KR100553387B1 - The method for determining glycosylated substances using the pba-magnetic bead and for detecting pba bound to a magnetic bead - Google Patents

Abstract

The present invention relates to a method for detecting a sugar chain substance using PBA-magnetic beads and a method for confirming magnetic bead binding of PBA, and more specifically, including beads and iron magnetized by an external magnetic field including iron oxide and the like. The present invention relates to a method for detecting sugar chains using PBA-magnetic beads and a method for identifying magnetic bead binding of PBA using MALDI spectra, comprising phenylboronic acid (PBA).

The present invention provides a method for detecting a sugar chain substance in a mixture, wherein PBA-magnetic beads are fixed by fixing phenylboronic acid (PBA) to beads magnetized by an external magnetic field including iron oxide and the like. Preparing a; Coupling a sugar chain material in the mixture to the boronic acid group of the phenylboronic acid to prepare a sugar chain material-bead complex; Separating the sugar chain material-bead complex from the mixture under an external magnetic field; And detecting the sugar chain substance by applying the separated sugar chain substance-bead complex to a MALDI-TOF MS to provide a sugar chain substance detection method using PBA-magnetic beads. The PBA-magnetic beads according to the present invention can easily and highly separate sugar chain materials such as glycoproteins. In addition, the present invention provides a method for identifying magnetic bead binding of PBA using the MALDI spectrum.

PBA, magnetic beads, MALDI, glycoprotein

Description

The method for determining glycosylated substances using the PBA-magnetic bead and for detecting PBA bound to a magnetic bead}

1 is a graph showing the MALDI spectrum of glycoproteins detected by APBA-polystyrene magnetic beads.

FIG. 2 is a graph showing MALDI spectra of peptide sequences obtained from HbA1c, fibrinogen, and RNase B isolated from APBA-polystyrene magnetic beads. FIG.

Figure 3 is a graph showing the MALDI spectrum of APBA liquid (a), APBA-polystyrene magnetic beads (b) and polystyrene magnetic beads alone (c).

Figure 4 is a photograph showing a confocal scanning microscope image when the protein is bound to APBA-polystyrene magnetic beads.

The present invention relates to a method for detecting a sugar chain substance using PBA-magnetic beads and a method for confirming magnetic bead binding of PBA, and more particularly, to phenyl on a magnetized bead containing iron oxide. The present invention relates to a method for detecting sugar chains using PBA-magnetic beads and a method for confirming magnetic bead binding of PBA using MALDI spectrum, and more specifically, phenylboron A method for detecting sugar chains using PBA-magnetic beads that can easily separate an acid-detected glycoprotein by an external magnetic field, and a method for identifying magnetic bead binding of PBA, which is a fine molecule, easily by using MALDI spectrum will be.

Although glycosylation is the most common form of post-transcriptional modification in eukaryotic proteins, analysis of glycated proteins is very difficult. This is because glycosylation sites of glycoproteins do not have a color development site and are not easily ionized by mass spectrometry, and signals are usually suppressed by glycoproteins. Glycoprotein detection method using the antibody has the disadvantage of expensive and complicated detection process. In addition, when using a PBA known as a chemical substance that binds to sugar, when the conventional PBA is separated into a fixed bead, the operation such as centrifugation is further required, and after the separation, a large amount of impurities are contained to separate the pure glycoprotein. There is a difficult problem. Therefore, the analysis of glycoproteins will be more effective if the glycoproteins can be effectively separated.

Therefore, the inventors have found that the use of magnetic beads facilitates the separation of pure glycoproteins. There are many ways to fix PBA to magnetic beads, but it is not known how to directly determine whether PBA, a fine molecule, is binding to magnetic beads. The inventors have completed the present invention by devising a method for confirming the binding of PBA to magnetic beads by first applying PBA-magnetic beads to MALDI.

Accordingly, it is an object of the present invention to provide a sugar chain substance detection method using PBA-magnetic beads in which magnetic beads are immobilized on PBA to separate and separate the beads to which glycoproteins are bound by an external magnetic field.

It is another object of the present invention to provide a sugar chain substance detection method using PBA-magnetic beads which can easily convert sugar chain substances such as glycoproteins into ionized form.

It is still another object of the present invention to provide a method of confirming binding of PBA to magnetic beads using MALDI spectrum of PBA-magnetic beads.

In order to achieve the above object, the present invention is a method for detecting a sugar chain material in the mixture, bead magnetized by an external magnetic field containing phenylboronic acid (PBA), iron oxide (iron oxide), etc. inside Fixing to to prepare PBA-magnetic beads; Coupling a sugar chain material in the mixture to the boronic acid group of the phenylboronic acid to prepare a sugar chain material-bead complex; Separating the sugar chain material-bead complex from the mixture under an external magnetic field; And applying the separated sugar chain material-bead complex to MALDI-TOF MS to detect the sugar chain material, thereby providing a sugar chain material detection method using PBA-magnetic beads.

The beads are preferably chemical polymers, more preferably polystyrene. In addition, the phenylboronic acid is 3-aminophenylboronic acid (3-aminophenylboronic acid) and the bead is preferably a surface modified with a carboxyl group (-COOH).

Glycoproteins such as hemoglobin A1c (HbA1c), fibrinogen, and RNase B are preferable as the sugar chain material detected by binding to the PBA-magnetic beads.

In addition, the present invention provides a method for detecting a glycoprotein in a mixture, in which phenylboronic acid (PBA) is immobilized on a bead magnetized by an external magnetic field, including iron oxide, etc., inside the PBA- Preparing magnetic beads; Binding a glycoprotein in a mixture to the boronic acid group of the phenylboronic acid to prepare a glycoprotein-bead complex; Separating the glycoprotein-bead complex from the mixture by applying an external magnetic field; Separating and digesting the protein portion of the complex with a peptide; And it provides a method for detecting glycoproteins using PBA-magnetic beads, characterized in that for detecting the glycoproteins by applying the decomposed complex to peptide mass finger printing (peptide mass finger printing). The beads are preferably chemical polymers, more preferably polystyrene. In addition, the phenylboronic acid is 3-aminophenylboronic acid (3-aminophenylboronic acid) and the bead is preferably a surface modified with a carboxyl group (-COOH).

In addition, the present invention provides a method for identifying magnetic bead binding of PBA using the MALDI spectrum.

Hereinafter, the present invention will be described in detail.

Boronic acid reversibly reacts with specific geometric positions—particularly the hydroxyl groups of cis-1,2-diols of substances containing diols such as nucleotides or glycoproteins. In the present invention, PBA-magnetic which can detect the sugar chain material by fixing the phenylboronic acid (PBA) to the magnetic beads and specifically binding to the sugar chain material, and then by Matrix Assisted Laser Desorption Ionization (MALDI) Provided is a method for detecting sugar chain material using beads.

Magnetic beads can be very effectively used to purely separate materials because they can be easily washed and separated from external magnetic fields. Therefore, when the PBA is immobilized on the magnetic beads, the sugar chain substance is detected as PBA, and the external magnetic field is applied to the magnetic beads to which the PBA is immobilized to purify sugar chain substances such as glycoproteins and carbohydrates bound to the PBA. It can be easily separated. In addition, the magnetic beads are small and heavy, so that the separation is easier, and the size of the beads is small, thereby reducing the concentration and amount of the sample. The size of the magnetic beads is 2.6um-3.0um can be used commercially available beads.

PBA is a chemical that is very small compared to beads. Therefore, in the past, it was difficult to confirm whether PBA is firmly bonded to the beads, and the present inventors fixed the PBA to magnetic beads and applied the MALDI MS (Mass spectrometry) for the first time to confirm the binding of PBA and magnetic beads. PBA-magnetic beads according to the present invention and a method for detecting sugar chains using the same. 1 to 4 show the test results confirming that the binding of the PBA and the magnetic bead well. In particular, the FITC-tagged proteins were immobilized on PBA-magnetic beads and then viewed by confocal laser microscopy. The fluorescence images of the glycoproteins (g: HbA1c in FIG. 3, c: in FIG. RNase B) and the nonglycosylated protein (nonglycoprotein (d: BSA in FIG. 3, e: Myoglobin) in FIG. 3) demonstrate that the efficiency of immobilization on the magnetic beads is different (a in FIG. 3 is PBA bead).

Since the PBA has a property of ionizing itself, it is possible to identify the sugar chain material bonded to the magnetic beads in the MALDI plate even without a matrix. In addition, when the sugar chain material is a glycoprotein, the magnetic beads may be measured by separating the proteins bound to the magnetic beads by directly putting them in a MALDI plate or giving salting out or pH changes. In addition, the protein bound to the magnetic beads using a protease such as trypsin is cut into peptides and then taken to the supernatant to go through the desalting process, and then the glycoprotein can be confirmed again by peptide mass finger printing. have.

As the magnetic beads, beads having a property of being magnetized by an external magnetic field by including iron oxide or the like inside can be used. Beads having the above properties are preferably used beads made of chemical polymers, and most preferably polystyrene beads.

PBA can be fixed to the magnetic beads in various ways.

As the most common example, APBA-magnetic beads can be prepared by combining the carboxyl groups of polystyrene magnetic beads whose surface has been converted to carboxyl groups with the amino groups of APBA, 3-aminophenylboronic acid. The process of binding the sugar chain material to the APBA-magnetic beads and the beads and the process of applying the binding material to the MALDI plate are as follows.

In addition, in the case of using a magnetic bead whose surface is converted to an amino group, PBA having an amino group is used, and a linker material such as bissulfosuccinimidyl suberate (Bis (sulfosuccinimidyl) suberate) may be used. PBA-magnetic beads can be prepared by linking amino groups. The following is an expression of the method. (An octagonal figure represents a PBA.)

In addition, magnetic beads having an epoxy group may be combined with PBA including an amine group, a hydroxyl group, and a thiol group to prepare PBA-magnetic beads. The following is an equation representing the method.

In addition, in the case of using magnetic beads having modified aldehyde surfaces, PBA-magnetic beads can be prepared by combining with PBA having a primary or secondary amino group. The following is an equation representing the method.

In addition, PBA-magnetic beads can be prepared using magnetic beads modified with IDA and magnetic beads modified with silica.

Hereinafter, the present invention will be described with reference to specific experimental examples, but the present invention is not limited to these examples.

Example 1 Detection of Glycoproteins Immobilized on PBA-magnetic Beads Using MALDI-TOF

a. Preparation of PBA-Magnetic Beads

As a test material, 3.0um -COOH-terminated magnetic beads were purchased from Dynal Biotech (Oslo, Norway), aminophenylphenylonic acid, 1-ethyl-3- (dimethylaminopropyl) carbodiimide (1-ethyl-3- (dimetylaminopropyl) carbodiimide (EDAC), N-hydroxysuccinimide (NHS), 2- [N-morpholino] ethane sulfonic acid (2- [N-Morpholino] ethane sulfonic acid, MES) It was purchased from Sigma.

100 μl of -COOH-terminated magnetic beads were washed twice with 0.01 M NaOH and three times with DI water to minimize non-specific binding (collecting the beads after collecting the beads to remove supernatant). Thereafter, 50 mg / ml NHS and 50 mg / ml EDC dissolved in 25 mM MES (pH 5) were mixed 1: 1 and placed in the washed beads and allowed to react at room temperature for 30 minutes. The beads having activated carboxyl group were washed twice with 25 mM MES and soaked in 40 μl of 25 mM MES. Then, 60 μl of 1 mg / ml PBA dissolved in the same buffer was mixed, followed by an overnight reaction at 4 ° C. after one hour reaction at room temperature. Thereafter, the supernatant was removed and reacted with 0.05 M ethanolamine (Ethanolamine, pH 8) for 15 minutes to prevent the reaction of non-reacted groups. Finally, washed four times with PBS and stored at 2 ~ 8 ℃.

b. Preparation of PBA-magnetic Beads and Glycoprotein Complexes

Glycoproteins HbA1c, ribonuclease B, and fibrinogen were each dissolved in DI water to make 50 μM. 20 μl of the stored PBA-magnetic beads was removed, the supernatant was removed, and 20 μl of the prepared glycoprotein was added. After reacting for 90 minutes at room temperature, the mixture was washed several times with DI water.

c. Detection of Proteins by PBA-magnetic Beads

After mixing 1 μl of PBA beads and 1 μl of matrix (matrix, sinnapinic acid) to which glycoproteins HbA1c, ribonuclease B, and fibrinogen were combined, 1 μl of them was placed in a MALDI plate and the MALDI spectrum was taken.

For HbA1c, the spectra of unglycosylated hemoglobin a unit (mb) at m / e 15130.37, and the spectrum of Hb b unit glycosylated at m / e 16034.6 (Fig. 1a) ). Ribonuclease B showed spectra for the five glycosylated forms of ribonuclease B corresponding to GlcNAc2Man5 and GlcNAc2Man9 with different masses of mannose units (162 Da). (B) of FIG. 1. In the case of fibrinogen, there is a limit in ionization due to its large molecular weight, but when the fibrinogen is immobilized in the magnetic beads according to the present invention, the ion molecule is detected (FIG. 1C).

Example 2 Detection of Glycoproteins Immobilized on PBA-magnetic Beads by Peptide Mass Fingerprinting

After PBA-beads combined with glycoproteins were completely dried in an evaporator, 20 μl of 5-20 ng / μl of trypsin dissolved in 25 mM ammonium bicabonate was added for overnight reaction. The supernatant was then taken through a desalting process and mixed with the matrix to take MALDI spectra. The peptide mass obtained was applied to Mascot as a database to find a peptide sequence.

From the MALDI spectrum of the supernatant obtained by combining the glycoproteins with PBA-magnetic beads and digesting them (FIG. 2), HbA1c (FIG. 2 a), RNase B (FIG. 2 b) and fibrinogen (FIG. 2) c) was able to identify the peptide sequence in the ratio shown in Table 1, respectively.

Glycoprotein Types Sequence confirmation rate Peptide Sequence Detected Hemoglobin beta subunit variant (Fragment) 82.6%                                              NVDEVGGEAL GRLLVVYPWT QRFFESFGDL STPDAVMGNP K VKAHGK KVL GAFSDGLAHL DNLKGTFATL SELHCDKLHV DPENFR LLGN VLVCVLDHHF GK EFTPPVQA AYQKVVAGVA NALAHKYH Pancreatic ribonuclease precursor (EC 3.1.27.5) 66.7% MALK SLVLLS LLVLVLLLVR VQPSLGK ETA AAKFERQHMD SSTSAASSSN YCNQMMKSRN LTKDRCK PVN TFVHESLADV QAVCSQKNVA CKNGQTNCYQ SYSTMSITDC R ETGSSK YPN CAYKTTQANK HIIVACEGNPAS YVPVHF Fibrinogen beta chain precursor 33.8% QFPTDYDEGQ DDRPKVGLGA RGHRPYDKKK EEAPSLRPVP PPISGGGYRA RPATATVGQK KVERKPPDAD GCLHADPDLG VLCPTGCKLQ DTLVRQERPI RKSIEDLRNT VDSVSR TSSS TFQYITLLK N MWK GRQNQVQ DNENVVNEYS SHLEK HQLYI DETVKNNIPT KLRVLRSILE NLRSKIQKLE SDVSTQMEYC RTPCTVTCNI PVVSGKECEK IIRNEGETSE MYLIQPEDSS KPYRVYCDMK TEK GGWTVIQ NR QDGSVDFG RKWDPYKQGF GNIATNAEGK K YCGVPGEYW LGNDRISQLT NMGPTKLLIE MEDWKGDKVT ALYEGFTVQN EANK YQLSVS KYKGTAGNAL IEGASQLVGE NR TMTIHNSM FFSTYDRDND GWK TTDPRKQ CSK EDGGGWW YNRCHAANPN GRYYWGGAYT WDMAK HGTDD GVVWMNWQGS WYSMKKMSMK IRPYFPEQ

(Bold alphabet: sequence found through peptide mass fingerprinting)

Example 3 It was confirmed that PBA was fixed to magnetic beads by comparing MALDI spectra of PBA liquid, PBA-magnetic beads, and magnetic beads alone.

PBA alone, PBA-polystyrene magnetic beads according to Example 1, and polystyrene magnetic beads to which PBA was not fixed were prepared.

PBA solution was placed in a MALDI plate without a matrix and MALDI spectra were taken (FIG. 3A). In addition, PBA-magnetic beads were placed in an aluminum sample plate without a matrix, and MALDI spectra were taken (Fig. 3b). Then, magnetic beads alone were placed in a MALDI plate without a matrix, and MALDI spectra were taken (FIG. 3C). The spectrum of PBA liquid peaked at m / e 137.1 (Fig. 3a), and the spectrum of PBA-magnetic beads was the spectrum of m / e 137.1 (Fig. 3a), which is the peak of PBA liquid, and magnetic beads alone (Fig. Since c) of all 3 is shown, it can be seen that PBA is fixed to magnetic beads.

[Test Example 1] Image comparison when protein is immobilized on PBA-magnetic beads

The fluorescent material FITC (Fluorescein isothiocyanate) was attached to glycoproteins and general proteins, and then bound to PBA-magnetic beads in the same manner as in Example 2. Then placed on a slide glass of confocal scanning microscope and magnified 200 times to confirm the image.

Images of confocal scanning microscopy appeared only when glycoproteins were immobilized on PBA-magnetic beads (b and c in FIG. 4).

As described above, the PBA-magnetic beads according to the present invention can separate sugar chain materials such as glycoproteins easily and with high purity. In addition, there is an advantage that can be easily applied to the MALDI-TOF glycoproteins can be easily converted into an ionized form through the enzyme treatment.

And, by taking the MALDI spectrum of the PBA-magnetic beads, it can be confirmed whether the PBA is fixed to the magnetic beads.

Claims (12)

In the method for detecting a glycoprotein in a mixture, Preparing PBA-magnetic beads by immobilizing phenylboronic acid (PBA) on polystyrene beads magnetized by an external magnetic field including iron oxide therein; Binding a glycoprotein in a mixture to the boronic acid group of the phenylboronic acid to prepare a glycoprotein-bead complex; Separating the glycoprotein-bead complex from the mixture by applying an external magnetic field; and Method for detecting a glycoprotein using PBA-magnetic beads, characterized in that for detecting the glycoprotein by applying the separated glycoprotein-bead complex to MALDI-TOF MS. delete delete The method of claim 1, wherein the phenylboronic acid is 3-aminophenylboronic acid (3-aminophenylboronic acid) and the bead glycoprotein detection method using PBA-magnetic beads, characterized in that the surface is modified with a carboxyl group (-COOH) . delete According to claim 1, wherein the glycoprotein is glycoprotein detection method using PBA-magnetic beads, characterized in that selected from the group consisting of hemoglobin A1c, fibrinogen and RNase B. In the method for detecting a glycoprotein in a mixture, Preparing PBA-magnetic beads by immobilizing phenylboronic acid (PBA) on polystyrene beads magnetized by an external magnetic field including iron oxide therein; Binding a glycoprotein in a mixture to the boronic acid group of the phenylboronic acid to prepare a glycoprotein-bead complex; Separating the glycoprotein-bead complex from the mixture by applying an external magnetic field; Separating and digesting the glycoprotein portion of the complex with a peptide; And The glycoprotein detection method using PBA-magnetic beads, characterized in that for detecting the glycoprotein by applying the digested glycoprotein portion to MALDI peptide mass finger printing. delete delete The method of claim 7, wherein the phenylboronic acid is 3-aminophenylboronic acid (3-aminophenylboronic acid) and the beads are glycoprotein detection method using PBA-magnetic beads, characterized in that the surface is modified with a carboxyl group (-COOH) . The method of claim 7, wherein the glycoproteins are hemoglobin A1c, fibrinogen (fibrinogen) and RNase B, characterized in that the glycoprotein detection method using PBA-magnetic beads. delete

Images