KR102196653B1 - Quantitative analysis method of non-human sialic acid using Cucurbituril[7] - Google Patents

Abstract

In the present invention, after forming [CB[7] + sialic acid + NH ₄ +H] ²⁺ complex ions using the electrospray ionization method, the analytical performance of non-human sialic acid can be dramatically improved using mass spectrometry. It relates to a method for quantitative analysis of non-human sialic acid using cucurbituril [7].
When the quantitative analysis method of non-human sialic acid using cucurbituril [7] according to the present invention is used, there is an effect of enabling accurate relative and absolute quantification of sialic acid through a simple method, thereby reducing the analysis time and cost of non-human sialic acid. It can be drastically reduced.

Description

Quantitative analysis method of non-human sialic acid using Cucurbituril[7]}

The present invention relates to a method for quantitative analysis of non-human sialic acid using cucurbituril [7].

Among the sialic acids glycosylated in proteins, N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) occupy the highest proportions, and their structures are very similar, so they are distinguished by the presence or absence of a hydroxyl group. do. Due to this structural similarity, when Neu5Gc and Neu5Ac are present in a mixture, the physical properties are similar and the elution time on liquid chromatography overlaps. Therefore, in order to quantitatively analyze them, the derivatization process of Neu5Gc and Neu5Ac. This is essential.

Meanwhile, cucurbit[7]uril, CB[7]) is one of the cucurbituril homologues, which are synthetic polymers. Cucurbituril homologues are composed of glycouril monomers, and there are CB[6], CB[7] and CB[8] depending on the number of monomers. These homologues are amber models with a hole in the middle, which can accommodate multiple guest molecules that fit the size of the middle. At both entrances of the hole, a partially negatively charged carbonyl group exists in accordance with the number of monomers, and the inside of the hole is hydrophobic.

In addition, the electrospray ionization mass spectrometer is a device that combines the electrospray ionization technology and the mass spectrometer technology. First of all, a mass spectrometer refers to an instrument capable of using mass spectrometry (MS). Mass spectrometry is a method of measuring the amount of ions according to each m/z by ionizing a sample and collecting it, separating and detecting individual ions according to the mass to charge ratio ( m/z ). to be. In addition, electrospray ionization is a soft ionization technology to generate ions to be detected in a mass spectrometer. As a problem that fragment ions are generated by splitting of polymers or non-covalent complexes during the ionization process does not occur, life, medicine It is widely used in the field.

Under the above-described technical background, the present inventors have conducted extensive research to solve the problem of the conventional Neu5Gc and Neu5Ac quantitative analysis method, and as a result, CB[7] and ammonium acetate (ammonium acetate, NH ₄ OAc) were mixed in a mixed solution of Neu5Gc and Neu5Ac. Thus, when performing the electrospray ionization mass spectrometry, it was confirmed that quantitative analysis was possible without a separate derivatization process, and the present invention was completed.

Republic of Korea Patent Publication No. 10-2017-0076236

In the present invention, after forming [CB[7]+sialic acid+NH ₄ +H] ²⁺ complex ions using the electrospray ionization method, the analytical performance of non-human sialic acid can be dramatically improved by using mass spectrometry. We intend to provide a method for quantitative analysis of non-human sialic acid using cucurbituril[7].

The present invention in order to solve the above problems,

(a) mixing cucurbituril[7] and an ammonium salt in a sialic acid solution to form a mixture; (b) forming [CB[7] + sialic acid + NH ₄ +H] ²⁺ complex ions by ionizing the mixture by electrospray ionization; And (c) quantitatively analyzing the complex ions using mass spectrometry. It provides a quantitative analysis method of non-human sialic acid using cucurbituril [7].

According to the present invention, the sialic acid may be N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), or a mixture thereof.

According to the present invention, the ammonium salt may be at least one selected from the group consisting of ammonium acetate, ammonium chloride, ammonium carbonate, and ammonium hydroxide.

According to the present invention, the [CB[7] + sialic acid + NH ₄ +H] ²⁺ complex ions in the (b) step are formed in both ports of cucurbituril [7] by electrospray ionization of the mixture. The gas phase host-guest interaction of either the first inlet region and the protonated acetamide group of sialic acid and the second inlet region and the other one of the two portals of cucurbituril [7] and ammonium It may be characterized in that it is formed by a gas phase host-guest interaction of ions and hydroxyl groups of sialic acid.

According to the present invention, in the quantitative analysis of the sialic acid, the sialic acid solution of step (a) may be characterized in that a plurality of different sialic acids to be analyzed are mixed.

According to the present invention, the quantitative analysis of sialic acid can be performed through a calibration curve derived by a standard addition method.

When the quantitative analysis method of non-human sialic acid using cucurbituril [7] according to the present invention is used, there is an effect of enabling accurate relative and absolute quantification of sialic acid through a simple method, thereby reducing the analysis time and cost of non-human sialic acid. It can be drastically reduced.

1 is a schematic diagram of a host-guest strategy for sialic acid analysis. (a) shows the structures of CB[7], Neu5Gc and Neu5Ac, (b) is a schematic diagram of the host-guest chemistry used in the present invention, (c) is CB[7], Neu5Gc and Neu5Ac. The mass spectrum result of the mixture is shown.
2 shows the results of quantification of sialic acid using gaseous host-guest chemistry. (a) is a calibration curve showing the Complexation proportion of Neu5Gc and Neu5Ac at different total sialic acid concentrations ([SA] _t ). (b) shows the result of quantifying the sialic acid binary mixture based on the calibration curve of (a). The x-axis represents the concentration of each sialic acid in the binary mixture ([SA] _sol ), and the y-axis represents the concentration of the sialic acid binary mixtures ([SA] _det ) inferred through the analysis method according to the present invention. (c) shows the results of quantifying a single sialic acid solution based on the calibration curve of (a). The x-axis and y-axis represent the concentration of sialic acid in the solution ([SA] _sol ) and the concentration of sialic acid inferred through the analysis method according to the present invention ([SA] _det ), respectively.
3 shows gas-phase complexation of sialic acid and CB[7]. (a) The results of the IM spectrum of CB[7]-sialic acid complex ions and free CB [7] ions are shown. The experimental collision cross section of each ion is indicated at the top of each IM peak. (b) shows two quantization structures of the acetamide group of Neu5Gc and Neu5Ac. (c) shows the representative structures of complex ions of CB[7]-Neu5Gc (top) and CB[7]-Neu5Ac (bottom). (d) shows the relative binding energy (ΔBEs) of sialic acid ions. ΔBEs represents the difference in binding energy of O-protonated Neu5Gc ions. (e) shows the expanded structure of the representative structure.
Figure 4 shows the quantitative results of sialic acid in the glycoprotein using gaseous host-guest chemistry. All proteins are omitted from this figure. The value of μg _{sialic acid} /mg _{protein in} cetuximab (cet) and mol _{sialic acid} /mol _{protein in} bovine mucin IS (mucin) is the result of the solution state of the cet sample and the unknown sequence of mucin, resulting in the mass of cet and the molar concentration of mucin. Was excluded because it could not be obtained. The reference concentration was obtained by averaging the previously reported concentrations.
5 shows the results of mass spectrometry when CB[7] and NH ₄ OAc were added to the sialic acid binary mixture.

Hereinafter, the present invention will be described in more detail.

As described above, among the sialic acids glycosylated in proteins, N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) occupy the highest proportions, and their very similar structures make it difficult to quantify. In addition, conventional techniques for quantitative analysis of sialic acid have a disadvantage that a derivatization process of sialic acids that requires a long analysis time is required.

Accordingly, in the present invention, after forming [CB[7]+sialic acid+NH ₄ +H] ²⁺ complex ions using the electrospray ionization method, the analytical performance of non-human sialic acid is dramatically improved using mass spectrometry. We intend to provide a method for quantitative analysis of non-human sialic acid using cucurbituril [7] that can be used.

1 is a schematic diagram of a method for quantitative analysis of sialic acid using a host-guest strategy according to the present invention.

Referring to FIG. 1, a method for quantitative analysis of non-human sialic acid according to the present invention includes the steps of: (a) mixing cucurbituril[7] and an ammonium salt in a sialic acid solution to produce a mixture; (b) forming [CB[7] + sialic acid + NH ₄ +H] ²⁺ complex ions by ionizing the mixture by electrospray ionization; And (c) quantitatively analyzing the complex ions using mass spectrometry.

At this time, the sialic acid is not particularly limited as long as it is a sialic acid known in the art, but may be preferably N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), or a mixture thereof.

In addition, the ammonium salt is not limited thereto as long as it contains an ammonium ion, but may be preferably at least one selected from the group consisting of ammonium acetate, ammonium chloride, ammonium carbonate and ammonium hydroxide.

In addition, the [CB[7] + sialic acid + NH ₄ +H] ²⁺ complex ion in the step (b) is one of the two portals of cucurbituril [7] by electrospray ionization of the mixture. The gas phase host-guest interaction of the first inlet region and the protonated acetamide group of sialic acid and the second inlet region, the other of the two portals of cucurbituril [7], and ammonium ions, sialic acid It can be formed by gaseous host-guest interactions of the acid's hydroxyl groups.

In addition, in the present invention, when the sialic acid to be analyzed is mixed, it can be quantified for each sialic acid. In this case, the sialic acid solution of step (a) contains a plurality of different sialic acids to be analyzed, and the following examples This can be done through the calibration curve derived by the standard addition method as described in.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples and the like are for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Experimental method

material

Standards of N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac), cucurbituril[7], ammonium acetate (NH ₄ OAc), formic acid (FA), acetic acid (≥99%), Bovine mucin IS, bovine transferrin, human transferrin, human α1-acid glycoprotein, and erythropoietin expressed in HEK293 cells were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen chloride standard solution (6.0 M) was purchased from Honeywell Fluka (Morris Plains, NJ, USA). The first and second sodium phosphate were purchased from Biosesang (Seongnam, Gyeonggi, South Korea). Cetuximab (Erbitux) was donated by Korea University School of Medicine. A solid-phase extraction cartridge filled with porous graphite carbon (HyperSep Hypercarb SPE cartridge) was purchased from ThermoFisher Scientific (Waltham, MA, USA). As a solvent, HPLC-grade water and acetonitrile (ACN) (Avantor Performance Materials, Center Valley, PA, USA) were used.

Electrospray ionization mass spectrometry ( ESI -MS)

All MS experiments for measuring sialic acid in mixtures using host-guest chemistry were performed in positive ion mode, Agilent 6560 ion mobility quadrupole time-of-flight (IM-) with an electrospray ionization (ESI) source. QTOF) mass spectrometer.

For the ionization of the CB[7]-sialic acid complex for quantification of Neu5Gc and Neu5Ac, gas temperature, dry gas flow rate and nebulizer pressure were set to 325°C, 3 L/min and 20 psig, respectively. Sheath gas flow rate and temperature were set to 8 L/min and 100°C, respectively. VCap, nozzle voltage, fragment voltage, and Oct 1 RF Vpp were also set to 3500, 2000, 350 and 700V, respectively. The IM experiment was performed in a drift cell filled with helium, and the drift cell pressure was set to 3.950 Torr.

Computing calculation and the theoretical of the calculated structure CCSs Calculation

All MD simulation results were obtained using GROMACS 4.5.5. For simulation, CHARMM General Forcefield (Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, IJ Comput.Chem. 2010, 31 (4), 671-690.) was used. The initial structure of the CB[7]-sialic acid complex ion was constructed using Hyperchem 7.5 (Hypercube Inc., Gainesville, FL, USA). MD simulation was performed to obtain the lowest energy structure of the complex ions in the gas phase as a candidate structure. Annealing simulations were performed between 300K and 600K to prevent trapping in certain structures. The last frame of 300 annealing cycles (90 ns) was extracted, and five structures were selected for each complex ion based on the potential energy. The selected structure was further optimized through DFT calculation using the Becke three-parameter and the Q-Chem 4.1 calculation package (Q-Chem Inc., Pittsburgh, PA, USA). The theoretical collision cross section of the complex ions was calculated using a modified trajectory method (Tangvoranuntakul, P.; Gagneux, P.; Diaz, S.; Bardor, M.; Varki, N.; Varki, A.; Muchmore, E. Proc. Natl. Acad. Sci. USA 2003, 100 (21), 12045-12050.). Briefly, this CCS calculation method combines the Exp-6 vdW interaction potential with the vdW interaction variable of the Molecular Dynamics Forcefield (MMFF94). The vdW interaction parameters are linearly adjusted for accurate CCS prediction of various ions.

Glycoprotein ( glycoprotein ) Isolation of my sialic acid

The following separation process was performed to confirm the content of Neu5Gc and Neu5Ac in the glycoprotein. First, 0.2-1.0 milligrams of glycoprotein was dissolved in 120 microliters of HPLC grade aqueous solution. In the case of Cetuximab, since a sample in solution was provided, the above solution was used for the experiment. Thereafter, 100 microliters of 4 molar concentration of acetic acid was mixed with 100 microliters of gastric glycoprotein, and the solution was placed in an oven at 80° C. for 3 hours and 30 minutes. The remaining 20 microliters of glycoprotein solution was used to check the concentration of glycoprotein in the solution through UV-Vis. Next, a PGC SPE cartridge was used to purify the sample placed in the oven. Before injecting the protein sample, 3 millimeters of 80% ACN containing 0.05% TFA were injected into the cartridge to wash the filler, and then 2 millimeters of an HPLC grade aqueous solution was injected to prepare an environment in which separation can proceed. After the sample was injected, 1 millimeter of an aqueous HPLC solution, 10% ACN containing 0.05% TFA, and 20% ACN containing 0.05% TFA were sequentially injected. After the sample was injected, each elution was received in an e-tube and lyophilized, and the dried sample was prepared by dissolving in 100 microliter HPLC aqueous solution. Finally, sialic acids in the prepared sample were fractionated using LCMS-8050 to which a Shim-pack GIS C18 column was connected. The solvents used to fractionate the sialic acids are 0.1% aqueous formic acid solution (A) and 0.1% formic acid ACN (B), and the degree of gradient of the solvents is as follows: 0-3.8 min, 0% B; 3.8-8.6 min, solvent B increases linearly from 0% to 60%; 8.6-10.6 min, 60% B; 10.6-11.6 min, solvent B decreases linearly from 60% to 0%; 11.6-25.0 min, 0% B. The finally obtained fractionated solution was freeze-dried, and then used for quantification of sialic acid in a target glycoprotein.

Results and Discussion

ESI -Through MS, of sialic acid using CB[7] Supramolecular Quantification

As a result of mixing CB[7] (10 μM) and ammonium acetate (NH ₄ OAc) (200 μM) in a mixed solution of Neu5Gc and Neu5Ac and analyzed through an electrospray ionization mass spectrometer, both Neu5Gc and Neu5Ac were found in the gas phase. During the ESI, it was confirmed that the complex ions were double-charged with CB [7], and specifically, Neu5Gc and Neu5Ac formed a complex ion with CB[7] and one ammonium ion and a proton, respectively (Figs. 1c and 5 ). Several complex ion peaks of Neu5Gc and Neu5Ac were observed overlapping each other in the spectrum (Fig. 1c). Two complex ion peaks, [CB[7]+Neu5Gc+NH ₄ +H] ²⁺ (observed: m/z 753.2409; calculated: m/z 753.2344) and [CB[7]+Neu5Ac+NH ₄ +H] ²⁺ (observed: m/z 745.2442; calculated: m/z 745.2458) did not overlap their monoisotope peaks. In order to derive a calibration curve for quantification of a sialic acid binary mixture, a sialic acid mixture solution was prepared by preparing the concentrations of the two sialic acids as shown in [Table 1] below, and then using the following Equations 1 to 3 After deriving the complex formation ratio of the sialic acid binary mixture solutions, the concentration ratios in the solution of each sialic acid were compared together and shown as a calibration curve in FIG. 2(a):

[Neu5Gc]+[Neu5Ac] (μM) [Neu5Gc]
(μM) [Neu5Ac]
(μM) [Neu5Gc]+[Neu5Ac] (μM) [Neu5Gc]
(μM) [Neu5Ac]
(μM) 0.5 0.05 0.45 20 2 18 0.15 0.35 6 14 0.25 0.25 10 10 0.35 0.15 14 6 0.45 0.05 18 2 1.0 0.1 0.9 50 5 45 0.3 0.7 15 35 0.5 0.5 25 25 0.7 0.3 35 15 0.9 0.1 45 5 2.0 0.2 1.8 75 7.5 67.5 0.6 1.4 22.5 52.5 1.0 1.0 37.5 37.5 1.4 0.6 52.5 22.5 1.8 0.2 67.5 7.5 5.0 0.5 4.5 100 10 90 1.5 3.5 30 70 2.5 2.5 50 50 3.5 1.5 70 30 4.5 0.5 90 10 10 One 9 3 7 5 5 7 3 9 One

[Equation 1]

I _Neu5Gc = I _{m /z 753.24}

[Equation 2]

_{I Neu5Ac = I m / z 745.24} - 0.170 × I m / z 744.24

[Equation 3]

Complexation proportion for A = I _A ÷ (I _A + I _B )

Here, I _Neu5Gc and I _Neu5Ac represent the intensity of monoisotopic complex ion peaks of Neu5Gc and Neu5Ac containing protons and ammonium cations. Each I _{m /z} symbol represents the intensity of a monoisotope peak with m/z described in the subscript. I _Neu5Gc was obtained from the monoisotope peak intensity of [CB[7]+Neu5Gc+NH ₄ +H] ²⁺ (Equation 1). CB [7] the strength of the complex ion is -Neu5Ac [CB [7] + Neu5Ac + NH 4 + H] of a single isotope peak intensity of ^{2+ [CB [7] + Neu5Gc} + NH 4 + H-H2O] 2+ By subtracting the tri-isotropic peak intensity (observed: m/z 744.2398; calculated: m/z 744.2381), the increase in I _Neu5Ac as a result of the overlap between the triple isotope peak and the single isotope peak is obtained. It was obtained by exclusion (Equation 2). The intensity ratio of the triple isotope peak of the CB [7]-dehydrated Neu5Gc complex to the monoisotope peak of 0.170 was calculated from the isotope pattern of the complex ions. Relative ratio of complex formation to sialic acid (Neu5Gc or Neu5Ac) was calculated using Equation 3. Next, the rate of complex formation of Neu5Gc and Neu5Ac in the binary mixture during ESI was investigated. As shown in FIG. 2A, it was observed that the ratio of the complex formation in the binary mixture solution and the concentration ratio of the two sialic acids had a strong correlation.

The sum of the concentrations of the two sialic acids and the ratio of the concentration were different, and the linearity between the concentration ratio and the ratio of complex formation was confirmed. 50, 75, 100 μM), and when the concentration ratio of Neu5Gc to Neu5Ac is between 10-90% (10, 30, 50, 70, 90%), they have excellent linearity regardless of the total concentration of sialic acid and CB[7]. Was seen (R ² = 0.999) (Fig. 2A). In addition, the complexing preferences of the two sialic acids for CB[7] were very similar. When the concentration ratio of the two sialic acids was 1:1, the measured complexing ratios of Neu5Gc and Neu5Ac using ESI-MS were 47.2, respectively. ±0.9% and 52.8±0.9% (Fig. 2a). In addition, the limits of quantification of Neu5Gc and Neu5Ac were 1.60 pmol and 0.65 pmol, respectively, and the confirmed linear range was measured to be 5-9,000 pmol.According to the present invention, accurate quantitative analysis of sialic acid without derivatization process It was confirmed that this is possible.

Next, mix the standard mixture of Neu5Gc and Neu5Ac with known concentrations in the mixture of Neu5Gc and Neu5Ac whose concentration is unknown, and then compare the complex formation ratio of the standard mixture and the complex formation ratio of the solution mixed with the unknown solution and the standard mixture. It was tested whether it was possible to quantify the contents of Neu5Gc and Neu5Ac. Specifically, FIG. 2B shows the results of quantifying sialic acid binary mixtures based on the calibration curve of FIG. 2A. The x-axis is the concentration of each sialic acid in the binary mixture, and the y-axis is the concentration of the sialic acid binary mixtures inferred by quantification. Sialic acids in the binary mixture were quantified using the standard addition method and Equation 4 below.

Here, I _A and I _B mean the intensity of each CB[7]-sialic acid complex ion in the sample solution to which the standard solution was added in the mass spectrometer. V ₀ , Vs, C _{A, s} and C _{B, s} refer to the volume of the sample before adding the standard solution, the volume of the standard solution added, and the concentration of sialic acid A or B in the standard solution, respectively. In the above experiment, 1-10 µl of a standard solution containing 50 µM of Neu5Gc and Neu5Ac was added to 50 µl of the solution to be analyzed. As a result of the measurement, it was confirmed that the determined sialic acid concentration and the concentration in the standard solution were almost identical (FIG. 2b).

Next, it was confirmed whether quantitative analysis was possible in the same way even in a solution in which only Neu5Gc or Neu5Ac was present. Specifically, FIG. 2C shows the results of quantifying a sialic acid solution based on the calibration curve of FIG. 2A. At this time, the x-axis and y-axis are the concentration of sialic acid inferred according to the concentration and quantification of sialic acid in the solution, respectively. Each sialic acid solution was quantified using the standard addition method and Equation 5 below, similar to the sialic acid binary mixture.

In the above experiment, 1-10 μl of a standard solution containing 50 μM of Neu5Gc and Neu5Ac was added to 100 μl of the solution to be analyzed. As a result of the measurement, it was confirmed that the determined sialic acid concentration almost coincided with the concentration in the solution (Fig. 2c).

In the gas phase Unique host between CB[7] and sialic acid- guest chemistry

The unique complexing properties of sialic acid and CB[7] were studied using ion mobility mass spectrometry (IM-MS). IM-MS can provide information about the structural characteristics of ions based on the collision cross-sectional area (CCS) determined from the speed of movement of ions under a weak electric field in a neutral buffer gas (ie, He). Figure 3a shows the ion arrival time distribution of [CB[7]+sialic acid+NH ₄ +H] ²⁺ and [CB[7]+2H] ²⁺ together with CCS values determined in ATDs (ion arrival time distributions). ). The complex ions of each of the two sialic acids were significantly higher in CCS (CCS _{CB [7] -Neu5Gc} = 227.9.0 ± 0.9) compared to the _unconjugated CB[7] ions (CCS _CB [7] = 207.0 ± 0.6 Å ² ). ^{_{Å 2; CCS CB [7]}} - shown ^{_{Neu5Ac = 226.0 ± 0.4 Å 2)}} . This means that a significant portion of the sialic acid guests are exposed outside the CB[7] cavity.

In the present invention, two possible protonated structures of sialic acid were studied in the complex of the proton of CB[7] and the ammonium cation of the protonated acetamide group (Fig. 3b, O- and N-protonation). Representative structures of CB[7]-sialic acid complex ions derived from molecular dynamics (MD) simulations and density function theory (DFT) calculations are shown in Fig. 3c. The theoretical CCSs values calculated using the trajectory (TJ) method using a combination of Exp-6 potential and MMFF94 parameters were consistent with the experimental CCS values. This indicates that the calculated structure can effectively reflect the gaseous structure of the complex ions. Through the representative structure of the CB[7]-sialic acid complex ion, it was confirmed that most of the sialic acid guests were encapsulated in the CB[7] cavity. The large CCSs value of the complex ions compared to the uncomplexed CB[7] ions is due to the exposure of the protonated acetamide group and part of the hexose ring of sialic acid from the CB[7] cavity. The complex was stabilized through an electrostatic interaction between the protonated acetamide group of sialic acid and the carbonyl group of the CB [7] portal.

Studies of the binding energies of representative structures of complex ions have provided other insights into the host-guest interactions of CB[7] and two sialic acids in the gas phase. The binding energy was obtained by subtracting the energy of the optimized structure of each component from the energy of the CB[7]-sialic acid complex ion. The binding energy of sialic acid and CB[7] was significantly different depending on the protonation position (Fig. 3d). In the case of Neu5Gc, it was found that the N-protonated structure favors the formation of a complex with CB [7] compared to the O-protonated structure (Fig. 3D). The calculated difference (ΔBEs) in the binding energy between the two structures in the CB[7] complex was 70 kJ/mol. In contrast, for Neu5Ac, the complexation of O-protonated Neu5Gc and CB[7] was energetically favored by 25 kJ/mol over N-protonated Neu5Gc.

Based on ΔBEs, the complexation preference of Neu5Gc and Neu5Ac ions for CB was evaluated in the order of N-protonated Neu5Gc> O-protonated Neu5Ac> N-protonated Neu5Ac> O-protonated Neu5Gc (Fig. 3d). In order to gain more insight into the complexation preference of sialic acid ions, we investigated the gaseous host-guest interaction between CB[7] and sialic acid in the complex. The protonated acetamide group strongly interacts with the carbonyl group of CB [7] regardless of the protonation position (Figs. 3c and e). In the case of O-protonated sialic acid ions, H _a and H _b (FIG. 3B) can form two hydrogen bonds with the carbonyl group of CB[7] (FIG. 3B). In addition, H _c and H _d of N-protonated sialic acid can interact with the CB [7] portal (Fig. 3b). These interactions have a decisive effect on the overall geometry of the CB[7]-sialic acid complex ions (Figs. 3c and e). In the case of O-protonated Neu5Ac, the acetamide group was adjacent to the CB[7] portal, and both H _a and H _b were capable of H-bonding interactions. In contrast, the N-protonated acetamide group of Neu5Ac is distant from the CB[7] inlet due to the partially negatively charged carbonyl oxygen of the acetamide group except for H _c and H _d and positively charged nitrogen. Is far away. Representative structures of CB[7]-Neu5Gc complex ions show that the structural characteristics of Neu5Gc in the CB[7] cavity are slightly different from those of Neu5Ac due to the presence of C5 hydroxymethyl groups (Figs. 3c and e). In the case of O-protonated Neu5Gc, the two hydrogen bonds of N-Ha...O and O-Hb...O interfere with the interaction between CB[7] and C5 hydroxymethyl groups, improving the structural stability of complex ions. Made it. This structure induces deeper insertion of O-protonated Neu5Gc ions into the CB[7] cavity, interfering with the effective electrostatic interaction of Neu5Gc ions and CB[7] at the opposite CB[7] inlet. In the case of N-protonated Neu5Gc, the C5 hydroxymethyl group interacted with the CB[7] inlet through the U-structure of the C5 functional group. The U-structured carbonyl oxygen was located farther than the N-protonated Neu5Ac complex ion, which explains the stronger complexation preference for N-protonated Neu5Gc.

In summary, from the above results, there is a distinct host-guest interaction between CB[7] and sialic acid ions, leading to different complexing preferences, and that a complex formation ratio having a linear relationship with their concentration ratio is derived. Can be seen.

Gas phase Host- guest Chemically Neu5Gc And Neu5Ac Quantitative analysis

Two kinds of protein therapeutics (cetuximab, human erythropoietin) and four kinds of glycoproteins (bovine mucin IS, bovine fetuin, human transferrin, human alpha-) were analyzed using the above-described quantitative analysis method using gaseous host-guest chemistry. 1 glycoprotein) content of Neu5Gc and Neu5Ac was quantified. First, the sialic acid of the glycoprotein was extracted using acid hydrolysis, a solid phase extraction cartridge and reverse phase chromatography. Next, supramolecular MS scanning of the sample was performed using CB[7] for sialic acid analysis. Figure 4 shows the quantitative results of sialic acid in each glycoprotein. As a result of the measurement, it was confirmed that the concentration of Neu5Ac and the glycoprotein-binding Neu5Gc present over a wide molar ratio can be accurately determined (from 0.07:1 to 21.25:1 of Neu5Gc:Neu5Ac). In addition, the literature on the conventional sialic acid quantification method (Ghaderi, D.; Taylor, RE; Padler-Karavani, V.; Diaz, S.; Varki, A. Nat. Biotechnol. 2010, 28 (8), 863- 867., Honda, S.; Suzuki, S. Anal.Biochem. 1984, 142 (1), 167-174.), it was confirmed that the relative error% was very similar to within 25%. In addition, it was confirmed that a trace amount of Neu5Ac in cetuximab, which was not confirmed in the previously reported literature, can be precisely quantified according to the present invention.

conclusion

In the present invention, the actual application of the host-guest principle was demonstrated by simply and accurately identifying and quantifying Neu5Gc and Neu5Ac using a CB[7] host. The unique host-guest chemistry between CB[7] and sialic acid in the gas phase was used for Neu5Gc analysis of glycoproteins by simple ESI-MS scanning. The distinct host-guest interaction between sialic acids and CB[7] induced a complexing ratio with a high linear relationship with the concentration ratio in solution. This linear relationship was used to develop an MS method to identify and quantify Neu5Gc and Neu5Ac in various glycoproteins with high sensitivity and simplicity. This approach provides excellent separation and quantification of non-human glycans, and is expected to be useful in controlling immunogenicity-related risks in biotherapeutic development.

Claims (6)

(a) mixing cucurbituril[7] and an ammonium salt in a sialic acid solution to form a mixture;
(b) forming [CB[7] + sialic acid + NH ₄ +H] ²⁺ complex ions by ionizing the mixture by electrospray ionization; And
(c) quantitatively analyzing the complex ions using mass spectrometry; Including,
In step (b), the gaseous host-guest interaction of the first inlet region, which is one of the ports of cucurbituril [7], and the protonated acetamide group of sialic acid, and cucurbituril [ [CB[7] + sialic acid + NH ₄ by gas-phase host-guest interaction between the second inlet region, which is the other of the two portals of [7], and ammonium ions and hydroxyl groups of sialic acid. +H] ^{2+ A} method for quantitative analysis of non-human sialic acid using cucurbituril [7], characterized in that it forms a complex ion. The method of claim 1,
The sialic acid is N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), or a mixture thereof, characterized in that the quantitative analysis method of non-human sialic acid using cucurbituril [7]. The method of claim 1,
The ammonium salt is one or more selected from the group consisting of ammonium acetate, ammonium chloride, ammonium carbonate, and ammonium hydroxide. Method for quantitative analysis of non-human sialic acid using cucurbituril [7]. delete The method of claim 1,
In the quantitative analysis of the sialic acid, the sialic acid solution of step (a) is a method of quantitative analysis of non-human sialic acid using cucurbituril [7], wherein a plurality of different sialic acids to be analyzed are mixed. The method of claim 1,
The quantitative analysis of sialic acid is a method for quantitative analysis of non-human sialic acid using cucurbituril [7], characterized in that it is performed through a calibration curve derived by a standard addition method.

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