the glycoprotein in step (1) comprises sialylated N-glycans. 4. The composition of claim 3, wherein the N-glycan is selected from the group consisting of GlcNAc ₂ Man ₃ GlcNAc ₂ Gal ₂ NeuAc ₂ , GlcNAc ₂ Man ₃ GlcNAc ₂ Gal ₂ , GlcNAc ₂ Man ₃ GlcNAc ₂ , GlcNAc ₂ Man ₅ GlcNAcGalNeuAc, GlcNAc ₂ Man ₅ GlcNAcGal, GlcNAc ₂ Man ₅ GlcNAc, GlcNAc ₂ Man ₃ and GlcNAc ₂ Man ₅ . delete 4. The method according to claim 1, wherein the quantitative analysis of step (7) comprises the steps (1) to (5) of recognizing the absolute amount of N-glycans in the mixed structure obtained from step (1) The N-glycans of the mixed structure are labeled with ¹² [C ₆ ] -2-AA and the N-glycans of the specific structure are labeled with ¹³ [C ₆ ] - 2-AA and measuring the mass spectrum using MALDI-TOF-MS, and then calculating an absolute peak area by calculating a peak area.

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