JPH08145948A - Mass spectrometry for reducible oligosaccharide - Google Patents

Abstract

PURPOSE: To effectively analyze the structure of reducible oligosaccharide by ionizing the oligosaccharide obtained by operating trialkyl (p-aminophenyl) ammonium salt at the reducing end of the oligosaccharide. CONSTITUTION: Oligosaccharide is obtained by reacting a large excess amount, preferably about 25 to 30 times of molar amount of trimethyl (p-aminophenyl) ammonium, chloride, etc., in normal solvent with reducible oligosaccharide. As the solvent, mixture liquid of organic acid such as acetic acid, etc., with water is, for example, used. The obtained oligosaccharide is ionized by a specific ionizing method, generated molecular ions and many fragment ions are separated according to the mass number/charge number (m/z), the strength of the ions are recorded, and the structure of the reducible oligosaccharide is more effectively analyzed from the information obtained from the mass spectra aligned in the order of the m/z.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a mass spectrometric method for reducing oligosaccharides and its use.

[0002]

2. Description of the Related Art Reducing oligosaccharides have a degree of polymerization (DP) of 2
To about several tens of sugars, and the anomeric carbon atom of the constituent monosaccharide located at the end thereof is not substituted (hence, it has a reducing property in an alkaline solution). The structures of such reducing oligosaccharides are extremely diverse and complicated. In recent years, it has been revealed that sugar chains of sugar chain-related compounds are involved in differentiation, proliferation, fertilization of living organisms, immunity, and interactions with cells, viruses, bacteria, etc. Research to use it is becoming popular. This is because the diverse structures of sugar chains are deeply related to various physiological actions and functions, and therefore it is extremely important to clarify the structures of the sugar chains. Therefore, recently, a method for analyzing reducing oligosaccharides with high sensitivity by using mass spectrometry has been used.

[0003]

The mass spectrometry of reducing oligosaccharides developed so far includes various reducing oligosaccharides such as methyl derivatives, acetyl derivatives, and 2-aminopyridine (PA) derivatives. A method of conducting mass spectrometry by introducing the derivative into a derivative and then ionizing it by the fast atom bombardment ionization (FAB) method is known, but in order to analyze reducing oligosaccharides having various and complicated structures, these are analyzed. Even the latest method is not enough in sensitivity,
Moreover, it was not always satisfactory in that there was little useful information on the obtained structure.

[0004]

Under these circumstances, the inventors of the present invention have conducted diligent studies, and as a result, even in the case of a small amount of sample, a certain reagent was made to act on the reducing end of the reducing oligosaccharide. The resulting oligosaccharide is ionized by a specific ionization method, and the molecular ion and a large number of fragment ions produced by the ionization are mass number / charge number (m /
z), the intensity of each ion is recorded and m / z
The inventors have found that useful information can be obtained from the mass spectra arranged in the order of, and have found that the structure of the reducing oligosaccharide can be analyzed more reliably from the information, and have completed the present invention. That is, the present invention is characterized by including a step of ionizing an oligosaccharide obtainable by causing a trialkyl (p-aminophenyl) ammonium salt to act on the reducing end of a reducing oligosaccharide by an atmospheric pressure ionization method. Mass spectrometry of the reducing oligosaccharide (hereinafter referred to as the mass spectrometry of the present invention), and further, the structure of the reducing oligosaccharide characterized by analyzing the structure of the reducing oligosaccharide from the information of the mass spectrum by the mass spectrometry. It provides an analytical method.

The present invention will be described in more detail below. First, the reducing oligosaccharide for analysis is subjected to a pretreatment in which a trialkyl (p-aminophenyl) ammonium salt is caused to act on the reducing end thereof so as to be suitable for mass spectrometry by the atmospheric pressure ionization method. The pretreatment is, for example, the following 2
Perform in a stepwise reaction. (1) First reaction Trialkyl (p-aminophenyl) on reducing oligosaccharide
The ammonium salt is usually reacted in a solvent. Examples of the solvent used in the reaction include a mixed solution of water and an organic acid such as acetic acid. The amount of water mixed here means a small amount of water necessary for dissolving the reducing oligosaccharide and the trialkyl (p-aminophenyl) ammonium salt, and is, for example, about 80% (volume ratio) or more. The amount can be increased. Examples of the trialkyl (p-aminophenyl) ammonium salt used in this reaction include trimethyl (p-aminophenyl) ammonium chloride, triethyl (p-aminophenyl) ammonium chloride, tributyl (p-aminophenyl) ammonium chloride and the like. And preferably trimethyl (p-aminophenyl)
Examples thereof include ammonium chloride. The amount of the reagent is a large excess amount, preferably about 15 times to about 30 times the molar amount of the reducing oligosaccharide. The treatment temperature is usually about 80 ° C to about 100 ° C, preferably about 85 ° C.
To about 95 ° C., and the treatment time is usually about 3 minutes to about 10 minutes. (2) Second reaction The reaction product obtained in the first reaction was trialkyl (p-
The aminophenyl) ammonium salt and the reducing agent are usually reacted in a solvent. As the solvent used in the reaction,
For example, a mixed solution of organic acid such as acetic acid and water may be used. The amount of water mixed here means a small amount of water necessary to dissolve the reducing oligosaccharide and the trialkyl (p-aminophenyl) ammonium salt, for example, 80%.
It is possible to increase the amount so as to be about (capacity ratio) or more. As the trialkyl (p-aminophenyl) ammonium salt used in this reaction, the same compound as used in the first reaction is used. The amount of the reagent is a large excess amount, preferably about 8 times the molar amount to about 1 to the reducing oligosaccharide.
It is a 5-fold molar amount. Further, as the reducing agent, for example, sodium borohydride or the like can be used. In the case of sodium borohydride, it is advisable to use a molar amount of about 65 to about 85 times with respect to the reducing oligosaccharide. The treatment temperature is usually in the range of about 80 ° C. to about 100 ° C., preferably about 85 ° C. to about 95 ° C., and the treatment time is usually about 45 minutes to about 90 minutes. After the treatment, the desired derivative can be obtained by concentrating under reduced pressure. Further, if necessary, it can be further purified by a usual method such as recrystallization or chromatography.

The trialkyl (p-
The aminophenyl) ammonium salt can be synthesized, for example, from a commercially available compound or a known compound by the following method. A dialkyl (p-aminophenyl) ammonium salt is reacted with benzyloxycarbonyl chloride to protect an amino group and then reacted with an alkyl halide to obtain a trialkyl [p- (benzyloxycarbonylamino) phenyl] ammonium salt. . These reactions are usually performed in a solvent. Examples of the solvent used in the above reaction include hydrophilic solvents such as methanol and ethanol. Examples of the dialkyl (p-aminophenyl) ammonium salt used in the above reaction include dimethyl (p-aminophenyl) ammonium chloride and diethyl (p-aminophenyl) ammonium chloride. Examples of the alkyl halide used in the above reaction include methyl iodide, ethyl iodide and the like. The amount of the reagent used in the above reaction is about 10 times to about 30 times, preferably about 15 times to about 20 times the molar amount of benzyloxycarbonyl chloride based on the dialkyl (p-aminophenyl) ammonium salt. The molar amount of the alkyl halide is about 5 times to about 15 times, and preferably about 7 times to about 10 times. The reaction temperature is usually about 50 ° C to about 100 ° C,
It is preferably in the range of about 80 ° C to about 90 ° C, and the reaction time is usually about 1 hour to about 2 hours. After the reaction,
The resulting trialkyl [p- (benzyloxycarbonylamino) phenyl] ammonium salt is treated with a conventional hydrogenation reaction to give the desired trialkyl (p-
Aminophenyl) ammonium salts can be obtained.
Further, if necessary, it can be further purified by a usual method such as recrystallization or chromatography.

The oligosaccharide thus prepared is subjected to mass spectrometry by introducing it into a mass spectrometer equipped with an interface of atmospheric pressure ionization method. Examples of the atmospheric pressure ionization method used in the present invention include an electrospray ionization method and an ion spray ionization method. A commercially available interface (electrospray ionization method) is, for example, an interface for API manufactured by Finnigan Mat. Further, as the mass spectrometer, a magnetic field type, a quadrupole type, an ion trap type, a Fourier transform-ion cyclotron resonance type mass spectrometer and the like can be mentioned, but a quadrupole type mass spectrometer is preferable. I can give you. More preferably, a tandem quadrupole mass spectrometer is used. In addition, as a commercially available quadrupole mass spectrometer, for example, TSQ-700 type chromatography
Examples include a tandem mass spectrometer (manufactured by Finnigan Mat). As a method for introducing the sample into the mass spectrometer equipped with the interface of the atmospheric pressure ionization method, for example,
Examples include high performance liquid chromatography and syringe pumps. A syringe pump is preferably used.

In this way, the oligosaccharide obtained by reacting the reducing end of the reducing oligosaccharide with a trialkyl (p-aminophenyl) ammonium salt is ionized by the atmospheric pressure ionization method, and a molecule produced by the ionization Ions and a large number of fragment ions are separated according to mass / charge number (m / z) and the intensity of each ion is recorded to provide useful information in a mass spectrum ordered in m / z (eg reducing end side). The structure (for example, the sequence of constituent monosaccharides) of the reducing oligosaccharide can be analyzed from the mass difference of each fragment ion). Further, if necessary, if a biochemical method using an enzyme or an antibody, an analysis method by a spectrum such as NMR, or an analysis by other constituent monosaccharide component analysis method is used complementarily, more reliable structure analysis can be performed. . In addition, in the mass spectrum of the conventional mass spectrometry method, analysis may be difficult due to the presence of matrix and impurity ions, but in the mass spectrum of the present invention, the signal peaks representing product ions are clear. Therefore, the analysis becomes easy. This is extremely useful for structural analysis of unknown reducing oligosaccharides.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

【Example】

Example 1 [Pretreatment Method of Reducing Oligosaccharide Using Trimethyl (p-aminophenyl) ammonium Salt] Trimethyl (p-aminophenyl) ammonium chloride (hereinafter referred to as TMAPA) was added to 350 μl of pure water containing 40 μl of acetic acid. .) 10 mg was dissolved. 2 mg of maltopentaose (used as a model compound) as a reducing oligosaccharide was added to the obtained solution, and the reaction was carried out at 90 ° C. for 5 minutes. After completion of the reaction, the reaction product was added to 350 μl of pure water containing 40 μl of acetic acid in advance and TMAPA.
4 mg and sodium borohydride 10 mg
Was added to the dissolved solution and reacted at 90 ° C. for 60 minutes.
After completion of the reaction, the reaction product is freeze-dried to give a trialkyl (p-aminophenyl) ammonium salt at the reducing end to obtain an oligosaccharide (hereinafter referred to as TMA).
It is described as a derivative with PA. Structural formula:

Embedded image ) Got.

Example 2 (Analysis of reducing oligosaccharides by various mass spectrometry methods) Maltopentaose was used as the reducing oligosaccharide. Derivatizing the reducing oligosaccharide with (1) methyl iodide,
(2) Derivatization with acetic acid, (3) Derivatization with TMAPA, (4) 2-aminopyridine (hereinafter referred to as PA).
Preparation of a methyl derivative, an acetyl derivative, a TMAPA derivative or a PA derivative obtained by derivatization with [[The derivatization method of (3) was described in Example 1). The derivatization methods other than (3) are described in the following reference examples. The prepared various derivatives and derivatized maltopentaose were mass analyzed by a fast atom bombardment ionization (FAB) method [positive / negative mode] or an electrospray ionization (ESI) method [positive mode]. Then, the ionization efficiencies of the prepared various derivatives and non-derivatized maltopentaose in each ionization method were compared. The results are shown in Table 1 (represented by the ratio of the ionization efficiency of various derivatives to the ionization efficiency when using non-derivatized maltopentaose as a sample). As is clear from Table 1, the mass spectrometric method of the present invention showed a remarkable improvement in sensitivity (in particular, a 5000-fold improvement in comparison with the non-derivatized maltopentaose-ESI method) as compared with other mass spectrometric methods. It was found that the mass spectrometry of the present invention can obtain a clear spectrum even with an extremely small amount of 20 fmol.

[0011]

[Table 1] ──────────────────────────────────── FAB ESI Note Positive Negative Positive ──── ──────────────────────────────── Methyl derivative 11 0.1-Acetyl derivative 60 0.1-TMAPA derivative 1 0.1 5000 The present invention PA derivative 3 1 100 derivatized maltopentaose 1 1 1 1 1 1 1 ────────────────────────────── ───────

Example 3 (Structural analysis based on mass spectrum information of derivative of TMAPA by mass spectrometry)
2 generated when ionized by SI interface
A mass spectrum of a product ion having a valence ion [M + Na] ^{2 +} as a parent ion was measured. The obtained mass spectrum is shown in FIG. As a result, in the obtained mass spectrum (FIG. 1), signal peaks representing sequence ions of both Y and Z series (see FIG. 3) having structural information from the reducing end of the reducing oligosaccharide were observed, From the mass difference between the sequence ions, the molecular ion [M
It was possible to obtain a lot of information on the chemical structure that the elimination in the hexose unit occurs from ⁺ ] (see Table 2 and FIG. 2). From this useful information, it is possible to analyze the structure of the reducing oligosaccharide. On the other hand, for the derivative described in Example 2 other than the derivative by TMAPA,
Mass spectra were measured by the same method as described above, but only mass spectra mainly representing Y series of sequence ions could be obtained, which was inferior in terms of usefulness of information on the chemical structure.

[0013]

[Table 2] ──────────────────────────────────── Y-series product ions Z-series product ions m / Z peak difference m / Z peak difference ──────────────────────────────────── [M ⁺ ] _{963 - 963 - Y 4 800 163} 784 179 Y 3 638 162 622 162 Y 2 476 162 460 162 Y 1 314 162 298 162 ────────────────────── ─────────────

Reference Example (Various Pretreatment Methods for Reducing Oligosaccharide) Maltopentaose was used as the reducing oligosaccharide. (A) Method of derivatization with methyl iodide Method of Ciucanu and Kerek [I.Ciucanu and F.Kerek, Carb
ohydrate Research, 131, 209 (1984)]. 50 mg (0.28 mmol equivalent amount) of reducing oligosaccharide D
After adding 1 ml of MSO and mixing, add 200 ml to this mixture.
mg of powdered NaOH (or 300 mg of powdered KOH)
Was added. 1 m of methyl iodide while stirring the mixed solution
After the addition of l, stirring was continued for 30 minutes (60 minutes in the case of KOH). After the reaction was completed, 1 ml of pure water was added, and the mixture was extracted 3 times with 1 ml of chloroform. The recovered chloroform fraction was washed 3 times with 1 ml of pure water, and then the chloroform fraction was concentrated to obtain the target methyl derivative (Formula 2).

Embedded image (B) Derivatization method by derivatization with acetic acid Method of DeU et al. [A. DeU and PRTiller, Biochem. Biophy
s.Res.Commun., 135,1126 (1986)]. After adding 500 μl of trifluoroacetic anhydride: acetic acid (2: 1) to 10 mg (equivalent to 0.011 mmol) of reducing oligosaccharide and mixing, the mixture was stirred at room temperature for 60 minutes. After completion of the reaction, nitrogen gas was flown to evaporate the solvent.
The extract solution recovered from the residue by three times extraction with 2 ml of dichloromethane was washed with 1 ml of pure water, and then the dichloromethane fraction was recovered. Nitrogen gas was passed through the collected dichloromethane fraction to evaporate the solvent to obtain the target acetyl derivative (Formula 3).

Embedded image (C) Method of derivatization with 2-aminopyridine Method of Kondo et al. [A.Kondo, J.Suzuki, N.Kuraya, S.Hase, IK
ate, T. Ikenaka, Agric. Biol. Chem., 54, 2169 (1990)]. This derivatization is based on a commercial kit (Takara Pal
Station Pyridyl amination reagent: for sugar chain analysis) and a commercially available device (Takara Pal Station model 4000: sugar chain pyridyl amination automatic device). Reducing oligosaccharide 45 ng-45 μg (50 pmol-50 nmol
Coupling reagent (2-Aminopyridine / acetic acid:
20 μl of 3 mg / ml) was added and the reaction was carried out at 90 ° C. for 60 minutes. After completion of the reaction, the reaction mixture was added with a reducing reagent (Borane-dim
20 μl of ethylamine complex / acetic acid) was added to 80
The reaction was carried out at 60 ° C for 60 minutes. After the reaction was completed, 20 μl of triethylamine / methanol and 40 μl of toluene were added and mixed, and then, while flowing a nitrogen gas into the mixture,
The mixture was dried under reduced pressure at 60 ° C. for 10 minutes. To the obtained residue, 20 μl of methanol and 40 μl of toluene were added and mixed again, and then the mixture was dried under reduced pressure at 60 ° C. for 10 minutes while flowing nitrogen gas. To the obtained residue, 50 μl of toluene was added and mixed again, and then the mixture was mixed with nitrogen gas while flowing nitrogen gas.
The derivative by PA (chemical formula 4) was obtained by drying under reduced pressure at 0 ° C. for 10 minutes.

[Chemical 4]

[0015]

The mass spectrometric method of the present invention makes it possible to detect reducing oligosaccharides and confirm the molecular weight thereof even in the case of an extremely small amount of sample. Further, it has become possible to analyze the structure of the reducing oligosaccharide from the useful information of the mass spectrum obtained by the analysis method.

[Brief description of drawings]

FIG. 1 is a divalent ion [M + N] produced when a derivative of TMAPA is ionized with an interface for ESI.
FIG. 3A is a diagram showing a mass spectrum of a product ion having a] ^{2 +} as a parent ion.

FIG. 2 is a diagram showing a chemical structural formula regarding sequence ions of both Y and Z series having structural information from the reducing end of the derivative by TMAPA. Here, "Y" means
It means the product ion of Y series, and "Z" means
It means the product ion of Z series.

FIG. 3 is a diagram showing a systematic nomenclature for each fragment ion of a reducing oligosaccharide in a mass spectrum. Fragment ions having a charge on the non-reducing end side are named A, B, and C, and fragment ions having a charge on the reducing end side are X, Y, and Z. A and X are fragment ions indicating ring cleavage, and indicate two bonds that are cleaved by superscripts separated by commas.

Claims (2)

[Claims] 1. A reducing property characterized by comprising a step of ionizing an oligosaccharide obtainable by reacting a reducing alkyl saccharide with a trialkyl (p-aminophenyl) ammonium salt at the reducing end by an atmospheric pressure ionization method. Mass spectrometry of oligosaccharides. 2. A method for analyzing a reducing oligosaccharide, which comprises analyzing the structure of the reducing oligosaccharide from the information on the mass spectrum of the reducing oligosaccharide according to claim 1.