WO2017039123A1 - Procédé d'analyse de n-glycanes dans des fluides biologiques complexes - Google Patents

Procédé d'analyse de n-glycanes dans des fluides biologiques complexes Download PDF

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Publication number
WO2017039123A1
WO2017039123A1 PCT/KR2016/005645 KR2016005645W WO2017039123A1 WO 2017039123 A1 WO2017039123 A1 WO 2017039123A1 KR 2016005645 W KR2016005645 W KR 2016005645W WO 2017039123 A1 WO2017039123 A1 WO 2017039123A1
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sample
protein
glycan
glycans
mass spectrum
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PCT/KR2016/005645
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English (en)
Korean (ko)
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장미
송성희
박형순
김양선
조응준
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주식회사 아스타
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Publication of WO2017039123A1 publication Critical patent/WO2017039123A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

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  • 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.
  • 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.
  • 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.
  • SPE Solid Phase Extraction
  • 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.
  • 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.
  • 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.
  • the protein comprising the first label glycan is horse radish peroxidase.
  • 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.
  • 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.
  • the second labeled glycan is Malto-hexose.
  • denaturing the protein in the sample comprises mixing the sample with a buffer solution comprising dithiothreitol and ammonium bicarbonate (NH 4 CO 3 ). .
  • 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.
  • 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 °C temperature.
  • 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.
  • 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.
  • eluting glycan from the sample comprises mixing the sample with a 20% concentration of acetonitrile solution.
  • 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.
  • FIG. 1 is a schematic diagram showing a pretreatment process of a sample for N-glycan analysis according to an embodiment.
  • FIG. 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 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.
  • FIG. 1 is a schematic diagram illustrating a pretreatment process of a sample for n-glycan analysis according to an embodiment
  • FIG. 2 is a flowchart of an N-glycan analysis method according to an 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
  • 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.
  • 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.
  • the 96 well plates described herein are merely exemplary, and the type of the well plates is not limited thereto.
  • the protein comprising the first labeled glycan is Horse radish peroxidase (HRP), a glycoprotein that is not present in an animal biological fluid.
  • HRP horse radish peroxidase
  • 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.
  • the type of protein containing the first labeled glycan is not limited to HRP.
  • 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.
  • NH 4 CO 3 ammonium bicarbonate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MALDI-TOF MS matrix assisted laser desorption / ionization time of flight mass spectrometry
  • 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.
  • 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.
  • n in the tables of the present specification means the number of samples to obtain data.
  • the intensity of the N-glycan main peak is large, especially when the concentration of dithiothreitol is 1mM, the highest peak is obtained.
  • the peak intensity is also affected by the concentration of acetonitrile (ACN) solution used for the elution, which will be described later in detail.
  • the glycan may be separated from the protein by mixing a sample in which the protein is denatured with an enzyme (S3).
  • 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.
  • 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 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.
  • the enzymatic reaction is carried out while irradiating microwaves to the 96 well plate.
  • Table 6 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 °C.
  • Table 7 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.
  • the enzyme reaction using ethanol to precipitate the protein and the precipitated protein can be removed from the sample (S4).
  • 450 uL of ethanol may be added to each well of the 96 well plate in which the enzyme reaction is completed.
  • 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.
  • 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.
  • 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.
  • SPE Solid Phase Extraction
  • 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.
  • the second labeled glycan may be Malto-hexose.
  • maltohexose prepared at a concentration of 100 ug / mL with water may be dispensed 5 uL into each well of a 96 well plate.
  • the kind of the second label glycan is not limited to maltohexose.
  • the glycan may be eluted from the sample by the SPE process (S6).
  • 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.
  • the sample contained in the 96 well plate is loaded into the 96 well type cartridge.
  • 500 uL of deionized water may be added to the sample, followed by agitation and centrifugation for 10 seconds.
  • 1 mL of deionized water may be flushed three times to a 96-well cartridge that has been loaded for washing.
  • 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.
  • the concentration of ACN solution for elution is 20%.
  • 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 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.
  • 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.
  • 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.
  • the glycan eluted by mass spectrometry can be analyzed (S7).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FT-ICR Fourier Transform Ion Cyclotron Resonance
  • 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
  • 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 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.
  • N-glycan analysis method has been described with reference to the flowchart shown in the drawings.
  • 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.
  • not all illustrated blocks may be required for the implementation of the methods described herein.
  • 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.

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Abstract

L'invention concerne un procédé d'analyse de N-glycanes dans des fluides biologiques complexes, qui peut comprendre les étapes consistant : à ajouter une protéine à un échantillon contenant des N-glycanes, la protéine comprenant des premiers glycanes marqueurs qui ont une masse différente des N-glycanes ; à dénaturer la protéine dans l'échantillon après l'étape consistant à ajouter la protéine ; à séparer les glycanes dans l'échantillon par rapport à la protéine à l'aide d'une enzyme après que la protéine a été dénaturée ; à éluer des glycanes provenant de l'échantillon dans lequel la protéine est séparée ; à obtenir un spectre de masse des glycanes élués ; et à déterminer l'efficacité ou la réaction de l'enzyme sur la base de la quantité des premiers glycanes marqueurs dans le spectre de masse. En utilisant le procédé, des N-glycanes purs peuvent être séparés et raffinés à partir de divers fluides biologiques complexes y compris le sérum, et la fiabilité des données peut être améliorée par en rendant maximale l'efficacité et la reproductibilité dans un temps court à l'aide d'une plaque à puits.
PCT/KR2016/005645 2015-09-03 2016-05-27 Procédé d'analyse de n-glycanes dans des fluides biologiques complexes WO2017039123A1 (fr)

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Citations (5)

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JP2004504621A (ja) * 2000-07-19 2004-02-12 バイオトロン・リミテッド ガンの診断におけるガンの標識形質を同定する方法および診断のための利用
KR100475642B1 (ko) * 2001-12-29 2005-03-10 한국생명공학연구원 암 발생 및 전이에 관여하는 단백질의 당쇄 변화를측정하여 암을 진단하는 방법 및 이를 이용한 진단킷트
JP5047790B2 (ja) * 2004-06-22 2012-10-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 癌を検出するためのオリゴ糖プロファイリング法
KR20130066481A (ko) * 2012-05-03 2013-06-20 주식회사 아스타 암 특이적 당쇄의 분석 방법 및 암 진단에서의 이의 이용
KR20140120651A (ko) * 2013-04-04 2014-10-14 충남대학교산학협력단 인간 혈청 당쇄 지도 및 이를 이용하여 혈청 시료를 검증하는 방법

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Publication number Priority date Publication date Assignee Title
JPH01237481A (ja) * 1988-02-09 1989-09-21 Furuno Electric Co Ltd 水中探知装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004504621A (ja) * 2000-07-19 2004-02-12 バイオトロン・リミテッド ガンの診断におけるガンの標識形質を同定する方法および診断のための利用
KR100475642B1 (ko) * 2001-12-29 2005-03-10 한국생명공학연구원 암 발생 및 전이에 관여하는 단백질의 당쇄 변화를측정하여 암을 진단하는 방법 및 이를 이용한 진단킷트
JP5047790B2 (ja) * 2004-06-22 2012-10-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 癌を検出するためのオリゴ糖プロファイリング法
KR20130066481A (ko) * 2012-05-03 2013-06-20 주식회사 아스타 암 특이적 당쇄의 분석 방법 및 암 진단에서의 이의 이용
KR20140120651A (ko) * 2013-04-04 2014-10-14 충남대학교산학협력단 인간 혈청 당쇄 지도 및 이를 이용하여 혈청 시료를 검증하는 방법

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