WO2017111219A1 - Method for qualitative and quantitative analysis of monosaccharides and peptides using cucurbituril[7] - Google Patents

Abstract

The present invention relates to a method for qualitative and quantitative analysis of monosaccharides using cucurbituril[7], and to a method for qualitatively and quantitatively analyzing monosaccharides in a gas phase by using electrospray ionization to form a cucurbituril[7]- monosaccharide complex ion to which an ammonium ion is bound through a gas-phase host-guest interaction, and then confirming a cleavage pattern of each complex ion through tandem mass spectrometry. According to the present invention, monosaccharides can be distinguished easily and quickly, and relative quantitation and absolute quantification of monosaccharides can also be performed. Thus, the analysis time and cost of monosaccharides can be drastically reduced. The present invention also relates to a method for qualitative and quantitative analysis of peptides using cucurbituril[7] and, more specifically, to a method for qualitative and quantitative analysis of peptides using cucurbituril[7], the method comprising the steps of: a) mixing cucurbituril[7] with a peptide solution to form a peptide-cucurbituril[7] complex; and b) analyzing the complex by mass spectrometry. According to the present invention, in the analysis of a peptide sample to be analyzed using various mass spectrometry-based methods, the analysis signal caused by a target sample can be remarkably improved by adding only cucurbituril[7] through a simple method, and the sequence coverage of the peptides to be analyzed can be improved.

Description

Qualitative and quantitative analysis of monosaccharides and peptides using cucurbituril [7]

The present invention relates to a qualitative and quantitative analysis method of monosaccharides using cucurbituril [7], and in detail, after forming a cucurbituril [7] -monosaccharide complex ion to which ammonia ions are bound using an electrospray ionization method, double mass spectrometry Tandem mass spectrometry relates to a method for qualitative and quantitative analysis of monosaccharides in a gas phase by identifying cleavage patterns of complex ions according to gas phase owner-guest interactions.

The present invention also relates to a method for qualitative and quantitative analysis of peptides using cucurbituril [7].

The development of qualitative and quantitative analysis of isomeric biomolecules is an important task in various fields such as chemistry and biology. Of the various isomeric biomolecules, monosaccharides are the most common isomeric biomolecules found anywhere in the body and are involved in various biological processes and diseases. Of note is that the errors related to the concentration of mono- and adjust a variety of diseases such as cancer, diabetes (Johnson et al, Am J. Clin Nutr 2007, 86, 899;..... Pitknen, E. Clin Chim Acta 1996 , 251 , 91).

However, monosaccharides have a problem in that qualitative and quantitative analysis is difficult due to the same mass and very similar structure. The most widely used sugar analysis method is a method using an oxidation-reduction reaction between enzymes and sugars (Wang, J. Chem . Rev. 2008 , 108 , 814). However, this method has a disadvantage in that the hydrogen ion concentration index or the temperature of the enzyme-containing solution must be adjusted to be always in the proper range in order to obtain high accuracy and precision.

In addition, in the case of the nuclear magnetic resonance device, it is easy to distinguish when monosaccharides are present, but when the monosaccharides are present in a mixed state, the spectral lines of the mixed monosaccharides are overlapped to increase the width of each monosaccharide mixture. In the case of liquid chromatography and gas chromatography, in order to have high sensitivity when analyzing monosaccharides, derivatization of columns and derivatives of monosaccharides are performed. The disadvantage is that a large number of sample preparation steps are required because of the need to perform the ignition.

Recently, methods for analyzing sugars using mass spectrometry (MS) for high sensitivity, low sample consumption, and fast analysis speed have been studied, but require derivatization of sugars (Gaucher SP et al. Anal. Chem. 1998 , 70 , 3009) or a number of experiments, such as energy-degrading MS (ERMS), are additionally required (Madhusudanan, KP J. Mass Spectrom . 2006 , 41 , 1096). Accordingly, there is a need for a rapid and convenient method for qualitative and quantitative analysis of monosaccharides.

In addition, with the development of soft ionization techniques, mass spectrometry (MS) is being used as a method for protein sequencing and detection. In a conventional method, proteins are first digested by enzymes and then analyzed by electrospray ionization MS (ESI-MS) or substrate-assisted laser desorption ionization MS (MALDI-MS) (Non Patent Literature 1). At this time, a plurality of peptides generated from the digestion provide abundant information about the protein primary structure (ie amino acid sequence). However, because various peptides compete for protons during the ionization process, those with low proton affinity show low ionization efficiency, which often makes MS analysis difficult and at the same time reduces sequence coverage (Non Patent Literature 2). Thus, for effective protein analysis, it is desirable to increase the ionization efficiency of the peptides of interest. In this regard, it is known that derivatization (Non-Patent Document 2) and instrumental modification (Non-Patent Document 3) can improve the ionization efficiency, but a convenient and widely accepted method has been reported to date. Nothing has happened.

Meanwhile, cucurbituril [ n ] (Cucurbit [ n ] urils, (CB [ n ] s, n = 5, 6, 7, 8, and 10) gives n glycoluril (C ₄ H ₂ N ₄ O ₂ ) repeat units And 2 n methylene (—CH ₂ —) linking units, and are neutral, water soluble host molecules used in various chemical fields (Non-Patent Document 4): cucurbituril [ n ] is a hydrophobic cavity encapsulating nonpolar groups, and A ureido carbonyl group capable of bonding with a cationic group includes two polar inlets located side by side (Non Patent Literature 5) Since Cougarbituril [ n ] has excellent properties as a host molecule, it is a solution phase (Non Patent Literature 6). ) And gas phase (Non-Patent Document 7) has been widely studied under various conditions.

The cucurbituril of [7] to form the N- terminal phenylalanine (Phe) residue and strong complex of peptides and proteins in solution _{^{(K a ~ 10 6 -10 7}} M -1) ( Non-Patent Document 6), which phenylalanine This is because the hydrophobic side chain of is well contained in the cavity of cucurbituril [7], and the N-terminal ammonium group has an effective ion-dipole interaction with carbonyl oxygen of cucurbituril [7] (see Fig. 1) (Non Patent Literature 8) . Interaction between cucurbituril [7] and phenylalanine is also preserved in the process of phase-conversion of the complex to the gas phase through ionization such as ESI or MALDI (Non-Patent Document 6). In addition, according to our previous studies using cucurbituril [6] and lysine (Lys) -containing peptides (Non Patent Literature 7), cucurbituril [n] as a host molecule can induce unique properties including selective fragmentation of peptides. In this regard, the study was carried out to improve mass spectrometry-based peptide and protein assays using cucurbituril [7].

[Non-Patent Document 1] Steen, H .; Mann, M. Nat. Rev. Mol . Cell Biol . 2004 , 5 , 699-711.

[Non-Patent Document 2] Mirzaei, H .; Regnier, F. Anal. Chem. 2006 , 78 , 4175-4183.

[Non-Patent Document 3] Heemskerk, AAM; Busnel, J.-M .; Schoenmaker, B .; Derks, RJE; Klychnikov, O .; Hensbergen, PJ; Deelder, AM; Mayboroda, OA Anal. Chem. 2012 , 84 , 4552-4559.

[Non-Patent Document 4] Kim, K .; Selvapalam, N .; Ko, YH; Park, KM; Kim, D .; Kim, J. Chem. Soc. Rev. 2007 , 36 , 267-279.

[Non-Patent Document 5] Assaf, KI; Nau, WM Chem . Soc . Rev. 2015 , 44 , 394-418.

[Non-Patent Document 6] Lee, HH; Choi, TS; Lee, SJC; Lee, JW; Park, J .; Ko, YH; Kim, WJ; Kim, K .; Kim, HI Angew . Chem . Int . Ed. 2014 , 53 , 7461-7465.

[Non-Patent Document 7] Lee, JW; Heo, SW; Lee, SJ; Ko, JY; Kim, H .; Kim, HI J. Am. Soc. Mass. Spectrom. 2013 , 24 , 21-29.

[Non-Patent Document 8] Chinai, JM; Taylor, AB; Ryno, LM; Hargreaves, ND; Morris, CA; Hart, PJ; Urbach, AR J. Am. Chem . Soc . 2011 , 133 , 8810-8813.

The present invention has been made to solve the above-described problems, in the present invention, after forming a cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound using an electrospray ionization method, a double mass spectrometry It is to provide a method of qualitative and quantitative analysis of monosaccharides by checking the cleavage pattern of each complex ion through.

In addition, in the present invention, in the analysis of peptide samples using various mass spectrometry-based methods, the addition of cooker bituril [7] can be added to cooker bituril [7], which can dramatically improve the analytical ability of the peptide to be analyzed. To provide a method for qualitative and quantitative analysis of peptides.

The present invention to solve the above problems,

(a) mixing cucurbituril [7] and an ammonium salt in an aqueous monosaccharide solution; (b) ionizing the mixture by electrospray ionization to form ammonium ions-linked cucurbituril [7] -monosaccharide complex ions; And (c) qualitative and quantitative analysis of the cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound using a Tandem mass spectrometry; Provide a quantitative analysis method.

According to one embodiment of the present invention, the monosaccharides contained in the monosaccharide solution are galactose (Galactose, Gal), glucose (Glucose, Glc), fructose (Fructose, Fru), mannose (Mannose, Man), allose (Allose) , All), Altrose (Alt), Gulose (Gulose, Gul), Taloose (Talose, Tal), Psicose (Psi), Sorboose (Sorbose, Sor), Takatose (Tagatose) , Tag), and Idoose (Idose, Ido) may be selected from the group consisting of.

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

According to another embodiment of the present invention, the formation of the cucurbituril [7] -monosaccharide complex ion to which the ammonium ion is bound in step (b) is formed in the gas phase of the cucurbituril [7] and the monosaccharide by electrospray ionization of the mixture. It may be formed by owner-guest interaction.

According to another embodiment of the present invention, the concentration ratio of the monosaccharide solution, cucurbituril [7] and the ammonium salt of step (a) may be 1: 1.5: 3 to 1: 6: 12.

According to another embodiment of the present invention, the qualitative and quantitative analysis of the monosaccharides is based on the mass / charge (m / z) of fragment ions generated from the cucurbituril [7] -monosaccharide complex ion to which the ammonium ion is bound. Analyze the ratio of the amount of ions according to the ratio of).

According to another embodiment of the present invention, when the quantitative analysis of the monosaccharides, the monosaccharide solution of step (a) may be a mixture of the monosaccharide selected as the analysis target monosaccharides and the internal standard.

At this time, the quantitative analysis of the monosaccharides may be analyzed through a calibration curve derived through an internal standard method.

In addition, the monosaccharide selected as the internal standard may be selected from the group consisting of allose (Allose, All), Altrose (Altrose, Alt), gulose (Gulose, Gul), Taloose (Talose, Tal) have.

According to another embodiment of the present invention, when the quantitative analysis of the monosaccharides, the aqueous monosaccharide solution of step (a) may be a mixture of a plurality of different analysis target monosaccharides.

In this case, the quantitative analysis of the monosaccharides may be analyzed through a calibration curve derived through a standard addition method.

In addition, the present invention to solve the above problems,

a) mixing a cucurbituril [7] with a peptide solution to form a peptide-cookerbituril [7] complex; And b) provides a method for qualitative and quantitative analysis of the peptide using cucurbituril [7] comprising the step of analyzing the complex by mass spectrometry.

According to one embodiment of the present invention, the complex may be formed by hydrophobic interaction and ion-dipole interaction between N-terminal phenylalanine residue of the peptide and cucurbituril [7].

According to another embodiment of the present invention, the peptide may be a peptide digested by pepsin.

According to another embodiment of the present invention, the peptide may be selected from the group consisting of insulin, B-chain of insulin, ubiquitin, myoglobin and mixtures thereof.

According to another embodiment of the present invention, the mass spectrometry is electrospray ionization mass spectrometry (ESI-MS), substrate-assisted laser desorption ionization mass spectrometry (MALDI-MS), ion mobility mass spectrometry (IM-MS), Or it may be based on tandem mass spectrometry based on collision-induced dissociation.

According to another embodiment of the present invention, the mixing ratio of the peptide solution and cucurbituril [7] in step a) may be 10 parts by weight to 1000 parts by weight of the cucurbituril [7] based on 100 parts by weight of the peptide solution.

The qualitative and quantitative analysis method of monosaccharides using the cucurbituril [7] according to the present invention can not only easily and quickly distinguish monosaccharides, but also have the effect of enabling the relative and absolute quantification of monosaccharides, and thus the analysis time and The cost can be drastically reduced.

In addition, according to the present invention, in analyzing a peptide sample to be analyzed using a variety of mass spectrometry, it is possible to dramatically improve the analysis signal caused by the target sample by adding only cucurbituril [7] by a simple method, The sequence coverage of the peptide of interest can be improved.

1 is a view schematically showing the qualitative and quantitative analysis of monosaccharides according to the present invention.

2 a to h) show the results of isothermal calorimetry calorimetry (ITC) measurement between cucurbituril [7] and monosaccharides in aqueous solution.

3 is a diagram showing an MS spectrum of a solution containing 20 μM of monosaccharides, 50 μM of cucurbituril [7] and 100 μM of ammonium acetate.

4 is a graph showing the ionic content ratio of fragment ions according to MS ² spectrum and normalized collision energy (CE _Norm ) of a cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound.

5 a) to c) show MS ² spectra of cucurbituril [7] -monosaccharide complex ions to which alkali metal (lithium, sodium, potassium) ions are bound.

6 a) to b) shows MS ² spectra obtained by measuring the cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound using Synapt G2 HDMS Q-TOF (a) and Q-EXACTIVE Orbitrap (b) mass spectrometers; The figure shown.

7 a) to b) show MS ² spectra obtained by measuring protonated monosaccharide ions (a) and monosaccharide ions (b) in which ammonium ions are bound by an LTQ Velos ion trap mass spectrometer.

8 a) to b) show a test curve (a) for each monosaccharide derived using the internal standard method and a test curve (b) for the monosaccharide binary mixture derived using the standard addition method.

9 is a diagram showing the structure and ion mobility spectrum distribution of the cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound.

Fig. 10 shows the geometry of the hydroxyl group (—OH) bonded to C6 of α and β monosaccharides in the cavity of cucurbituril [7].

11A to 11D show hydroxyl groups (-OH) bound to C1 (a), C2 (b), C3 (c) and C4 (d) of α- and β-type monosaccharides in the cavity of cucurbituril [7]. Figure showing the geometric structure of the.

Fig. 12 shows the geometry of the hydroxyl group (-OH) bonded to C1, C2, C3, C4, and C6 when the hydroxyl group bonded to C2 of α type Glc and Man is similarly arranged in the cavity of cucurbituril [7]. The figure shown.

FIG. 13 is a cucurbituril conjugated with ammonium ions formed using allose, all, altrose, alt, gulose, gulose and talos, as internal standards. MS ⁷ spectrum of the [7] -monosaccharide complex ion and normalized colloision energy (CE _Norm ) according to the ionic content ratio of the fragment ions.

FIG. 14 is a schematic diagram of the structure of cucurbituril [7] and a diagram in which cucurbituril [7] forms a complex with N-terminal peptide residues of peptides and proteins in solution.

15A to 15D are diagrams showing the addition of cookerbituril [7] to peptides digested with pepsin and the ESI-MS spectrum when not.

16a to 16d show the addition of cucurbituril [7] to peptides digested with pepsin and low-energy collision-induced dissociation spectra without.

17a to 17d show the addition of cucurbituril [7] to peptides digested with pepsin and MALDI-MS spectra without.

Fig. 18 is a schematic diagram showing the structure of a cucurbituril [7] peptide complex using IM-MS.

FIG. 19 is a schematic diagram illustrating the fragmentation pathway of ubiquitin4-15 peptide ions complexed with cucurbituril [7] and not.

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

As described above, monosaccharides have a problem in that qualitative and quantitative analysis is difficult due to the same mass and very similar structure, and conventional qualitative and quantitative analysis techniques of monosaccharides have many problems such as derivatization or energy-degrading MS (ERMS) of monosaccharides. There was a disadvantage that additional experiments were required, and additionally, derivatization of columns or monosaccharides was required for the analysis of mixtures of monosaccharides.

Accordingly, in the present invention, after forming a cucurbituril [7] -monosaccharide complex ion in which ammonium ions are bonded according to the gas phase owner-guest interaction of cucurbituril [7] and monosaccharides, Tandem Mass Spectrometry (MS ² ) In addition to the qualitative analysis of monosaccharides easily and quickly, it is intended to provide an effective method for qualitative and quantitative analysis of monosaccharides, which enables relative and absolute quantification of monosaccharides.

1 is a view schematically showing the qualitative and quantitative analysis of monosaccharides according to the present invention.

Referring to Figure 1, the qualitative and quantitative analysis of monosaccharides according to the present invention comprises the steps of: (a) mixing a cucurbituril [7] and an ammonium salt in a monosaccharide solution; (b) ionizing the mixture by electrospray ionization to form ammonium ions-linked cucurbituril [7] -monosaccharide complex ions; And (c) qualitatively and quantitatively analyzing the cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound by using dual mass spectrometry.

At this time, the monosaccharide is not particularly limited as long as it is known in the art, but preferably galactose (Galactose, Gal), glucose (Glucose, Glc), fructose (Fructose, Fru), mannose (Mannose, Man), allo Allose, All, Altrose, Alt, Gulose, Gul, Talos, Tal, Psicose, Psi, Sorbose, Sor, Takato It may be selected from the group consisting of (Tagatose, Tag), Idoose (Idose, Ido).

In addition, the ammonium salt is not limited thereto as long as it includes a salt containing ammonium ions, preferably may be one or more selected from the group consisting of ammonium acetate, ammonium chloride, ammonium carbonate, ammonium hydroxide.

In addition, the cucurbituril [7] -monosaccharide complex ion to which the ammonium ion is bound in step (b) is separated by the gas phase owner-guest interaction between the cucurbituril and the monosaccharide while the solvent contained in the monosaccharide solution is evaporated by electrospray ionization. Ammonium ions, cookerbituryl [7] and monosaccharides form complex ions.

In addition, the concentration ratio of the monosaccharide aqueous solution, cucurbituril [7] and the ammonium salt of step (a) is preferably 1: 1.5: 3 to 1: 6: 12. In this case, when the concentration ratio is less than the lower limit, there are many monosaccharides that do not bind, which may cause a problem in quantification. When the concentration ratio exceeds the upper limit, there may be a problem in measuring complex ions due to the increase in the number of cucurbituril ions to which the monosaccharide is not bound.

In addition, the qualitative and quantitative analysis of the monosaccharide according to the present invention is based on the ratio of the mass / charge (m / z) of the fragment ions (fragment ions) generated from the cucurbituril [7] -monosaccharide complex ion to which the ammonium ion is bound Analyzing the ratio; may be performed through. At this time, when qualitatively analyzing the monosaccharides, the ratio of the mass / charge (m / z) of the fragment ions generated from the cucurbituril [7] -monosaccharide complex ions to which ammonium ions are formed per monosaccharide is bound. By comparing the ionic content ratio according to the monosaccharides will be distinguished.

In addition, when the monosaccharide is quantitatively analyzed, the monosaccharide solution of step (a) is mixed with the analyte monosaccharide and a monosaccharide selected as an internal standard, and is described in the following Examples. Analytical monosaccharides can be quantified through the test curve derived from. In this case, the monosaccharide selected as the internal standard is hardly present in the body, and the monosaccharide is known to know the absolute amount, but is not necessarily limited thereto. Allose, All, Altrose, Alt, and Gulose ( Gulose, Gul), Talose (Talose, Tal) can be selected from the group consisting of.

In addition, the present invention can be quantified for each monosaccharide when the analyte monosaccharide is mixed, wherein the aqueous monosaccharide solution of step (a) is mixed with a plurality of different analyte monosaccharides, which will be described in the following examples. As described above, the assay curve derived through the standard addition method can be used to quantify the analyte monosaccharides.

In addition, the present invention provides a method for improving analytical performance of peptides that are hardly detected on mass spectrometry through selective non-covalent interactions between a cucurbituril [7] and a peptide having an N-terminal phenylalanine residue. The method according to the present invention, which promotes signal enrichment using such master-guest chemistry, is applicable to both ESI-MS and MALDI-MS, and has a particularly excellent effect on protein analysis including pepsin digestion process. This is because pepsin preferentially cleaves non-polar sites containing phenylalanine. In the present invention, this signal potentiating effect of cucurbituril [7] is used to utilize insulin B-chain (InsB; 3.4 kDa), insulin (Ins; 5.8 kDa), ubiquitin (Ubq; 8.6 kDa), and myoglobin (Myb; 17.0 kDa). Analysis was performed. Furthermore, according to the present invention, the sequence coverage of peptides associated with cucurbituril [7] was improved for double mass spectrometry experiments. Accordingly, in the present invention, signal enhancement and improved sequence coverage by cucurbituril [7] were analyzed based on charge stabilization properties, and thus an effective approach in analyzing them using mass spectrometry by targeting and capturing specific residues of peptides. It suggested that a method could be provided, and also proposed a mechanism for a novel fragmentation pathway of polypeptides induced by the host molecule.

Therefore, in the present invention,

a) mixing a cucurbituril [7] with a peptide solution to form a peptide-cookerbituril [7] complex; And b) provides a method for qualitative and quantitative analysis of the peptide using cucurbituril [7] comprising the step of analyzing the complex by mass spectrometry.

In the present invention, the peptide-cookerbituryl [7] complex may be formed by hydrophobic interaction and ion-dipole interaction between the N-terminal phenylalanine residue and the cucurbituril [7] of the peptide, and may be analyzed in the present invention. Is a variety of peptides digestible by pepsin, for example, but not limited to various peptides selected from the group consisting of insulin, B-chain of insulin, ubiquitin, myoglobin and mixtures thereof.

In addition, the qualitative and quantitative analysis methods according to the present invention are applicable to various mass spectrometry based methods, for example, electrospray ionization mass spectrometry (ESI-MS), substrate-assisted laser desorption ionization mass spectrometry (MALDI). -MS), ion mobility mass spectrometry (IM-MS), or tandem mass spectrometry based on collision-induced dissociation.

Meanwhile, the mixing ratio of the peptide solution and the cucurbituril [7] in step a) may be 10 parts by weight to 1000 parts by weight of the cucurbituril [7] with respect to 100 parts by weight of the peptide solution, and the content of cucurbituril [7] is 10 parts by weight. If it is less than the portion, there is a problem that complex formation with the peptide may not be efficient, and if it exceeds 1000 parts by weight, there is a problem that extra cooker bituril [7] is ionized, which is not preferable.

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

Qualitative and quantitative analysis of monosaccharides

material

D-type monosaccharides (D-Gal, D-Glc, D-Fru, D-Man, D-All, D-Alt, D-Tal), L-type monosaccharides (L-Gul), cucurbituril [7], acetic acid Ammonium, lithium acetate, sodium acetate and potassium acetate were purchased from Sigmal Aldrich (St. Louis, MO, USA). HPLC-grade water purchased from Avantor Performance Materials, Inc. (Center Vallet, PA, USA) was used as a solvent, and a stock solution of monosaccharides was prepared at least 100 mM, and the exact concentration of the stock solution was determined by nuclear magnetic resonance spectroscopy. magnetic resonance spectroscopy).

Isothermal calorimetry (ITC) measurement

All ITC experiments were measured using a VP-ITC calorimetry (Microcal, Northampton, MA, USA) at 25 ° C. 0.4 mM CB [7] solution in ITC cells was titrated with a syringe containing 10 mM aqueous solution of monosaccharides. The solution solution was degassed before the experiment, the number of injections was 40 and the injection volume was 6 μL. Initial equilibration time was 1000 s and injection interval was 300 s.

Double mass spectrometry

Dual mass spectrometry (Tandem MS, MS ² ) experiments for monosaccharide analysis were performed using a Thermo Scientific LTQ Velos Dual Ion Trap Mass Spectrometer (San Jose, CA, USA) with an ESI source. The spray voltage was 3.5 kV, the temperature of the capillary was 300 ° C., and the sheath gas flow was 18 (arbitrary units). The concentration of CB [7] was 150 μM and the concentrations of ammonium and metal salts were 300 μM. The concentration of D-allose for quantitative analysis of each monosaccharide was adjusted to 20 μM. Normalized collision energy (CE _Norm) value at the time of content by normalized collision energy (CE _Norm) and the standard addition method at the time of content by the internal standard method was set at 15% and 18%, respectively.

High resolution mass spectrometry experiments were performed with a Thermo Q-Exactive Orbitrap mass spectrometer. The spraying voltage was 4.00 kV and the capillary temperature was 200 ° C. Ion mobility measurement experiments were carried out using a Synapt G2 HDMS mass spectrometer with a Z-spray ESI source (Waters, Manchester, U.K.). Capillary, sampling cone, and extraction cone voltages were 2.00 kV, 20 V, and 2.00 V, respectively, and the source and desolvent temperatures were 100 and 150 ° C, respectively. The gas flows of helium IMS cells were 180 mL / min and 60 mL / min, respectively, and the propagation rates and heights were 280 m / s and 13 V, respectively. In addition, polyaniline was used to correct the arrival time for the impact cross section.

Molecular Dynamics (MD) Simulation and Density Functional Theory (DFT) Calculation

All MD simulations were measured using Gromacs 4.5.5. (Hess, B. et al. J. Chem . Theory Comput. 2008 , 4 , 435). The force field parameters of CB [7] and monosaccharides were adapted from the CHARMM general force field (CGENFF) (Vanommeslaeghe, K. et al. J. Comput. Chem. 2010 , 31 , 671).

The initial geometry of the CB [7] -monosaccharide complex ions was formed using Hyperchem 7.5 (Hypercube Inc., Gainesvile, FL, USA) to insert monosaccharides into the cavity of CB [7], forming a candidate structure in the gas phase. Simulated annealing was performed to obtain. The final frame of 300 annealing cycles (90 ns) was extracted and further structural optimization was performed for the five lowest energy structures of each composite ion. Structure optimization was performed using DFT calculations and Q-Chem4.1 computational package (Q-Chem Inc., Pittsburgh, PA, USA). Subsequently, isothermal annealing was performed at 600 ° C. for the optimized α complexes to observe the structural differences between the monosaccharides inside the cavities of CB [7].

In monosaccharide mixtures, MS ²  Quantitative Analysis of Monosaccharides Through Spectrum

First, the signal of the fragment peaks in the MS ² spectrum was integrated and the relative abundance ratio between the two fragment peaks ( m / z 662.8 and 671.8) was obtained. In the case of the mixed solution of monosaccharide mixed with Man and Fru, m / z 653.8 and 671.8 were used for quantification because the abundance ratios of Man and Fru m / z 662.8 per 671.8 were similar. The signal of the fragment peaks was integrated at 5 σ. Then, the α value of the two monosaccharides was calculated using the following equation (1) and Table 1 below.

[Equation 1]

m / z 590.7 599.2 653.8 662.8 671.8 680.8 All 0.09 0.02 0.07 0.08 0.73 0.01 Gal 0.01 0.01 0.01 0.01 0.71 0.25 Glc 0.10 0.04 0.01 0.44 0.36 0.05 Man 0.15 0.17 0.01 0.17 0.49 0.01 Fru 0.14 0.15 0.52 0.05 0.13 0.01

Quantitative Analysis of Monosaccharides Using the Slope of the Calibration Curve

The concentration of the analyte monosaccharide was calculated using the following [Equation 2], in order to quantify the concentration of the reference monosaccharide (c _Ref ) in the solution, the ratio of the α value of the analyte monosaccharide and the reference monosaccharide (y = α _A / α Slope of the calibration curve of _Ref ) and two monosaccharides.

[Equation 2]

In addition, in order to quantify the two monosaccharides (denoted A and B) in the monosaccharide binary mixture, the following [Formula 3] and [Formula 4] were used based on the standard addition method.

[Equation 3]

[Equation 4]

At this time, the concentration (c _{B, s} ) of the monosaccharide B added further was adjusted to 10 μM.

Review

Although master-guest chemistry using synthetic polymers sometimes distinguishes isomeric molecules (Han, C. et al. Sens . Actuator B- Chem . 2009 , 137 , 704), monosaccharide isomeric distinctions are still a challenge in master-guest chemistry. to be. The reason is that the hydrogen bond between the monosaccharide and the water molecule is broken when the synthetic polymer and the monosaccharide are bonded, resulting in an energy disadvantage. It is also because the fine structural difference between the monosaccharides may not be visible in the aqueous solution in the presence of such strong non-covalent bonds. Therefore, in the present invention, in the absence of water, the monosaccharide isomers will be able to bind with the synthetic polymer, and the structural difference between the monosaccharide isomers will be easily expressed through the owner-guest interaction with the synthetic polymer CB [7] in the gas phase. With this in mind, the present invention has been completed.

Monosaccharides to be analyzed in the present invention are not necessarily limited thereto, but the most common monosaccharide isomers in the human body are D-galactose (Gal), D-glucose (Glc), D-mannose (Man) and D-fructose. (Fru) was used, and the interaction between them and CB [7] in an aqueous solution was first investigated by thermal measurement using an isothermal titration calorimeter (ITC). As a result, it was confirmed that none of the monosaccharides showed significant interaction with CB [7] in aqueous solution (FIG. 2), probably because these monosaccharides are hydrophilic and have no cationic functional groups ( Lee, JW et al. J. Phys. Chem. B 2013 , 117 , 8855).

In contrast, in the present invention, it can be confirmed that CB [7] and monosaccharide complexes are formed through electrospray ionization (ESI) (FIG. 3), which removes the interaction between water and monosaccharides in ESI and enhances electrostatic interaction. For owner-guest union can be increased. CB [7] -monosaccharide complexes were observed as cationized forms with ammonium ions or alkali metal ions, depending on the type of salt added to the solution, by performing MS ² to CB [7]-with ammonium ions or alkali metal ions. Investigation of the cleavage pattern of monosaccharide complexes (FIGS. 4 and 5) shows that the complexes bound to alkali metals readily show the separation of monosaccharides from CB [7] upon collision activation, whereas the CBs bound to ammonium salts of different monosaccharides As shown in FIG. 4, the [7] -monosaccharide complex ion exhibited a cleavage pattern that was clearly distinguished over a wide range of MS ² parameters in the ion trap MS (LTQ Velos). Fragment peaks of CB [7] -monosaccharide complex ions bound by ammonium salts were observed at m / z 680.8, 671.8, 662.8, 653.8, 599.2 and 590.7, which correspond mainly to ammonia and water-depleted complex ions (FIGS. 4 and 6). ). In contrast, pure monosaccharide ions without CB [7] showed cleavage patterns that were difficult to distinguish between different monosaccharides. Protonated forms of Gal, Glc and Man showed very similar cleavage patterns and Gal and Man combined with ammonium salts had similar cleavage patterns in common (FIG. 7). These results show that CB [7] used in the present invention amplifies small differences in monosaccharide structure in the gas phase, yielding distinctive cleavage patterns and facilitating qualitative analysis through MS ² . In addition, these distinct cleavage patterns of CB [7] -monosaccharide complex ions were observed by three different mass spectrometers (ie, ion traps, orbitrap and Q-TOF mass spectrometers) employing different MS ² methods. Universality was demonstrated (FIG. 6).

In addition, in the present invention, as described above, the monosaccharides can be qualitatively distinguished using CB [7], and quantitative analysis of the monosaccharides is possible. Specifically, the cleavage pattern of the mixture of CB [7] -monosaccharide complex ions bound to ammonium salts can be separated by a linear combination of fragmentation patterns of each of the complex ions. It is noted, however, that the ratio of the complex ions of each of the monosaccharides obtained from this linear separation is not necessarily consistent with the ratio of monosaccharides in solution since it is affected by both the binding in the solution and the binding in the gas phase observed in MS. Should be. The complexity of the binding phenomena in ESI according to the present invention was greatly reduced because monosaccharides rarely interacted with CB [7] in solution. Accordingly, a simple relationship can be found between the ratio of monosaccharides present in solution and the ratio of monosaccharides present as complex ions in the gas phase. In the present invention, D-allose (All) was used as an internal standard as a non-natural monosaccharide standard showing a distinct pattern of cleavage in MS ² compared to natural monosaccharides, and CB [7] for quantitative analysis of monosaccharide isomers. A distinctive cleavage pattern of the complex was utilized (FIG. 13). The relative abundance of the cleavage peaks of five monosaccharides (four are analyte and one internal standard) is shown in Table 1 above.

On the other hand, this condition was used during MS ² experiments because complete fragmentation was observed at 15% of normalized collision energy (CE _{Norm .} ). The relative presence of cleavage peaks in the MS ² spectrum in a mixture of monosaccharides to be analyzed and the monosaccharide as an internal standard follows the relationship shown in Equation 1 below:

[Equation 1]

In Formula 1, α _A and α _Ref represent the fractions of the monosaccharides to be analyzed as the complex ions and the monosaccharides selected as the internal standard (reference). R _A , R _Ref and R _Mix are the relative presence of two cleaved peaks in the MS ² spectrum of the monosaccharide complex ion and the mixture of monosaccharide complex ion and monosaccharide complex ion selected as an internal standard, respectively (R = I _A / I _B ). Since the peaks at m / z 662.8 and 671.8 in this study provided the most reproducible results, these peaks were chosen to quantify the individual monosaccharides.

In the present invention, the gas phase fractions α _A and α _Ref of the monosaccharide complexes were obtained to quantify the monosaccharides to be analyzed in the range of 10-100 μM using Equation 1 above. It was confirmed that the ratio of α _A and α _Ref was proportional to the ratio of the monosaccharides selected as analyte and the monosaccharides in the solution. 8a shows a linear relationship between the ratio of monosaccharides in solution and the ratio of monosaccharides of CB [7] -monosaccharide complex ions bound to ammonium ions in the gas phase. The slopes of the calibration curves for Gal, Glc, Man and Fru were 0.489, 0.345, 0.631 and 0.189, respectively, indicating high linearity (R ² = 0.999). In addition, the detection limit (LOD) and quantification limit (LOQ) of the four monosaccharides were 0.46-0.90 g / mL and 1.39-2.72 g / mL, respectively (see Table 2). It is suggested that it is a very suitable method for determining the concentration of.

LOD (μg / ml) LOQ (μg / ml) Gal 0.62 1.88 Glc 0.46 1.39 Man 0.90 2.72 Fru 0.87 2.65

In the present invention, MS is based on a method of adding a standard to infer the concentration of the analyte from an experiment including adding one of the monosaccharides in the mixture as a standard to the analyte solution for quantification of the monosaccharide in the bicomponent monosaccharide mixed solution ^Two experiments were performed. At this time, CE _{Norm in} MS ² (LTQ Velos) _. Was set to 18%, and a relationship between each of the natural monosaccharides and the ratio of two monosaccharides similar to that obtained from Allose (All) (Fig. 8a) was obtained (Fig. 8b). Next, the concentration ratio of the said monosaccharide was obtained by calculating the value of two monosaccharides (indicated by A and B) in the mixture using the above [Formula 3]. Using this method, the mixture concentration of two monosaccharides could be calculated very accurately as shown in Tables 3 and 4 below.

[Glc]: [ ^a M] 5: 20 20: 5 ^a M Gal Man Fru Gal Man Fru [Glc] 5.1 ± 0.1 5.3 ± 0.6 5.1 ± 0.1 19.7 ± 1.9 19.7 ± 0.2 21.0 ± 0.3 [ ^a M] 18.8 ± 0.4 21.4 ± 1.9 22.2 ± 0.8 4.7 ± 0.4 5.9 ± 0.7 4.9 ± 0.7

^a M indicates monosaccharides, Gal, Man, and Fru.

[A]: [B] 5: 20 20: 5 A Gal Gal Man Gal Gal Man B Man Fru Fru Man Fru Fru [A] 4.9 ± 0.3 4.7 ± 0.3 4.6 ± 0.2 20.6 ± 0.9 19.1 ± 0.2 20.5 ± 1.1 [B] 19.2 ± 1.6 18.5 ± 1.2 19.0 ± 1.2 5.7 ± 0.2 6.1 ± 0.4 4.4 ± 0.5

On the other hand, in order to understand the different master-guest interactions of monosaccharides inside CB [7], the structural characteristics of CB [7] -monosaccharide complex ions bound to ammonium ions can provide the overall structural information of gas phase ions. Ion mobility MS was analyzed using Synapt G2 HDMS. It was confirmed that the arrival time of the complex ion was almost the same as the arrival time of the CB [7] ion to which the ammonium salt, which did not form a complex, was found to be present in the CB [7] cavity (FIG. 9). ). In addition, computational modeling using molecular dynamics (MD) simulations and density function theory (DFT) calculations confirmed that the monosaccharides were present in the CB [7] cavity in vacuum (FIG. 9). Furthermore, the geometries of monosaccharides in CB [7] are distinctly distinct, suggesting that different monosaccharides interact differently from CB [7].

Next, an isothermal annealing simulation was performed at 600 K for 150 ns to observe the geometry of the monosaccharides in CB [7] in the thermally activated gas phase. Gathering the complex ions' geometries, it was confirmed that each of the monosaccharides existed at different positions within the CB [7] cavity. The distance and angle between the saccharide hydroxyl group and the central plane of CB [7] are shown in FIGS. 10 and 11 below. As shown in FIGS. 10 and 11, the orientation of the hydroxyl groups bonded to C6 of the monosaccharide was different under thermal activation conditions, and the same tendency was observed for all other hydroxyl groups except for the hydroxyl groups bonded to C2. . The hydroxyl groups bonded to C2 of α-type Glc and Man showed similar embryonic shapes, but as shown in FIG. 12, all hydroxyl groups except C-linked hydroxyl groups were arranged differently from each other. It can be seen that α-type Glc and Man also exist differently in [7].

As described above, the qualitative and quantitative analysis of the monosaccharides using the cucurbituril [7] according to the present invention can easily and quickly distinguish monosaccharides without separate sample processing such as derivatization, which has been pointed out as a problem of the prior art. In addition, the effect of enabling relative and absolute quantification of monosaccharides can significantly reduce the analysis time and cost of monosaccharides.

Peptide  Qualitative and quantitative analysis

material

InsB was purchased from Anygen (Gwangju, Korea). Human recombinant Ins, Ubq from bovine erythrocytes, Myb from horse heart, pepsin from porcine gastric mucosa, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade water and acetonitrile were obtained from Avantor Performance Materials, Inc. (Center Valley, PA, USA) was purchased and used as a solvent. Cookerbituril [7] was purchased from CBTECH (Pohang, Korea) and a stock solution (1 mM) was prepared by dissolving cookerbituril [7] in water. Pepsin digests were prepared by incubating protein (100 μM) with pepsin (0.24 mg / mL) for 15 minutes at 37 ° C. in water containing 1% v / v formic acid. In ESI-MS, the sample was diluted 10-fold. Cucurbituril [7] was then added to the solution, and the solution was subjected to ESI-MS analysis. The concentration of cucurbituril [7] was optimized to 50, 35, 100, and 50 μM for 10 μM of InsB, Ins, Ubq, and Myb, respectively.

Electrospray Ionization Mass Spectrometry and Collision Induction harry  (Collision-Induced Dissociation)

For mass spectrometry experiments, in cationic mode, a Waters Synapt G2 HDMS quadrupole time-of-flight (Q-TOF) mass spectrometer with a Z-spray ESI source (Waters, Manchester, UK) was used. As experimental parameters, capillary voltage of 3.50 kV, sampling cone voltage of 10 V, extraction cone voltage of 5 V, source temperature of 100 ° C., and desolvation temperature of 150 ° C. were set.

For collision induced dissociation (CID) experiments, in cationic mode, a Thermo Scientific LTQ Velos dual ion trap mass spectrometer (San Jose, CA, USA) was used. As a parameter for ESI, a capillary voltage of 3 kV and a capillary temperature of 200 ° C were set. Each spectrum was obtained by averaging 200 scanned spectra obtained using an enhanced scan mode. The nomenclature for the detected ions and their fragment ions followed the method of Roepstorff and Fohlman (Roepstorff, P .; Fohlman, J. Biomed. Mass Spectrom . 1984 , 11 , 601). Left asterisk (*) subscripts indicated for peptide and fragment ions indicate the conjugate of cucurbituril [7]. For example, the b _n ²⁺ fragment ion complexed with cucurbituril [7] is named * b _n ²⁺ .

Substrate-Assisted Laser Desorption Ionization Mass Spectrometry

For MALDI-MS experiments, a Waters Synapt G2 HDMS mass spectrometer with a MALDI source using a 355-nm Nd: YAG laser was used (Waters, Manchester, UK). MALDI laser firing speed and energy were 1000 Hz and 350 (arbitrary units), respectively. The substrate solution was 20 mg / mL α-cyano-4-hydroxycinnamic acid (CHCA) in 77% acetonitrile, 22.9% water and 0.1% formic acid (v / v). The concentration of protein for pepsin digestion was 20 μΜ and pepsin digestion conditions were the same as those used in the ESI-MS experiments. The final concentration of cucurbituril [7] after addition to the digested samples was optimized to 20 μM for InsB and 40 μM for Ubq. A sample solution for MALDI-MS was then prepared by mixing the substrate and analyte solutions in a volume ratio of 1: 1. For analysis, sample solutions (2 μL) were spotted on the sample plate.

Ion Mobility Mass Spectrometry and Computer Modeling

For ion mobility MS (IM-MS) experiments, a Waters Synapt G2 HDMS device was used. Ion mobility measurements were performed using four device parameters. The gas flow, TWIMS wavelength velocity and wavelength height for helium cells are fixed at 180 mL min ^-1 , 300 ms ^-1 and 20.0 V, respectively, while the gas flow for drift cells is three different: 50, 60 or 80 mL min ^-1 . The conditions were used. Compensation of the experimental arrival time with the collision cross-sectional area (Ω _D ) values is based on polyalanine (Henderson, SC; Li, J .; Counterman, AE; Clemmer, DE J. Phys. Chem . B 1999 , 103 , 8780). -8785), melittin (Salbo, R .; Bush, MF; Naver, H .; Campuzano, I .; Robinson, CV; Pettersson, I .; Jorgensen, TJD; Haselmann, KF Rapid Commun . Mass Spectrom. 2012 , 26 , 1181-1193), and denatured Ubq (Bush, MF; Hall, Z .; Giles, K .; Hoyes, J .; Robinson, CV; Ruotolo, BT Anal. Chem . 2010 , 82 , 9557-9565) Was performed using the previously reported Ω _D values for. The corrected Ω _D values from the three measurements were averaged.

Computer modeling of cucurbituril [7] -peptide complexes was performed using GROMACS 4.5.5 (Hess, B .; Kutzner, C .; van der Spoel, D .; Lindahl, E. J. Chem. Theory Comput . 2008 , 4 , 435-447). For simulation, the CHARMM force field (Mackerell, AD; Feig, M .; Brooks, CL J. Comput . Chem . 2004 , 25 , 1400-1415; Bjelkmar, P .; Larsson, P .; Cuendet, MA; Hess, B Lindahl, E. J. Chem. Theory Comput . 2010 , 6 , 459-466 and CHARMM General Force Field (Vanommeslaeghe, K .; Hatcher, E .; Acharya, C .; Kundu, S .; Zhong, S Shim, J .; Darian, E .; Guvench, O .; Lopes, P .; Vorobyov, I .; Mackerell, AD J. Comput. Chem . 2010 , 31 , 671-690). Initial random coil structures of the peptides were generated using Hyperchem 7.0 (Hypercube Inc., Gainesville, FL. USA). Each of the peptide models was merged with a cucurbituril [7] molecule and annealing simulation 500 cycles were performed using the following profile: heating from 300 K to 800 K for 50 ps, constant temperature simulation at 800 K for 50 ps, 25 ps Cool from 800 K to 300 K, and equilibrate at 300 K for 25 ps. Structures were extracted from the last frame of each annealing cycle and considered as candidate structures. Five structures with the lowest potential energy were selected and the projection approximation model (Wyttenbach, T .; vonHelden, G .;) adopted in MOBCAL ( http://www.indiana.edu/~nano/ ). Batka, JJ; Carlat, D .; Bowers, MT J. Am. Soc . Mass Spectrom . 1997 , 8 , 275-282) were used to calculate their Ω _D values. No complexes and no Myb34-70 peptides were simulated because they have very high degrees of freedom and require long simulation times.

Results and review

With Cookerbituril [7] In complexation  ESI-MS signals of pepsin digested peptides with N-terminal phenylalanine are enhanced

15A-15D show the mass spectra of pepsin digested peptides with and without cucurbituril [7]. Table 5 also lists the peaks of the peptides with N-terminal phenylalanine and the ratio of their relative abundance in the presence and absence of cucurbituril [7].

Two peptide species with N-terminal phenylalanine (B1-11 and B1-13, separately colored in FIG. 15A) were observed on the digested InsB sample, but in the absence of cucurbituril [7], its abundance was low. By adding cucurbituril [7] to this sample, the abundance of InsB1-11 (FVNQHLCGSHL) and InsB1-13 (FVNQHLCGSHLVE) peptides increased ˜5 fold and ˜4 fold, respectively, with the cucurbituril [7] peptide complex peak on the mass spectrum It became the biggest. In addition to the InsB1-11 and InsB1-13 peptides described above, two peptides containing N-terminal phenylalanine were additionally generated when pepsin digested Ins (see FIG. 15B). The abundance of these two peptide ions was also increased ˜7 and ˜3 times by the addition of cucurbituril [7], respectively.

Analysis of the larger protein, Ubq, revealed that signals for peptides with N-terminal phenylalanine are rarely detectable initially (see FIG. 15C). The low signal of these peptide peaks also pose problems in conventional analytical procedures that further identify and characterize the sequences using dual mass spectrometry. Following the addition of cucurbituril [7], the Ubq4-15 peptide (FVKTLTGKTITL) peaks appeared to increase ˜9-fold in relative abundance, making analysis very easy. Similar reinforcing effects were observed for Myb (see FIG. 15D), and the abundance of Myb34-70 (FTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVL) peptide was increased by 2 times. Consistent signal potentiation observed for four different proteins means that combining pepsin digested peptides with cucurbituril [7] is an effective technique that is widely applicable.

In CID Cooker bituril [7] · Peptide  Enhanced sequence of complexes Coverage

Analyzing fragment ions using dual mass spectrometry is an important process for the precise characterization of peptide ions. Therefore, in this example, it was tested whether cookerbituril [7] .peptide complex ions can be accurately specified by using dual mass spectrometry (see FIGS. 16A to 16D). In addition, the sequence coverage of the complexed ions and the percentage of complementary b / y ion pair detection were compared to the uncomplexed ions, both of which are important for reliable analysis using dual mass spectrometry. In order to compare the efficiency of the double mass spectrometry, we compared the above with respect to cucurbituril [7] conjugated and unbounded ions and compared the ions with the most abundant peptide ions whose peaks do not overlap with other peaks. Are summarized in Table 6 below.

CID spectra of uncomplexed InsB1-11 ions were observed to consist of b- and y-type fragment ions, which are the fragments commonly found in low-energy CIDs (Paizs, B .; Suhai, S. Mass Spectrom . Rev. 2005 , 24 , 508-548). Similarly, the complexed InsB1-11 ions were also dissociated into b- and y-type fragment ions (FIG. 16A, bottom), through which additional sequence information can be obtained. Interestingly, up to 100% sequence coverage was obtained for the complexed InsB1-11 ions, but the uncomplexed ions did not fragment between Gly8 and Ser9 (inset of FIG. 16A). Furthermore, for complexed ions, there were a greater number of b / y complementary ion pairs due to the observation of additional * b ₃ , * b ₄ ²⁺ , and * b ₅ ²⁺ ions. These results indicate that cucurbituril [7] complexation can improve the results of double mass spectrometry of peptides. CIDs for other peptide ions and their complexes also produced more extensively fragmented b- and y-type fragment ions (see FIG. 16B-D and Table 6). In all cases, the cucurbituril [7] complex showed better results. For example, 19 out of 22 amide bonds were cleaved and 8 b / y complementary ion pairs were present in the uncomplexed InsA1-11 / B1-11 ions (FIG. 16B, top). In the complexed form, all the amide bonds were cleaved and there were 12 b / y complementary ion pairs (bottom of FIG. 16B).

Ubq4-15 peptide ions also exhibited complete sequence coverage, with a larger number of b / y complementary ion pairs observed in the complexed form (see Table 6). The sequence of the complexed Myb34-70 ions was too large in size to be completely covered, but nevertheless extensive fragmentation was observed (61% to 75%, see Table 6). Referring to Table 6, the complexed InsB1-13 and InsA1-13 / B1-13 ions also increased sequence coverage (83% to 100% and 83% to 92%, respectively) and larger b / y complementary ion pairs It can be seen that the ratio of these. The consistent improvement in fragmentation efficiency observed for the various peptides was achieved by complexing the peptides with cucurbituril [7], thus further fragmentation more than would normally be possible in the absence of cucurbituril [7], without the need for special modification or peptide modification using chemical reactions. And analysis is possible.

With Cookerbituril [7] In complexation  MALDI-MS signals of pepsin digested peptide with N-terminal phenylalanine are enhanced

The signal enhancing effect of cucurbituril [7] was also observed in the MALDI spectrum. Three peptides containing N-terminal phenylalanine, obtained from digestion of InsB, exhibited a ˜2 to ˜8-fold increase in their relative abundance when complexed with cucurbituril [7] (see FIG. 17A). ). InsB25-30 peptide, which was initially undetectable, also became apparently observable after the addition of cucurbituril [7]. Similarly, for Ubq4-15 peptides that were not detectable by MALDI-MS, the addition of cucurbituril [7] became very rich (FIG. 17B). Accordingly, the signal enhancing effect of cucurbituril [7] is not limited to ESI-MS, but also applicable to MALDI-MS, and it provides information on peptides that could not be easily detected in the absence of cucurbituril [7].

On cucurbituril [7]  By Mediated  Enhanced mass spectrometry signals and Understanding Fragmentation

It has previously been reported that ionization of cucurbituril [7] produces higher charged complex ions (Lee, JW; Lee, HHL; Ko, YH; Kim, K .; Kim, HI J. Phys. Chem . B 2015 , 119 , 4628-4636). This is because the functional groups of the positively charged guest molecules are stabilized by strong ion-dipole interactions with carbonyl groups at the inlet portion of cucurbituril [7], thus reducing the electrostatic repulsion between the two cationic functional groups. It is believed that similar charge strengthening effects occur in the present invention. For example, the most abundant state of charge of B-chain Ins peptides (B1-11 and B1-13) and Ins peptides (A1-13 / B1-11 and A1-13 / B1-13) containing N-terminal phenylalanine Are +2 and +3, respectively, in the absence of cookerbituril [7], but are added to +3 and +4, respectively, when cookerbituril [7] is added (see FIG. 15B). In the case of the Ubq4-15 peptide, +2 charged ions disappear by adding cucurbituril [7], and only +3 charged ions are observed (see FIG. 15C). These results clearly show that cucurbituril [7] causes an antistatic effect on the guest molecules. Previous studies have reported similar effects (Lee, JW; Lee, HHL; Ko, YH; Kim, K .; Kim, HI J. Phys. Chem . B 2015 , 119 , 4628-4636). On the other hand, high proton affinity is one of the most important factors for effective ionization by ESI and MALDI (Mirzaei, H .; Regnier, F. Anal. Chem . 2006 , 78 , 4175-4183) This is because water competes to obtain protons and ionize during the ionization process. Therefore, the charge enhancing effect by cucurbituril [7] increases the proton affinity of the peptides, which can be presumed to cause signal enhancement of each peptide ion.

In order to understand cucurbituril [7] peptide complex ions in more detail, the present invention investigated the structure of cucurbituril [7] peptide using IM-MS (see FIG. 18). It can provide structural information. According to the IM-MS results, both complexed and uncomplexed peptide ions are generally present in one or two structures. The theoretical collision cross-sectional area (Ω _D ) values for the lowest energy structures of the composite ions obtained through computer modeling generally show good agreement with the experimental data. Looking at the structure of the theoretical models, it is shown that cucurbituril [7] binds to the N-terminal phenylalanine of the peptides. Interestingly, these models also draw cucurbituril [7], in addition to the phenylalanine N-terminus, to attract other positively charged residues towards them to form a 'peptide tweezer' structure (FIG. 18). Structural features of these peptides caused by cucurbituril [7] have not been reported previously, meaning that cucurbituril [7] has excellent charge stabilizing ability.

Based on the above results, the inventors determined that cucurbituril [7] promotes fragmentation of peptides during the dual mass spectrometry because it effectively interacts with the functional groups of the peptides. For example, FIG. 16C shows that cleavage in the C-terminal amide backbone of Lys residues does not occur in the CID of uncomplexed Ubq4-15 peptide ions. This is in contrast to the cleavage of the C-terminal amide bonds in the presence of Lys (Paizs, B .; Suhai, S. Mass Spectrom . Rev. 2005 , 24 , 508-548). However, since the cleavage of the amide bond C-terminus to the Lys residue must be preceded by the neutralization of the Lys side chain and the proton transport to the amide bond, these processes are unlikely to occur for uncomplexed Ubq4-15 ions. Can be. In contrast, experimental results according to the present invention show that fragmentation at these sites is possible when the peptide complexes with cucurbituril [7] (FIG. 16C, bottom). The results show that cucurbituril [7] can facilitate the neutralization of Lys residues and the proton transport to its C-terminal amide bonds. Thus, the inventors have discovered that more efficient fragmentation is achieved by promoting the migration of the protons while the cucurbituril [7] interacts with the positively charged Lys side chains, allowing the protons to move to the amide bond C-terminus to the Lys residue. It is determined to cause (see Fig. 19).

Taken together, the present invention provides an improved mass spectrometry based analysis method for proteins by improving the sequence coverage in signal and double mass spectrometry of peptides digested with puckerin by Cookerbituril [7]. We have demonstrated that by capturing N-terminal phenylalanine with cucurbituril [7], we can provide a general approach to selectively improve the analytical performance in mass spectrometry of pepsin digested peptides. Signal enhancement by the addition of cucurbituril [7] may be attributed to the increased proton affinity. In addition, improved sequence coverage by cucurbituril [7] can be judged because cucurbituril [7] interacts strongly with residues charged at varying amounts during CID to enable several fragmentation pathways. The method according to the invention does not require the preparation of new instruments, chemical labels or special samples, and can be carried out by simply adding a small amount of commercially available cucurbituril [7] to the sample. Thus, given the simplicity and generality of the present invention, the present invention can provide a very effective means for general purpose protein analysis.

According to the present invention, qualitative analysis, relative quantification and absolute quantification of monosaccharides can be efficiently performed, and the analysis time and cost of monosaccharides can be drastically reduced.

In addition, according to the present invention, in analyzing a peptide sample, by adding only cucurbituril [7] by a simple method, the analysis signal caused by the target sample can be significantly improved, and the sequence coverage of the peptide to be analyzed can be improved. have.

Claims (17)

(a) mixing cucurbituril [7] and an ammonium salt in an aqueous monosaccharide solution; (b) ionizing the mixture by electrospray ionization to form ammonium ions-linked cucurbituril [7] -monosaccharide complex ions; And Qualitative and quantitative determination of monosaccharides using cucurbituril [7], comprising the steps of: qualitative and quantitative analysis of the cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound using a dual mass spectrometry; Analytical Method. The method of claim 1, The monosaccharides are galactose (Galactose, Gal), glucose (Glucose, Glc), fructose (Fructose, Fru), mannose (Mannose, Man), allose (Allose, All), Altose (Altrose, Alt), Gulose (Gulose, Gul), Taloose (Talose, Tal), Pseose (Psicose, Psi), Sorbose (Sorbose, Sor), Takatose (Tagatose, Tag), Idoose (Idose, Ido) Qualitative and quantitative analysis of monosaccharides using cucurbituril [7], characterized in that selected from the group. The method of claim 1, The ammonium salt is qualitative and quantitative analysis method of monosaccharides using cucurbituril [7], characterized in that at least one selected from the group consisting of ammonium acetate, ammonium chloride, ammonium carbonate, ammonium hydroxide. The method of claim 1, The formation of the cucurbituril [7] -monosaccharide complex ion to which the ammonium ion is bound in step (b) is formed by the gas phase owner-guest interaction between cucurbituril [7] and the monosaccharide by electrospray ionization of the mixture. Qualitative analysis of monosaccharides using Cooker bituril [7]. The method of claim 1, The qualitative and quantitative analysis method of monosaccharides using cucurbituril [7], wherein the concentration ratio of the monosaccharide aqueous solution, cucurbituril [7] and ammonium salt in step (a) is 1: 1.5: 3 to 1: 6: 12. The method of claim 1, Qualitative and quantitative analysis of the monosaccharides comprises analyzing the ratio of the amount of ions according to the ratio of mass / charge (m / z) of fragment ions generated from the cucurbituril [7] -monosaccharide complex ion to which ammonium ions are bound. Qualitative and quantitative analysis method of monosaccharides using a cucurbituril [7] comprising the above. The method of claim 6, The qualitative and quantitative analysis method of monosaccharides using cucurbituril [7] characterized in that the monosaccharide solution of step (a) is a monosaccharide to be analyzed and a monosaccharide selected as an internal standard when quantitatively analyzing the monosaccharides. The method of claim 7, wherein The quantitative analysis of the monosaccharides qualitative and quantitative analysis method of the monosaccharides using cucurbituril [7], characterized in that the analysis through the calibration curve derived through the internal standard method (Internal standard method). The method of claim 7, wherein The monosaccharide selected as the internal standard is selected from the group consisting of allose (Allose, All), Altrose (Altrose, Alt), gulose (Gulose, Gul), Taloose (Talose, Tal) Qualitative and quantitative analysis of monosaccharides using Cooker bituril [7]. The method of claim 6, The qualitative and quantitative analysis method of monosaccharides using cucurbituril [7], wherein the monosaccharide aqueous solution of step (a) is mixed with a plurality of analysis target monosaccharides in the quantitative analysis of the monosaccharides. The method of claim 10, The quantitative analysis of the monosaccharides qualitative and quantitative analysis method of the monosaccharides using the cucurbituril [7], characterized in that the analysis through the calibration curve derived through the standard addition method (standard addition method). a) mixing a cucurbituril [7] with a peptide solution to form a peptide-cookerbituril [7] complex; And b) analyzing the complex by mass spectrometry; Qualitative and quantitative analysis of peptides using cucurbituril [7]. The method of claim 12, The complex is qualitative and quantitative analysis method of the peptide using cucurbituril [7], characterized in that formed by the hydrophobic interaction and ion-dipole interaction between the N-terminal phenylalanine residue of the peptide and cucurbituril [7]. The method of claim 12, The peptide is a qualitative and quantitative analysis method of the peptide using cucurbituril [7], characterized in that the peptide is digested by pepsin. The method of claim 14, The peptide is a qualitative and quantitative analysis method of the peptide using cucurbituril [7], characterized in that selected from the group consisting of insulin, B-chain of insulin, ubiquitin, myoglobin and mixtures thereof. The method of claim 12, The mass spectrometry may be electrospray ionization mass spectrometry (ESI-MS), substrate-assisted laser desorption ionization mass spectrometry (MALDI-MS), ion mobility mass spectrometry (IM-MS), or collision-induced dissociation Qualitative and quantitative analysis of peptides using cucurbituril [7], characterized in that based on tandem mass spectrometry. The method of claim 12, The mixing ratio of the peptide solution and the cucurbituril [7] in step a) is 10 parts by weight to 1000 parts by weight of the cucurbituril [7] with respect to 100 parts by weight of the peptide solution. Analytical Method.

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