JP5821741B2 - Mass spectrometry of neutral sugar chains - Google Patents

Description

The present invention relates to a method for mass spectrometry of sugar chains. More specifically, the present invention relates to structural analysis of neutral sugar chains using mass spectrometry.
The present invention is applied in the medical and drug discovery fields where biomarker searches and the like are performed.

  Patent Document 1 (Japanese Patent Laid-Open No. 2008-261824) and Patent Document 2 (Japanese Patent Publication No. 2005-536759) describe an ionic liquid composed of tetramethylguanidine ion and p-coumarate ion as a matrix for mass spectrometry. Mass spectrometry is disclosed for use as (ie, liquid matrix).

  Neutral sugar chains by mass spectrometry using non-patent document 1 (Anissa W. Wong et al. Anal. Chem. 71 (1999) 205-211) with sulfate ions added to DHAP and harmine as a matrix. It is described that negative ionization is possible. Further, Non-Patent Document 2 (Anissa W. Wong et al. Anal. Chem. 72 (2000) 1419-1425) describes a mass spectrometry method using an acidic substance such as an alkyl sulfonate in place of the above-described sulfate ion. Has been.

Non-patent document 3 (Yang Cai et al. Anal. Chem. 75 (2003) 1638-1644) is negatively ionized by adding Cl ⁻ to a low molecular weight neutral sugar chain, and the structure by MS ² analysis Analyzing. It has been reported that Cl ^- addition occurred when 3-aminoquinoline, 4-aminophenol, harmine was used as the matrix. Furthermore, it is described that it is most preferable to use harmine as the matrix.
In Non-Patent Document 4 (Jiang, Y. and Cole, RB Journal of the American Society for Mass Spectrometry 16 (2005) 60-70) related to Non-Patent Document 3, the negative ion of the above-mentioned low molecular weight neutral sugar chain It is described that an anion suitable for acquisition of MS ² spectrum is Cl ⁻ , acetate ion, or F ⁻ .

Non-Patent Document 5 (Tohru Yamagaki et al. Anal. Chem. 77 (2005) 1701-1707) uses harmine or norharman as a matrix to a low molecular weight neutral sugar chain and adds Cl ⁻ to make it negative. It is described to ionize. In addition, it is described that linkage analysis of sugar chains was performed from the MS ² spectrum of the negative ions.

Non-patent document 6 (Paula Domann et al. Rapid Communications in Mass Spectrometry 26 (2012) 469-479) examined combinations of matrix type and anion type to negatively ionize N-type sugar chains with MALDI. From the viewpoint of MS ¹ and MS ² analysis, it is concluded that the combination of THAP (2,4,6-trihydroxyacetophenone) and NO ₃ ^— is the best. In Non-Patent Document 6 and related Non-Patent Document 7 (Harvey, DJ Journal of American Society for Mass Spectrometry 16 (2005) 622-630), MS ² spectrum of I ^- adduct ion of neutral N-type sugar chain Describes that it is almost impossible to obtain structural information of sugar chains.

JP 2008-261824 A JP 2005-536759 A

Analytical Chemistry, 1999, Vol. 71, p. 205-211 Analytical Chemistry, 2000, Vol. 72, p. 1419-1425 Analytical Chemistry, 2003, Vol. 75, p. 1638-1644 Journal of the American Society for Mass Spectrometry, 2005, Vol. 16, p. 60-70 Analytical Chemistry, 2005, Vol. 77, p. 1701-1707 Rapid Communications in Mass Spectrometry, 2012, Vol. 26, p. 469-479 Journal of the American Society for Mass Spectrometry, 2005, Vol. 16, p. 622-630

Since neutral sugar chains do not have a site that can be easily deprotonated, they are unlikely to become negative ions. For example, in the methods of Patent Document 1 and Patent Document 2, a neutral sugar chain that has not been derivatized cannot be negatively ionized.
Even if the sugar chain is deprotonated, the obtained deprotonated substance is unstable and thus causes fragmentation immediately. For this reason, in order to negatively ionize neutral sugar chains relatively stably, a technique for ionization as an anion adduct has been developed.

A technique of adding an anion to a relatively large molecular weight neutral sugar chain such as an N-type sugar chain and detecting it as a negative ion has been mainly studied by electrospray ionization (ESI). Although some anionic species would destabilize the negative ^{ions, Cl -, Br -, I} -, H 2 PO 4 -, HSO 4 - and NO ₃ ^- anion, such as is possible to generate a stable anion adduct Are known. The negative ionization of neutral sugar chains in ESI has been energetically studied, including fragmentation.

  Unlike the mode in which the positive ion fragmentation of the N-type sugar chain is one in which the constituent monosaccharides of the sugar chain are desorbed one after another, the negative ion fragmentation in the N-type sugar chain is a mode in which a fragment reflecting the branch structure is generated. Therefore, it is particularly useful for structural analysis. However, since ESI generates multivalent ions, the peaks are dispersed and it is said that it is not suitable for glycan profiling. Moreover, it is known that the negative ion of the sugar chain containing an anion adduct is unstable, and fragmentation is likely to occur depending on the ionization conditions and the voltage gradient in the mass spectrometer.

  On the other hand, in matrix-assisted laser desorption / ionization (MALDI), only monovalent ions are generated, so sugar chain profiling can be easily performed. Furthermore, with the advent of mass spectrometers such as MALDI-IT (ion trap) -TOF (time-of-flight) or MALDI-Q (quadrupole) -TOF, MALDI-IT-Orbitrap in recent years, Detection and structural analysis techniques have become useful techniques. As described above, negative ion fragmentation of N-type sugar chains provides useful information for structural analysis, but in MALDI, efficient negative ionization of neutral sugar chains has been considered particularly difficult.

  One of the main reasons is the existence of the matrix. The matrix usually has the ability to supply protons. Accordingly, it has been considered that efficient negative ionization is difficult because protons supplied from the matrix neutralize the anion and prevent anion addition to the sugar chain. One solution is to use an anion having a low ability to capture protons (that is, an anion having a low gas-phase basicity (GB)). It is reported that the sugar chain can be negatively ionized by adding a sulfate anion.

However, the use of low GB anions poses difficulties in MS ² analysis using collision-induced dissociation (CID). Here, in the CID of the anion-added ion of the neutral sugar chain, ideally, the initial reaction is that the anion first extracts a proton from the neutral sugar chain to form a deprotonated form of the sugar chain. Since this deprotonated glycan is very unstable, fragmentation occurs immediately, and as a result, a peak reflecting the structure information of the glycan is observed in the CID spectrum. However, since an anion with a low GB such as a sulfate anion cannot extract a proton from a sugar chain, the sugar chain itself is neutrally lost, and only the anion peak is observed in the CID spectrum. Naturally, the structural information of the sugar chain cannot be obtained. On the other hand, by raising the laser power to cause dehydration condensation between sulfuric acid and sugar chains, structural information is obtained by MS ² analysis using ions in the sulfated derivatized state as precursor ions. The fragmentation mechanism of derivatized sugar chains is very different from the ideal negative ion fragmentation mechanism of neutral sugar chains, and product ions useful for structural analysis reflecting branch structures cannot be obtained.

As described above, the use of anions having a low GB interferes with MS ² analysis.
Conversely, the use of high GB anions makes it easier for the anions to neutralize upon receiving protons from the matrix, preventing the anion from being added to the glycan, and thus the efficiency of the glycan during the MS ¹ analysis stage. Makes good negative ionization impossible.
MALDI measurement for the purpose of structural analysis of neutral sugar chains has such a dilemma.

One solution is to use a basic matrix that does not readily release protons, that is, a matrix that has a high GB in the deprotonated state. In Non-Patent Document 3, a harmine matrix was used to negatively ionize a neutral sugar chain. harmine is one of the most GB matrix, Cl neutral sugar chain with this property ^- the PSD analysis was performed by adding. The same analysis was performed in Non-Patent Document 5.
However, these methods have a problem in terms of sensitivity even if a high GB GB Cl ^- anion can be added to the sugar chain. Moreover, the application to an N-type sugar chain is not made.

Recently, in Non-Patent Document 6, the matrix and the anion is considered for negative ionization of N-type sugar chain, from the viewpoint of both MS ¹ and MS ² analysis, THAP matrix and NO ₃ ^- use was optimal for It has been reported.
THAP is a matrix with a high GB in a deprotonated state, but not as harmine. However, even in this case, it cannot be said that the problem of sensitivity has been solved. Specifically, the detection limit of a standard N-type sugar chain is 2 pmol when MALDI-MS manufactured by Bruker is used. This sensitivity is much worse than the detection limit of the positive ion mode.

  Accordingly, an object of the present invention is to provide a MALDI mass spectrometry method capable of detecting an anion adduct ion from a neutral sugar chain with high sensitivity.

  The present inventors have found that the object of the present invention can be achieved by using a liquid matrix and specific acidic substances and salts thereof as additives for the liquid matrix, and have completed the present invention.

The present invention includes the following inventions.
(1)
Mixture comprising an ionic liquid as the matrix, a material selected from tetrafluoroboric acid and its salts or Ranaru group as a matrix additive, a neutral molecule selected from the group consisting of sugar chains and sugar chain derivative as a sample Mass spectrometry of neutral sugar chains, in which the molecular weight-related ions added to the molecules of the sample by the anions constituting the additive substance are detected.

  By further subjecting the molecular weight related ions to mass spectrometry, product ions that give structural information on sugar chains can be detected.

( 2 )
Before SL ionic liquid, an ion of the amine, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxy acetophenone, alpha-cyano-4-hydroxycinnamic acid, 2- (4-hydroxyphenyl) benzoic The method of (1), comprising an ion of an acid selected from the group consisting of an acid and coumaric acid.
( 3 )
Wherein the amine is selected from the group consisting of 1,1,3,3-tetramethylguanidine and n- butylamine, method (2).

( 4 )
The method according to any one of claims (1) to ( 3 ), wherein the salt of tetrafluoroboric acid is an ammonium salt.

The present invention enables MALDI mass spectrometry capable of detecting anion-added ions with high sensitivity from a neutral sugar chain. Anion adduct ions allow acquisition of structural information in MS ² analysis.

G ₃ CA is used as the liquid matrix, and (a) PF ₆ ⁻ , (b) BF ₄ ⁻ , (c) HSO ₄ ⁻ , (d) I ⁻ , (e) NO ₃ ⁻ , (f) as matrix additives. It is an MS ¹ spectrum of neutral sugar chain NA2 (100 fmol) obtained by using an anionic substance of Br ⁻ , (g) SCN ⁻ , (h) H ₂ PO ₄ ⁻ or (i) Cl ⁻ . It is a MS ¹ spectrum of neutral sugar chain NA2 obtained by using liquid matrix G ₃ CA and using NO ₃ ⁻ as a matrix additive and having a detection limit concentration (b) and a concentration 10 times the detection limit (a). It is a MS ¹ spectrum of neutral sugar chain NA2 obtained using a liquid matrix G ₃ CA and using BF ₄ ⁻ as a matrix additive and having a detection limit concentration (b) and a concentration 10 times the detection limit (a). FIG. ₃ is an MS ² spectrum of an anionic adduct neutral sugar chain NA2 obtained using a liquid matrix G ₃ CA and (a) PF ₆ ⁻ , (b) BF ₄ ⁻ or (c) HSO ₄ ⁻ as a matrix additive. . FIG. 2 is an MS ² spectrum of an anionic adduct neutral sugar chain NA2 obtained by using a liquid matrix G ₃ CA and using (d) I ⁻ , (e) NO ₃ ⁻ or (f) Br ⁻ as a matrix additive. It is an MS ² spectrum of an anionic adduct neutral sugar chain NA2 obtained using a liquid matrix G ₃ CA and using (g) SCN ⁻ , (h) H ₂ PO ₄ ⁻ or (i) Cl ⁻ as a matrix additive. .

[1. Mass spectrometry target]
The object to be subjected to sample analysis is a sugar chain. The sugar chain molecules include both those having a natural structure (sugar chain) and those having a non-natural structure (sugar chain derivative), provided that they are neutral, ie, have no charge. The sugar chains may be obtained by, for example, isolation of various organisms from the living body, and may be chemically or enzymatically used, for example, when the sugar chain part is released from a complex carbohydrate. It may be artificially prepared by a technique.

As a more specific example of the sugar chain derivative, there is a neutralized acid sugar chain. Acidic sugar chains contain acidic sugars such as sialic acid, sulfate group-containing sugars and phosphate group-containing sugars as constituent sugars. Examples of neutralization include modification with acidic sugar neutral groups and acidic sugars. Examples include the release of sugar. Examples of modification include alkyl esterification.
As a more specific example of the sugar chain derivative, one labeled with a reducing end can be mentioned. Examples of labeling include the introduction of a label group by reductive amination represented by pyridylamination.

  The sugar chains in the present invention are preferably sugar chains derived from glycoproteins. More specifically, there are an N-linked type bonded to an asparagine residue and an O-linked type bonded to a serine or threonine residue. The present invention is particularly suitably used for analysis of N-linked sugar chains.

  The N-linked sugar chain contains a branched pentasaccharide of Manα1 → 6 (Manα1 → 3) Manβ1 → 4GlcNAcβ1 → 4GlcNAc as a common mother nucleus. The N-linked sugar chain further has a structure of a sugar chain that binds to the outside of this pentasaccharide mother nucleus, and a high mannose-type five sugar mother nucleus in which only an α-mannosyl residue is bound to the five sugar mother nucleus. A complex type in which 1 to 5 side chains starting from N-acetylglucosamine are bonded to two α-mannosyl residues, and a side chain similar to the complex type is attached to the Manα1 → 3 side of the pentasaccharide mother nucleus, and Manα1 → On the 6th side, it is classified into three groups of hybrids having a structure of a hybrid of high mannose type and complex type having 1 to 2 α-mannosyl residues. The complex type sugar chain and the hybrid sugar chain further have an α-fucosyl residue bonded to the C-6 position of the root N-acetylglucosamine residue and the C of the β-mannosyl residue of the pentasaccharide mother nucleus. There is a diversity of structures depending on the presence or absence of an N-acetylglucosamine residue bonded to the -4 position or a residue bonded to galactose.

  The molecular weight of the sugar chains in the present invention is, for example, 300 to 6,000, preferably 900 to 5,000, more preferably 1,200 to 4,000.

[2. Liquid matrix]
In the present invention, an ionic liquid is used as the matrix. An ionic liquid is a substance that exists in a liquid state at room temperature and is actually a salt. In the present invention, a matrix that is an ionic liquid is referred to as a liquid matrix.
More specifically, an ionic liquid composed of amine ions and acidic group-containing organic substance ions is used as the liquid matrix. Any of these amines and acidic group-containing organic substances absorbs laser light having a wavelength selected from the ultraviolet to visible regions.

  The amines are 1,1,3,3-tetramethylguanidine (TMG), n-butylamine (BA), ethylamine, N, N-diethylamine (DEA), N, N-diethylaniline, N, N-diethyl. Methylamine, diethylbenzeneamine, N, N-dimethylamine, triethylamine, tri-n-butylamine, tri-n-propylamine, ethanolamine, polyether tailed triethylamine, polyester tailed triethylamine, nitrophenol, aniline, 2,4 -Dinitroaniline, 2-nitrophenyl octyl ether, pyridine, 2-pyridinepropanol (2PP), 2-ethylpyridine (2EP), 2-amino-4-methyl-5-nitropyridine, 3-aminoquinoline (3AQ), 3-hydroxypyrid 1-methylimidazole, 1-butyl-3-methylimidazole, 1- (1-hydroxypropyl) -3-methylimidazole, 1,3-dimethylimidazole, 1,5-diaminonaphthalene, 6-aza-2-thiothymine , Coumarin, 6,7-dihydroxycoumarin, 1,8-dihydroxy-9 [10H] -anthracenone, carbolines (Norharman, Harman, Harmine, Harmol, Harmarin, Halmarol, etc.) and the like.

  On the other hand, the acidic substance-containing organic substance includes p-coumaric acid (p-CA), α-cyano-4-hydroxycinnamic acid (4-CHCA), α-cyano-3-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid (DHB), 4-hydroxybenzoic acid, p-hydroxybenzoic acid, p-hydroxyphenylpyruvic acid, 3-hydroxypicolinic acid, 3,5-dimesoxy-4-hydroxycinnamic acid (sinapic acid) 4-hydroxy-3-mesoxycinnamic acid (ferulic acid), caffeic acid (3,4-dihydroxycinnamic acid), 5-mesoxysalicylic acid, 2- (4-hydroxyphenylazo) benzoic acid (HABA), Nicotinic acid, picolinic acid, 3-aminopicolinic acid, 3-hydroxypicolinic acid, 2-aminobenzoic acid, 3-amino-4-hydroxybenzoic acid, 6-aza Selected from 2-thiothymine, 2,4,6-trihydroxyacetophenone (THAP), 1,4-dihydro-2-naphthoic acid, 3-indoleacrylic acid, indole-2-carboxylic acid, thioglycolic acid, etc. .

[3. Matrix additive]
The matrix additive is basically a substance containing an anion having a gas phase basicity (GB) higher than the gas phase basicity (GB) of the deprotonated sugar chain. By using such a matrix additive, product ions can be generated efficiently. Conversely, when a substance comprising an anion having a gas phase basicity lower than the gas phase basicity of the deprotonated glycan is used as a matrix additive, the anion of the additive in MS ² analysis is used. Since loss occurs preferentially, sugar chain product ions cannot be obtained efficiently.

Here, the gas phase basicity of the deprotonated glycan can vary depending on the glycan.
For example, there is a report that a sugar chain composed of two sugar residues has a gas phase basicity of 1373 kJ / mol (Anal. Chem. 75 (2003) 1638-1644 (Non-patent Document 3)). In addition, there are reports that anions suitable for MS ² spectrum acquisition of this sugar chain are Cl ⁻ , acetate ions, and F ⁻ (Journal of the American Society for Mass Spectrometry 16 (2005) 60-70 (Non-patent Document 4)). ). Accordingly, as anions of the matrix additive in the present invention, the basicity of the gas phase to the extent that Cl ⁻ and F ⁻ have (specifically, Cl ⁻ of 1373 kJ / mol, acetate ion of 1427 kJ / mol, F ⁻ of At least 1529 kJ / mol) is acceptable.

On the other hand, no detailed report has been made on the gas phase basicity of sugar chains having a large molecular weight, such as N-linked sugar chains. However, suitable anions for MS ² spectrum acquisition of N-linked glycans (ie, which could give rise to glycan fragments) are Br ⁻ and NO ₃ ⁻ , while depending on the use of I ⁻ It has been reported that it is almost impossible to obtain structural information from the MS ² spectrum of N-linked sugar chains (Rapid Communications in Mass Spectrometry 26 (2012) 469-479 (Non-Patent Document 6) and Journal of American Society for Mass Spectrometry 16 (2005) 622-630 (non-patent document 7)). Thus, as the anion of the matrix additive in the present invention, Br ^- (specifically, Br ^- of 1332kJ / mol, NO ₃ ^- of 1330kJ / mol) degree of vapor phase basicity with the ^- or NO ₃ and What you have is at least acceptable. In addition, as an aspect of the present invention, the basicity of gas phase (specifically, 1294 kJ / mol) to the extent that I ⁻ can be eliminated as an anion of the matrix additive in the present invention can be eliminated.

Specifically, the matrix additive in the present invention is selected from the group consisting of tetrafluoroboric acid (HBF ₄ ) and salts thereof, acidic substances having a gas phase basicity of 1300 to 1550 kJ / mol, and salts thereof.
Specific examples of the acidic substance having a gas phase basicity of 1300 to 1550 kJ / mol include an anion selected from the group consisting of NO ₃ ⁻ , Br ⁻ , SCN ⁻ , H ₂ PO ₄ ⁻ , Cl ⁻ and F ^−. Examples include acidic substances.

In the present invention, the case where the matrix additive is tetrafluoroboric acid (HBF ₄ ) and / or a salt thereof, and the case where the matrix additive is an acidic substance having a gas phase basicity of 1300 to 1550 kJ / mol and / or a salt thereof. Each have a particularly suitable combination with a liquid matrix.

When tetrafluoroboric acid (HBF ₄ ) and / or a salt thereof is used as the matrix additive, a wide variety of ionic liquids are acceptable as the liquid matrix. Among them, for example, 1,1,3,3-tetramethylguanidine (TMG), n-butylamine (BA), N, N-diethylamine (DEA), 2-pyridinepropanol (2PP), 2 as amine ions. -In some cases, ions of ethylpyridine (2EP) and 3-aminoquinoline (3AQ) may be suitably selected, and as ions of the organic substance containing an acidic group, p-coumaric acid (p-QA), 2,5- Dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (4-CHCA), 2,4,6-trihydroxyacetophenone (THAP), and 2- (4-hydroxyphenylazo) benzoic acid (HABA) ) May be suitably selected. More specifically, a combination of TMG and 4-CHCA, THAP, HABA, or p-CA, a combination of BA and DHB or 4-CHCA, a combination of DEA and 4-CHCA, 2PP and DHB, 4- A combination of CHCA or THAP, a combination of 2EP and DHB, a combination of 4-CHCA or THAP, and a combination of 3AQ and DHB or 4-CHCA are preferably selected.

  When an acidic substance having a gas phase basicity of 1300 to 1550 kJ / mol and / or a salt thereof is used as a matrix additive, an ion of TMG as an ion of amino, an ion of p-CA or HABA as an ion of an organic substance containing an acidic group Is preferably selected. In particular, a combination of TMG ions and p-CA ions is preferably selected.

  When the matrix additive is a salt, examples of the counter cation include ammonium ions and metal ions (alkali metal ions, alkaline earth metal ions, etc.). In the present invention, an ammonium salt is preferably used.

[4. Liquid matrix, mixture containing additives and sugar chains]
In the present invention, a mixture containing a liquid matrix, an additive, and sugar chains is prepared on a target plate for mass spectrometry. This mixture is subject to laser irradiation.
It does not specifically limit as a preparation method of a mixture, Those skilled in the art can select suitably. Usually, liquid droplets containing a liquid matrix, additives, and sugar chains in a suitable solvent are prepared on a target plate for mass spectrometry, the solvent is removed, and residual droplets are formed as a mixture to be laser-targeted. obtain. Removal of the solvent includes natural drying and reduced pressure drying.

The liquid mixture containing a liquid matrix, an additive, and sugar chains can be prepared by mixing a liquid matrix solution, an additive solution, and a solution containing a sugar chain (sample solution) in any order.
The solvent of the mixed solution is not particularly limited, and those skilled in the art can appropriately select those used in normal mass spectrometry. For example, it is selected from the group consisting of acetonitrile, water, trifluoroacetic acid, methanol and ethanol. Preferably, an acetonitrile aqueous solution is used. The concentration of acetonitrile in this case is, for example, 10 to 90% by volume, preferably 40 to 60% by volume.

  A person skilled in the art can appropriately determine the concentration and mixing amount of each of the liquid matrix solution, the additive solution, and the solution (sample solution) containing sugar chains for preparing the mixed solution. For example, when prepared as a mixed solution, the concentration of the liquid matrix in the mixed solution may be adjusted to, for example, 0.1 mg / mL to 1 g / mL, preferably 1 mg / mL to 100 mg / mL. it can. The concentration of the additive in the mixed solution may be, for example, 1 μmol / L to 1 mol / L, preferably 0.1 mmol / L to 100 mmol / L. The concentration of sugar chains in the mixed solution may be, for example, 0.1 amol / μL to 1 nmol / μL, preferably 10 amol / μL to 10 pmol / μL. For each remaining droplet of the mixture on the target plate, the amount of sugar chains can be 0.1 amol to 1 μmol, or 50 amol to 100 fmol, or 100 amol to 1 fmol.

[5. Mass spectrometry]
Using a mass spectrometer combined with a MALDI ion source, a mixture containing an ionic liquid, a matrix additive, and sugar chains is subjected to mass spectrometry (specifically, MS ¹ analysis). A molecular weight related ion (anionic adduct ion) in which an anion constituting a substance is added to a molecule of a sugar chain is obtained. At this time, it is preferable that generation of fragment ions of molecular weight related ions is suppressed.

Furthermore, in measurement of molecular weight related ions for obtaining structure information (specifically, MS ² analysis), product ions that give sugar chain structure information can be obtained. More specifically, in the molecular weight related ion, an anion withdraws a proton from the sugar chain to form a deprotonated form of the sugar chain. Next, the deprotonated form of the sugar chain undergoes fragmentation, producing a product ion that gives sugar chain structure information.

The molecular weight-related ion cleavage method is appropriately selected by those skilled in the art from existing or new techniques used in tandem mass spectrometry. More specifically, post source decay (PSD), low energy collision Low-energy collision induced dissociation (low-energy CID), high-energy collision induced dissociation (high-energy CID), infrared multi-photon dissociation (IRMPD), And photo-induced dissociation (PID). Examples of a mass spectrometer capable of performing low-energy CID, high-energy CID, IRMPD, and PID include a collision chamber or a mass spectrometer having a quadrupole or ion trap having a collision chamber function. Specifically, ion trap mass spectrometer, triple quadrupole mass spectrometer, quadrupole-time-of-flight mass spectrometer, ion trap-time-of-flight mass spectrometer, quadrupole-ion trap type Mass spectrometer, quadrupole-Fourier transform mass spectrometer, ion trap-Fourier transform mass spectrometer, quadrupole-orbitrap mass spectrometer, ion trap-orbitrap mass spectrometer, flight time-flight A time-type mass spectrometer is exemplified. Specific examples of a mass spectrometer capable of performing PSD include a time-of-flight mass spectrometer and a time-of-flight mass-time mass spectrometer.
In the case of MS ² analysis involving aggressive cleavage operations such as CID, IRMPD, and PID, anion adduct ions are selected as precursors and fragmentation is performed. In the case of PSD measurement, excessive internal energy is given to the anion adduct ion generated by the MALDI ion source by increasing the laser power, etc., until the anion adduct ion is accelerated toward the analysis part and reaches the detector. In the meantime, fragmentation occurs due to the excess internal energy.

In the present invention, since an anion constituting the additive substance having a gas phase basicity in a specific range (1300 to 1550 kJ / mol) is used, anion desorption from molecular weight related ions is effectively suppressed. And useful product ions can be preferably obtained. Surprisingly, even when BF ₄ ⁻ is used as an anion constituting the additive substance, useful product ions can be obtained despite low gas phase basicity.

In the present invention, product ions obtained from molecular weight-related ions are obtained in a mode useful for sugar chain structure analysis. In other words, useful product ions that provide glycan structure information can be obtained with favorable sensitivity, and unwanted product ions that can occur due to excessive fragmentation, for example, that hinder these useful product ions are preferably suppressed. It is done. For example, in an N-linked sugar chain, D ions, E ions, C ions, and / or ^2,4 A ions derived from ring cleavage, which are particularly important for structural analysis, can be obtained. If present, the appearance of unnecessary product ions is preferably suppressed in the mass range in which the peaks of D ions and E ions appear.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.
As the liquid matrix, five kinds of ionic liquids shown in Table 1 were prepared. These ionic liquids are reported to be suitable for ionization of sugar chains.
Specifically, a methanol solution of the acid (acidic group-containing organic substance) and amine in Table 1 is prepared, and the quantitative relationship described in the preparation method in Table 1 is obtained so that the molar ratio in Table 1 is obtained. The solution was mixed and subjected to sonication for 3 minutes. Subsequently, methanol and unreacted reagents were removed overnight using SpeedVac ^(R) . Each obtained liquid matrix was dissolved in 50 v / v% acetonitrile (ACN) aqueous solution so as to be 100 mg / mL to form a stock solution, and stored in a -20 ° C environment. In Table 1, DHBB, GTHAP, G ₂ CHCA, G ₂ HABA, and G ₃ CA are liquid matrices comprising combinations of DHB and BA, THAP and TMG, CHCA and TMG, HABA and TMG, and CA and TMG, respectively. (The same applies to Table 2 described later).

As matrix additives, nine ammonium salts whose anions are PF ₆ ⁻ , BF ₄ ⁻ , HSO ₄ ⁻ , I ⁻ , NO ₃ ⁻ , H ₂ PO ₄ ⁻ , Br ⁻ , SCN ⁻ , and Cl ⁻ , respectively. Prepared. Each additive was dissolved in a 50 v / v% ACN aqueous solution to prepare a 2.5 mM solution, and 4 times the amount (v / v) was added to the liquid matrix stock solution to obtain an additive mixed matrix solution. Therefore, the anion concentration in the additive mixed matrix solution is 2 mM. In addition, when the anion concentration was examined in the range of 0.2 to 200 mM, it was confirmed that about 2 mM was optimal for the effect of the present invention for any anion, and this concentration was adopted in this example.

  As a sample, double-stranded neutral sugar chain NA2 was used. NA2 is as shown in the following formula. A sample solution of 200 fmol / L was prepared by dissolving NA2 in water. As the MALDI plate, μ-Focus MALDI plate (Hudson Surface Technology) was used.

On the MALDI plate, 0.5 μL of the additive-mixed matrix solution is mixed with 0.5 μL of the sample solution (neutral sugar chain NA2 concentration in the sample solution is 200 fmol / μL), dried at room temperature for about 20 minutes, and mass spectrometry measurement ( MALDI-QIT-TOFMS apparatus: AXIMA Resonance (registered trademark), manufactured by Shimadzu Corporation, the same applies hereinafter. After performing MS ¹ analysis without a cleavage operation, low-energy CID was performed using the detected molecular weight-related ions (anionic adduct ions) as precursors, and MS ² analysis was performed. In addition, anion adduct ions can be cleaved by other cleavage methods, and an example of actual cleavage and analysis by high-energy CID is Rapid Communications in Mass Spectrometry 26 (2012) 469-479 (Non-patent Document 6). It is described in.
The droplets adhering to the plate were not completely dried, and a relatively uniform liquid state was maintained for a long time even under high vacuum after being introduced into the mass spectrometer.

The results of examining the negative ionization of neutral sugar chain NA2 of 100 fmol are shown in Table 2, and the following explanation is added to the annotations in the figure.
(*) A value with an asterisk indicates a calculated value. Other values are experimental values.
Specifically, the calculated values are quoted from Koppel, IA et al. Journal of the American Chemical Society 122 (2000) 5114-5124, and the experimental values are from the website NIST Chemistry WebBook (http: //webbook.nist. quoted from gov./chemistry/).
(A) [M + anion] ^- + if peak is S / N <20, +++ if 20 <S / N <100, +++ if 100 <S / N <250, 250 < In the case of S / N, it was written as ++++.
(B) DoF indicates the degree of fragmentation in MS ¹ analysis. The degree of fragmentation, ^{2, 4} A ₆ fragment ion peak intensity _{^{(I [2,4 A 6] -}} ) and [M + anion] ^- of the peak intensity ^{(I [M + anion] -} ) and on the basis of Calculated by the following formula:
DoF = I [ ^2,4 A ₆ ] ^- / (I [M + anion] ^- + I [ ^2,4 A ₆ ] ^- )
When fragmentation did not occur, it was expressed as-when DoF <10%, Middle when 10% <DoF <50%, and High when 50% <DoF.
(C) [M + anion] - in the CID spectra (MS ² spectra), useful product ions for structural analysis, such as ^{2, 4} A-ion and D-ion in the spectrum is observed with sufficient strength ● is marked with ●.

As shown in Table 2, an anion (specifically, PF ₆ ⁻ , BF ₄ ⁻ , HSO ₄ ⁻ , I ⁻ ) having a low gas phase basicity (GB) was used as an additive. If, in combination with any liquid matrix [M + anion] ^- peak were obtained strongly. ^{_{(Specifically, Br -, NO 3 -,}} H 2 PO 4 -, SCN -, Cl -) GB high anion additive when using, in particular, G ₃ CA are combined as a liquid matrix In some cases, an anionic adduct of a sugar chain could be obtained with a relatively good strength. In addition, fragmentation in MS ¹ analysis was also effectively reduced. G ₃ CA is used as the liquid matrix, and (a) PF ₆ ⁻ , (b) BF ₄ ⁻ , (c) HSO ₄ ⁻ , (d) I ⁻ , (e) NO ₃ ⁻ , (f) as matrix additives. FIG. 1 shows an MS ¹ spectrum of an anionic adduct of neutral sugar chain NA2 (100 fmol) obtained by using an anionic substance of Br ⁻ , (g) SCN ⁻ , (h) H ₂ PO ₄ ⁻ or (i) Cl ⁻ . Shown in

Or NO ₃ ^{- -} BF ₄ as an anion additives used, the detection limit in the case of using a G ₃ CA as a liquid matrix, 1 fmol (NO ₃ ^- When using a) and 100 amol (BF ₄ ^- use the (Table 3 below). Further, in each case, MS ¹ spectra obtained by mass spectrometry of NA 2 having a detection limit concentration (b) and a concentration 10 times the detection limit (a) are shown in FIG. 2 and FIG.

In the prior art, for example, the document Rapid Communications in Mass Spectrometry 26 (2012) 469-479, the sensitivity of negative ions of N-type sugar chains is about 2p to 350 fmol when using THAP to which NO ₃ ^- is added. It is reported. That is, the detection sensitivity in this document is inferior to 1/1000 times the detection limit of the present invention.

For a specific comparison of the effects of the present invention and the prior art, the following experiment was conducted by the same method as the above prior art document.
The additive anion BF ₄ ⁻ or NO ₃ ⁻ which gave particularly good results in the present invention was added to the solid matrix THAP to prepare an additive mixed solid matrix solution (solvent: 50 v / v% ACN aqueous solution). In the additive-mixed solid matrix solution, the anion concentration was 20 mM and the THAP concentration was 10 mg / mL. Note that, when the anion concentration was examined in the range of 1 to 200 mM, it was confirmed that about 20 mM was optimal for the effect of the conventional technology, and this concentration was adopted in the comparative experiment.
On the MALDI plate, 0.5 μL of the additive mixed solid matrix solution was mixed with 0.5 μL of the sample solution, dried at room temperature for about 20 minutes, and mass spectrometry was performed. As a result, in this comparative experiment, the detection limit was about 10 fmol (Table 3 below). That is, the detection sensitivity in the comparative experiment was 1/100 or less of the detection limit of the present invention.

(A) PF ₆ ⁻ , (b) BF ₄ ⁻ , (c) HSO ₄ ⁻ , (d) I ⁻ , (e) NO ₃ ⁻ , (f) Br ⁻ , (g) SCN ⁻ , (h) H ₂ PO ₄ ^- or (i) Cl ^- performs CID anionic adduct ions resulted from NA2 of 100 fmol as precursor with the anionic material, were obtained MS ² spectra. The obtained MS ² spectrum and the analysis result are shown in FIGS.

As shown in FIG. 4 (a) (for comparison), PF ₆ ^- was not possible to obtain useful MS ² spectra from adduct ions. This PF ₆ ^- is believed to be because it can not be pulled out GB low of a proton from a sugar chain.
As shown in FIG. 4 (c) (for comparison), HSO ₄ ^- In MS ² analysis of adduct ions, unlike the MS ² analysis of other anions adduct ions, product ions peaks observed containing anion However, the peak intensity was significantly lower than the intensity of the precursor ion, which was not practical at all. This is probably because anion desorption occurs preferentially without extracting protons from the sugar chain.

Further, as shown in FIG. 5 (d) (for comparison), I ^- although MS ² spectra acquired from adduct ions was possible, it was necessary to integrate the large number of times the spectrum. This is also considered to be because anion desorption occurs preferentially. Moreover, in the obtained spectrum, fragments in the low m / z region were strongly detected, and a large number of product ions that were considered to be multiple cleavages were observed in the m / z 700-1200 region. In this region, D ions and E ions that are particularly important for structural analysis are often observed, which is disadvantageous for spectral interpretation. Therefore, I ^- adducts are not suitable for structural analysis.
More than, PF ₆ ^- adduct ion, HSO ₄ ^- adduct ion and I ^- adduct ions has been found to be not useful for structural analysis.

Meanwhile, Br in FIG. 5 (f) ^- The MS ² analysis of adduct ions, the same spectrum pattern as FIG. 5 (d) (comparative) was obtained. However Br ^- adduct ions during MS ² spectra acquired I ^- so many integration is not required and relatively efficiently product ions occurs.

Further, as shown respectively in FIG. 5 (e), FIG. 6 (g), FIG. 6 (h) and Figure ^{_{6 (i), NO 3 -}} , SCN -, H 2 PO 4 - and Cl ^- from adduct ions MS ² spectra were obtained with good and good S / N. These MS ² spectral patterns were nearly the same, and there was no inefficiency in product ion generation as in the comparative I ^- adduct ion of FIG. 5 (d). Further, Br in FIG. 5 (f) ^- not peak seems more cleavage also observed as in adduct ions clearly be observed D ions and E ion was found to be a more useful manner in structural analysis . The negative ions analysis sensitive neutral oligosaccharides, NO ₃ in FIG ion generation amount was higher among these anionic adduct ions 5 (e) ^- it was found to be aspects of the adduct ions are particularly useful.

Furthermore, BF ₄ in FIG. 4 (b) ^- in the embodiment of the adduct ions, BF ₄ ^- in spite GB is low, surprisingly, efficiently good S / N, the acquisition of MS ² spectra It was possible. Particularly preferred embodiment FIG. 5 NO ₃ of (e) ^- is more in aspects of the adduct ions, although higher efficiency in product ions generated, BF ₄ in FIG. 4 (b) ^- in the embodiment of the adduct ions, a certain When integrated, a spectrum with sufficient quality for structural analysis was obtained. On the other hand, in this embodiment, NO ₃ ^- than aspects of adduct ions, BF ₄ ^- Sensitivity in MS ¹ analysis of adduct ions is high. Specifically, the detection sensitivity in MS ¹ analysis is an order of magnitude higher than in the case of NO ₃ ⁻ . Thus, the profiling of the glycan MS ¹ analysis sensitive BF ₄ in ^- done on the basis of the adduct, after obtaining the MS ² spectra of somewhat strong peak, if the MS ² analysis requires sensitivity NO ₃ ^- It is also possible to use an adduct.

Claims (4)

Mixture comprising an ionic liquid as the matrix, a material selected from tetrafluoroboric acid and its salts or Ranaru group as a matrix additive, a neutral molecule selected from the group consisting of sugar chains and sugar chain derivative as a sample Is used for mass spectrometry, and a molecular weight-related ion analysis is performed in which an anion constituting the additive substance detects a molecular weight-related ion added to a molecule of the sample. Before SL ionic liquid, an ion of the amine, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxy acetophenone, alpha-cyano-4-hydroxycinnamic acid, 2- (4-hydroxyphenyl) benzoic 2. The method of claim 1 comprising an acid ion selected from the group consisting of acid and coumaric acid. Wherein the amine is selected from the group consisting of 1,1,3,3-tetramethylguanidine and n- butylamine, method of claim 2. The salt of tetrafluoroboric acid is an ammonium salt The method of any of claims 1-3.