JP7594307B2 - Method for direct analysis of glycoconjugate polymer sugar chains by MALDI-TOFMS and solid matrix composition for use therein - Google Patents

Description

The present invention provides a method for analyzing glycan components such as high molecular weight glycoconjugates with high accuracy and sensitivity by MALDI-TOFMS without the need for cleavage procedures from the aglycone components, and a matrix system suitable for such a method. In particular, the present invention provides a series of matrix systems applicable to the analysis of any type of glycoconjugate, such as glycoproteins and lipid-bound glycans.

Glycoconjugates such as glycoproteins and glycolipids play important biological roles in nature. For example, differences in glycan structures presented extracellularly are recognized by the immune system and presented on the cell surface as blood types, which are used as indicators for blood transfusions, and serotypes, which are used to identify pathogenic microorganisms. It is known that glycans presented on asparagine residues (N-linked glycans), which are known as the main post-translational modification of proteins presented extracellularly, differ between glycans presented on the surface of normal cells and those presented on the surface of abnormal cells (e.g., cancer cells) (Figure 1). In other words, carbohydrates presented on the cell surface are biomarkers for diseases, infections, and immune mechanisms. Therefore, the development of a rapid and highly accurate method for analyzing glycoconjugates is a very important theme, and analysis is being carried out using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, in MALDI-MS, the ionization efficiency of glycans is low compared to other biomolecules, and therefore analysis of glycoconjugates in biomolecules requires a series of pretreatment steps, such as cleaving the glycans from the aglycone components (proteins, lipids, etc.), separating the glycan components, and chemically modifying the glycans to improve their ionization efficiency.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOFMS) is a rapid and powerful method for sensitive and high-resolution analysis of biomolecules. Carbohydrates and glycoconjugates are commonly analyzed by MALDI-TOFMS using 2,5-dihydroxybenzoic acid (DHB) as the solid matrix. However, when ionizing glycoconjugate macromolecules containing aglycone components such as glycoproteins and glycolipids, the diversity of both glycans and aglycone components leads to complex peak patterns that are difficult to interpret. In addition, when analyzing biopolymers, the signal intensity becomes weak due to the decrease in the number of molecules per unit mass, the decrease in ionization efficiency, and the decomposition of the target molecules due to both in-source decay (ISD) and post-source decay (PSD), making their detection difficult. In addition, when glycoconjugates are fragmented by ISD and PSD, pseudo-molecular ions are generated, which enable the analysis of the component structure and sequence information in the analyte, but the ionization efficiency of glycan components is lower than that of other components, making it difficult to identify the structure and sequence information of glycans. In fact, it has been reported that when ISD and PSD analysis of glycoproteins is performed using a general matrix for MALDI-TOFMS such as DHB, only peptide fragment signals are obtained, and terminal sequence information of the protein, which is the aglycon component, is obtained, but the ion signals indicating glycan information disappear [Non-Patent Document 1].

To solve these problems, it has been reported that the aglycone components of glycoconjugates are decomposed by enzymes or acid-base treatment, the glycan component fragments are separated, and the glycan components are chemically modified to improve the ionization efficiency before being analyzed by mass spectrometry. This modification strategy is particularly effective for glycomics-type research using mass spectrometry with separation methods such as solid-phase capture.
However, in order to simplify and speed up analytical processes (e.g., identification and diagnosis of microorganisms), there is a demand for direct analysis without decomposition and removal processes of aglycone components. In this sense, the decomposition (fragmentation) of the analyte molecule and acquisition of internal structural information in mass spectrometry using ISD and PSD are extremely effective. However, due to the low ionization efficiency of glycans, the glycan components of polymeric glycoconjugates were thought to be unsuitable for ISD and PSD analysis targeting fragment ions.

Recently, various arylamine-type basic substances have been reported as additives in matrices that form ionic liquids and homogeneous crystalline forms with the analytes on the MALDI target plate, improving the sensitivity and reproducibility of the analyte glycans [Non-patent literature 2-8].

For example, a system using α-cyano-4-hydroxycinnamic acid (CHCA) as a solid matrix and adding 3-aminoquinoline as an arylamine-type basic substance (3AQ/CHCA) is known to form a homogeneous ionic liquid and provide highly sensitive aminoquinoline-labeled glycans on a MALDI target plate [Non-Patent Document 9]. Although such homogeneous ionic liquid systems often improve both ionization efficiency and analyte signal reproducibility compared to normal solid matrices, the ionic liquid spot on the target plate becomes dome-shaped due to surface tension. In this way, the ions are ejected at various angles, which causes variations in flight time and tends to broaden the peak shape of the ion signal, as does DHB, which tends to form large heterogeneous crystals on the target plate. It is also known that aniline and its N-methyl derivatives can form microcrystals or ionic liquids with DHB and ionize unmodified carbohydrates for detection by MALDI-TOFMS.
In addition, an oxime-forming reaction by combining the reducing end of a glycan with an O-substituted hydroxylamine (aminooxy group) has been reported as a glycan labeling and capture reaction. In particular, a process using a hydroxylamine-type labeling reaction as a key for glycan structural analysis has been reported [Patent Documents 1 to 10, Non-Patent Documents 10 to 12].

Patent No. 5147815 Patent No. 5467815 Patent No. 5368725 Patent No. 5682767 Patent No. 4566604 Specification Patent No. 5289707 Patent No. 5485449 Patent No. 5301705 Specification Patent No. 5301706 Patent No. 5301708 Specification International Publication No. 2020/153502

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When structural analysis of glycans bound to glycopeptides or glycoproteins with higher molecular weights and complex three-dimensional structures is performed by mass spectrometry, the glycopeptides or glycoproteins are fragmented. Since the ionization efficiency of amino acid sequences is higher than that of glycans, peptide fragments are observed, but the ionization of the glycan portion is suppressed, making analysis of the glycans difficult.
In fact, when the glycoprotein RNase B is analyzed using typical matrices such as sinapic acid (SA), 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid (CHCA), only peptide fragments are observed, and no glycan fragments are generated [Non-Patent Document 1].
Therefore, the standard procedure for glycan analysis is to (i) excise the glycans from the peptide or protein (chemical or enzymatic cleavage), (ii) separate the glycans (hydrophilic chromatography, lectins, chemoselective modification, etc.), and (iii) improve the ionization efficiency (anomeric modification, complete methylation, carboxylic acid modification, etc.) before analysis.

In recent years, ionic liquid matrices in which counter salts have been added to the matrix have come to be widely used. In response to this, the present inventors developed an "aniline derivative/DHB/alkali metal matrix composition" as an optimal solid matrix for the soft ionization method of unmodified sialylated glycans, and further discovered that labeling of glycans and high-sensitivity soft ionization can be achieved simultaneously by using a matrix containing an aminooxy group-containing aromatic derivative instead of an aniline derivative [Patent Document 11, Non-Patent Document 13]. These matrices have been demonstrated to increase the ionization efficiency of glycopeptide (e.g., egg yolk-derived sialylglycopeptide (SGP)) glycans.

However, it has not been confirmed whether the matrix composition developed by the present inventors is also effective for the analysis of glycoproteins and lipid-bound glycans with higher molecular weights and more complex structures. In addition, even if the subject of analysis is the above-mentioned SPG, there is compatibility between the subject of analysis and the matrix composition, and there are matrix combinations that result in no signal when metal ions such as sodium ions are present in the system [Patent Document 11, Non-Patent Document 13].

Currently, rapid testing of pathogenic bacteria is considered important in terms of preventing the spread of infectious diseases. Escherichia coli, a well-studied pathogenic bacteria, is a gram-negative bacterium. Gram-negative bacteria have a glycolipid called lipopolysaccharide on the outermost part of their cell wall, the end of which has a repeating polysaccharide called O antigen, and the repeating pattern varies even within the same bacterial species. When we become infected with bacteria, our bodies develop immunity to these O antigens. Even bacteria of the same species have various forms of O antigens (called serotypes). Therefore, it is used to classify bacterial strains, and it has been reported that O1, O2, O5, O6, O18, O25, O26, O55, O74, O91, O103, O104, O105, O111, O113, O114, O115, O117, O118, O119, O121, O128, O143, O145, O153, O157, O161, O165, O172, etc. can become pathogenic E. coli. Among them, enterohemorrhagic E. coli O26, O111, and O157 are well known.

In medical settings, MALDI-TOFMS analysis is used to directly analyze, classify, and identify microorganisms (microbial bodies) such as bacteria and fungi, and such MALDI-TOFMS instruments have been approved as medical devices. However, current MALDI-TOFMS analysis is limited to detecting proteins derived from bacterial bodies and cannot identify serotypes, so it can only broadly classify bacterial bodies into types (e.g., E. coli, Vibrio cholerae, etc.) and cannot classify strains within the same species (e.g., E. coli O6, O157, etc.).

Lipopolysaccharide (LPS) present on the surface of these gram-negative bacteria consists of lipid A, which is the base component, and O antigens that bind to it. It has been reported that the structure of lipid A can be directly analyzed by negative ion mode MALTI-TOFMS [Non-Patent Document 14]. On the other hand, since the structure of O antigens cannot be directly determined, conventional analytical techniques that require molecular probes such as antibodies and oligo DNA primers are used for strain classification, and in order to respond to a wide range of infectious diseases, it is necessary to prepare probe molecules corresponding to all expected strains in advance. Therefore, to determine the strain, it is necessary to send the sample to a testing institution that has a stock of probe molecules and perform the test again. IR biotyping technology has been proposed as a non-probe analysis technology for O antigens that can be performed at the speed required by medical sites, but it has not been widely used due to its low resolution. In addition, there are no examples of direct sequence analysis of O antigens by mass spectrometry.

The structure of the O antigen has been determined by combining a method in which lipopolysaccharide obtained by phenol extraction is fully methylated and then acid-hydrolyzed, and the binding positions are estimated from the methylation patterns of monosaccharides obtained by gas chromatography or mass spectrometry, with a method in which samples obtained through processes such as mild acid hydrolysis or enzymatic decomposition are observed by NMR or other methods [Non-Patent Document 20] (Figure 6g-3). However, this analysis method requires advanced techniques including pretreatment and a sample amount suitable for NMR, making it difficult to generally use in the medical field or food inspection. Furthermore, a direct structural analysis method for O antigens that meets this need has not yet been reported.

In other words, the objective of the present invention is to construct a series of matrix compositions for directly analyzing the glycan component structures of glycoproteins and lipid-bound glycans without preliminary treatments such as glycosylation or modification.

The method for direct analysis of complex carbohydrate polymer glycans of the present invention can analyze any type of complex carbohydrate, such as glycoproteins and lipid-bound glycans.
The present inventors have already developed a method for the direct analysis of glycopeptides. In the present invention, however, the present inventors have succeeded in selectively ionizing and directly analyzing glycan component fragments of glycoproteins, which have larger molecular weights and more complex structures than glycopeptides, and lipopolysaccharides, which are glycolipids with large molecular weights.
The target high molecular weight complex carbohydrates include ribonucleases (RNases) and lipopolysaccharides. Ribonucleases are enzymes that catalyze the reaction of decomposing ribonucleic acid into oligonucleotides or mononucleotides, and include RNase A, RNase B, RNase C, RNase T1, and RNase I. For example, RNase B has a sugar chain consisting of two GlcNAc and 3 to 9 mannose units on its surface (Fig. 6g-1).

Furthermore, the direct analysis method of complex carbohydrate polymer glycans of the present invention is also applicable to protein mixtures containing various proteins, such as quail egg white, and complex mixtures at the biological level, such as gram-negative bacteria and their extracts.

In the present invention, in the structural analysis of glycans of glycoproteins and lipid-bound glycans, which have higher molecular weights and more complex structures than glycopeptides, it was found that a matrix composition in which alkali metal ions were added to an aniline derivative/DHB matrix system selectively improved the ionization efficiency of glycan fragments, and that by adjusting the laser intensity, it became possible to perform mass analysis of the internal structure of the glycan, and that ionization of aglycone sites such as peptides in complex carbohydrates was suppressed.
Furthermore, it was found that the addition of 1,5-diaminonaphthalene (DAN) to the above-mentioned matrix composition promotes the fragmentation of glycans, forming glycan fragment peaks with smaller molecular weights and showing molecular weight differences equivalent to monosaccharide components, thereby improving the resolution and accuracy of selective analysis of glycan components in complex glycopolymers.

Furthermore, when the aniline derivative was replaced with benzylhydroxylamine, a hydroxylamine compound whose conjugate acid has a pKa of 4.0 to 5.0, without changing the ratio of the above-mentioned matrix system, it was found that the same ionization efficiency was obtained for lipid-bound glycans as with the above-mentioned matrix, while fragmentation of the glycans was suppressed, resulting in the formation of glycan fragment peaks with larger molecular weights than when the aniline derivative was used.

Furthermore, without changing the ratio of the above-mentioned matrix system, DHB was replaced with acidic matrices commonly used in MALDI-MS, such as α-cyano-4-hydroxycinnamic acid (CHCA), which is used in the analysis of peptides and proteins, and its CHCA derivatives 4-chloro-α-cyanocinnamic acid (ClCCA), 4-bromo-α-cyanocinnamic acid (BrCCA), methyl α-cyano-4-hydroxycinnamate (CNME), 2,4,6-trihydroxyacetophenone (THAP), and picolinic acid (3PA). It was found that all of these matrices exhibited selective ionization ability for glycans and preferentially produced glycan fragment peaks in the analysis of complex carbohydrates such as glycoproteins.

The inventors focused on the fact that 3-amino-4-hydroxybenzoic acid (AHBA) has the characteristics of both 2,5-dihydroxybenzoic acid (DHB), which has a carboxyl group and a hydroxyl group in one molecule, and aniline derivatives, which have an amino group. They performed MALDI-ISD analysis of a glycoprotein (RNase B) with N-linked glycans using alkali metal-added AHBA as a matrix, and demonstrated that selective endocyclic dissociation of the reducing terminal sugar residues of N-linked glycans and selective ionization of the sugar residues can be achieved.

AHBA was reported by Mock et al. in 1991 as a matrix capable of ionizing free glycans in the early days of the development of the MALDI-TOF MS method [Non-Patent Document 15]. However, compared with DHB reported by Hillenkamp et al. at the same time [Non-Patent Document 16], its ionization efficiency (detection sensitivity) for neutral glycans was one order of magnitude lower, and it fell out of use [Non-Patent Documents 17, 18].

In the present invention, it was discovered that by adding an alkali metal such as sodium to an aminohydroxybenzoic acid derivative such as 3-amino-4-hydroxybenzoic acid (AHBA), which has a carboxyl group, a hydroxyl group, and an amino group in one molecule as a matrix system, it is possible to directly analyze not only N-linked glycoproteins but also O-linked glycoproteins. This has made it possible to directly analyze the proteins and glycans of microorganisms (bacteria, fungi, etc.) using MALDI-TOFMS analysis, and to not only classify bacterial species but also identify the serotype of the bacterial species.

That is, the present invention comprises the following aspects.
1) A solid matrix composition that enables selective and highly sensitive analysis of ISD and PSD fragment glycans of polymeric glycans and polymeric glycoconjugates in matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOFMS analysis);
2) A selective MALDI-TOFMS analysis method for glycan fragments derived from polymeric glycans and polymeric glycoconjugates and a MALDI-TOF/TOFMS analysis method for glycan components of polymeric glycans and polymeric glycoconjugates using the solid matrix composition according to the present invention;
3) An analytical measurement plate for use in a MALDI-TOF/TOFMS analysis method using the solid matrix composition according to the present invention.

In a first aspect of the present invention, there is provided a solid matrix composition comprising one or more groups of compounds having a conjugate acid selected from an acidic group, an aromatic basic residue, and a hydroxylamine-type basic residue, the conjugate acid having a pKa of 4.0 to 5.0, and an aromatic hydroxyl group; and a monovalent metal ion.

In one specific example of the first aspect of the present invention, a mixture containing an acidic solid matrix, a basic molecule selected from aromatic basic molecules and hydroxylamine-type basic molecules, the conjugate acid of which has a pKa of 4.0 to 5.0, and, optionally, a monovalent metal ion is used as the solid matrix composition. It is also preferable to further add 1,5-diaminonaphthalene (DAN) as a fragmentation promoter.

The acidic solid matrix preferably contains a carboxylic acid and has a pKa of 1.0 to 6.0, or contains an aromatic hydroxyl group and has a pKa of 6.0 to 10.0. Examples of typical matrix compounds used in the MALDI method include 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), CHCA derivatives 4-chloro-α-cyanocinnamic acid (Cl-CCA), 4-bromo-α-cyanocinnamic acid (Br-CCA), α-cyano-4-hydroxycinnamic acid methyl (CNME), 2,4,6-trihydroxyacetophenone (THAP), and picolinic acid (PA).

Examples of the monovalent metal ions include alkali metal ions such as lithium ions, sodium ions, potassium ions, rubidium ions, and cesium ions.

Examples of basic compounds whose conjugate acids have a pKa of 4.0 to 5.0 include aromatic basic molecules such as aniline, N-methylaniline (NMA), N,N-dimethylaniline (DMA), and 3-aminoquinoline (3AQ), and hydroxylamine-type basic molecules such as O-benzylhydroxylamine (BOA).

It is preferable that the amount of monovalent metal ion added is 1 to 50 mol % relative to 100 mol % of the acidic solid matrix.

It is preferable that the amount of the basic compound whose conjugate acid has a pKa of 4.0 to 5.0 is 10 to 150 mol % relative to 100 mol % of the acidic solid matrix.

Additionally, the fragmentation promoter may include 1 to 100 mol % of 1,5-diaminonaphthalene relative to 100 mol % of the acidic solid matrix.

In another specific example of the first aspect of the present invention, there is provided an amphoteric solid matrix having a carboxyl group, an aromatic hydroxyl group, and an aromatic basic residue whose conjugate acid has a pKa of 4.0 to 5.0 in one molecule, the amphoteric solid matrix being represented by the formula (1):

（式中、Ｒ^１～Ｒ^５は、それぞれ、独立して、水素原子、水酸基、アルキル基、エーテル基、カルボキシル基および／またはアミノ基であって、Ｒ^１～Ｒ^５のうちいずれかふたつは、それぞれ、水酸基およびアミノ基である。）で表されるアミノヒドロキシ安息香酸；ならびに１価の金属イオンを含む混合物を固体マトリックス組成物として採用する。

(wherein R ¹ to R ⁵ are each independently a hydrogen atom, a hydroxyl group, an alkyl group, an ether group, a carboxyl group, and/or an amino group, and any two of R ¹ to R ⁵ are each a hydroxyl group and an amino group), and a mixture containing a monovalent metal ion is used as the solid matrix composition.

The aminohydroxybenzoic acid has the following formula:

で表される化合物からなる群から選択されるアミノヒドロキシ安息香酸である。

The aminohydroxybenzoic acid is selected from the group consisting of compounds represented by the formula:

3-Amino-4-hydroxybenzoic acid (AHBA) is an example of an amphoteric solid matrix that contains an aromatic hydroxyl group and an aromatic basic residue whose conjugate acid has a pKa of 4.0 to 5.0 within one molecule.

Examples of the monovalent metal ions include alkali metal ions such as lithium ions, sodium ions, potassium ions, rubidium ions, and cesium ions.

It is preferable that the amount of monovalent metal ion added is 1 to 50 mol % relative to 100 mol % of the amphoteric solid matrix.

In the second aspect of the present invention, a target plate that can be expected to have a concentration effect by water repellent treatment or the like is used in the glycan fragment selective MALDI-TOF mass spectrometry, thereby controlling the sample concentration position and enabling high sensitivity automatic analysis of a trace amount of specimen. In addition, in order to fully obtain the effects of the present invention, it is essential that the matrix system and the measurement target form a cloudy white solid. According to the present invention, the matrix has properties different from those of conventional DHB, and is a microcrystalline or amorphous solid, and the measurement target is uniformly distributed in the target area, improving measurement reproducibility. In other words, matrices with ionic liquid properties and matrices that form relatively large crystals are not subject to the present invention.
Furthermore, by performing TOF/TOF analysis, it becomes possible to analyze fragment information such as the internal structure of the glycan.

In a third aspect of the present invention, an aqueous solution of the acidic solid matrix composition of the present invention is spotted onto a target plate and dried to form a matrix layer on at least a portion thereof.

By using a MALDI-TOFMS analysis in which the solid matrix composition of the present invention is appropriately combined with 1,5-diaminonaphthalene (DAN) as a fragmentation promoter, glycans contained in glycoproteins and lipid-bound glycans can be analyzed with high accuracy in reflectron mode without any modification.

Schematic diagram of glycans bound to the surface of normal and diseased cells. FIG. 1 is a schematic diagram showing compounds used in the preparation of a matrix composition. Schematic diagram showing compounds used in the preparation of the matrix composition. Schematic diagram showing compounds used in the preparation of the matrix composition. Schematic diagram showing compounds used in the preparation of the matrix composition. Schematic diagram of the analysis target. [Example 1] Spectral pattern derived from the decomposition of the analyte RNase B in reflectron mode MALDI-TOFMS using matrix composition 1 (DHBNa-aniline). [Example 1-2] TOF/TOF degradation pattern derived from analyte RNase B in LIFT-TOF/TOF MS mode using matrix composition 1 (DHBNa-aniline). [Example 2] Degradation pattern of the analyte RNase B in reflectron mode MALDI-TOF MSF using matrix composition 2 (DHBNa-aniline+DAN). [Example 2-2] TOF/TOF degradation pattern derived from analyte RNase B in LIFT-TOF/TOF MS mode using matrix composition 2 (DHBNa-aniline+DAN). [Example 3] Decomposition pattern of the analyzed quail egg white (glycoprotein mixture) in reflectron mode MALDI-TOF MS using matrix composition 2 (DHBNa-aniline+DAN). [Example 4] Degradation pattern of the analyte RNase B in reflectron mode MALDI-TOF MS using matrix composition 3 (CHANa-3AQ). [Example 5] Decomposition pattern of the analyte O157-derived lipopolysaccharide in reflectron mode MALDI-TOF MS using matrix composition 1 (DHBNa-aniline). [Example 5-2] Internal structure pattern of O-antigen repeating glycan sequence in the analyzed O157-derived lipopolysaccharide in LIFT-TOF/TOF mode using matrix composition 1 (DHBNa-aniline). [Example 6] Decomposition pattern of the analyte O157-derived lipopolysaccharide in reflectron mode MALDI-TOF MS using matrix composition 2 (DHBNa-aniline+DAN). [Example 6-2] Internal structure pattern of O-antigen repeating glycan sequence in the analyzed O157-derived lipopolysaccharide in LIFT-TOF/TOF mode using matrix composition 2 (DHBNa-aniline + DAN). [Example 7] Decomposition pattern of the analyte O157-derived lipopolysaccharide in reflectron mode MALDI-TOF MS using matrix composition 4 (DHBNa-BOA). [Example 7-2] Internal structure pattern of O-antigen repeating glycan sequence in the analyzed O157-derived lipopolysaccharide in LIFT-TOF/TOF mode using matrix composition 4 (DHBNa-BOA). [Example 8] In reflectron mode MALDI-TOF MS using matrix composition 5 (DHBNa + DAN), (a) degradation pattern of lipopolysaccharide in the supernatant of the treated O6 bacterial body to be analyzed; (b) degradation pattern of lipopolysaccharide in the supernatant of the treated O157 bacterial body to be analyzed. [Example 9] Effect of added sodium amount on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using matrix composition 1 (DHBNa-aniline). [Example 10] Effect of added sodium amount on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using matrix composition 2 (DHBNa-aniline+DAN). [Example 11] Effect of the amount of DAN added on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using matrix composition 5 (DHBNa-DAN). [Example 12] Effect of added sodium amount on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using matrix composition 6 (DHBNa). [Reference Example] Spectral pattern derived from the decomposition of the target RNase B in reflectron mode MALDI-TOF MS using 1,5-diaminonaphthalene (DAN). [Example 13] Effect of adding 20 mol% DAN on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using a matrix composition containing sodium and aniline in α-cyano-4-hydroxycinnamic acid (CHCA). [Example 14] Effect of adding 20 mol% DAN on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using a matrix composition containing sodium and aniline in 2,4,6-trihydroxyacetophenone (THAP). [Example 15] Effect of adding 20 mol% DAN on the spectral pattern derived from the degradation of the target RNase B in reflectron mode MALDI-TOF MS using a matrix composition containing sodium and aniline in picolinic acid (PA). [Example 16] Effect of adding 20 mol% DNA on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using a matrix composition containing α-cyano-4-hydroxycinnamate (CNME) with sodium and aniline. [Example 17] Effect of adding 20 mol% DAN on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using a matrix composition containing 4-chloro-α-cyanocinnamic acid (Cl-CCA), sodium, and aniline. [Example 18] Effect of adding 20 mol% DAN on the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using a matrix composition containing 4-bromo-α-cyanocinnamic acid (Br-CCA), sodium, and aniline. [Example 19] Possibility of direct analysis of N-linked glycans in the spectral pattern derived from the degradation of the analyte RNase B in reflectron mode MALDI-TOF MS using a matrix composition containing sodium in 3-amino-4-hydroxybenzoic acid (AHBA). [Comparative Examples 1 to 4] In reflectron mode MALDI-TOF MS using a matrix composition containing 3,5-diaminobenzoic acid (DABA), the effects of (a) DABA alone, (b) the addition of sodium, (c) the addition of sodium and DABA, and (d) the addition of sodium and DHB on the spectral pattern derived from the degradation of the analyte RNase B. [Example 20] Possibility of direct analysis of O-linked glycans in the spectral pattern derived from the decomposition of the target porcine stomach mucin in reflectron mode MALDI-TOF MS using a matrix composition containing sodium in 3-amino-4-hydroxybenzoic acid (AHBA).

In a first aspect of the present invention, a matrix system and its composition are provided that realizes selective analysis of glycan fragments of high molecular weight glycoconjugates in MALDI-TOFMS analysis.
The matrix system of the present invention is intended to realize selective analysis of glycan fragments of high molecular weight complex carbohydrates in MALDI-TOFMS analysis, and is composed of acidic molecules commonly used as matrices in the MALDI method (2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and CHCA derivatives such as 4-chloro-α-cyanocinnamic acid (Cl-CCA), 4-bromo-α-cyanocinnamic acid (Br-CCA), α-cyano-4-hydroxycinnamic acid methyl ester (CNME), 2,4,6-trihydroxyacetophenone (THAP), and picolinic acid (PA)) and aromatic basic molecules whose conjugate acids show a pKa of 4.0 to 5.0 (aniline, N-methylaniline (NMA), and and N,N-dimethylaniline (DMA)) or an aminooxy group-containing aromatic derivative selected from the group consisting of aniline derivatives (hereinafter, including aniline, collectively referred to as "aniline derivatives" or an aminopyridine derivative selected from 3-aminoquinoline (3AQ)) or a hydroxylamine-type basic molecule (O-benzylhydroxylamine (BOA)) whose conjugate acid shows a pKa of 4.0 to 5.0; 1,5-diaminonaphthalene (DAN) that promotes in-source and post-source decomposition; and an alkali metal ion selected from the group consisting of lithium ion, sodium ion, potassium ion, rubidium ion, and cesium ion.

The above aromatic basic molecules are used in the composition because, when mixed with DHB, they suppress the crystal growth of DHB, form a uniform matrix layer, and reduce background noise in the MALDI-TOFMS spectrum.

In the composition, the amount of aromatic basic molecule added is preferably 10 mol% or more relative to the acidic molecular component such as DHB, and more preferably an equimolar amount or more (100 mol% or more) relative to the acidic molecular component such as DHB. In the present invention, excessive addition of aromatic basic molecule does not affect the effect of the present invention, but if a large excess of aromatic basic molecule is present during the volatilization process of the excess aromatic basic molecule, the concentration process up to the measurement in the high vacuum device will be delayed, so it is desirable to keep the amount of aromatic basic molecule added to DHB in small excess (preferably 150 mol% or less, more preferably 120 mol% or less).

The addition of 1,5-diaminonaphthalene (DAN) to the composition is not essential, but it promotes in-source and post-source degradation, making it easier to explore the internal structure of glycan fragments. The amount of DAN added can replace aromatic basic molecules at any ratio, and it is preferable to add up to 100 mol% of the acidic molecular components such as DHB, and more preferably up to 60 mol% of the acidic molecular components such as DHB.

In the composition, the above alkali metal ions are used because they are stable under laser irradiation, have low corrosiveness, and are easily vaporized as counter ions and adduct ions when added to a matrix. Although not limited thereto, salts of lithium, sodium, potassium, rubidium, and cesium, such as carbonate, bicarbonate, hydrochloric acid, and acetate, are preferably used as sources of the above alkali metal ions.

In the composition, the amount of alkali metal ions added is 0.1 to 50 mol% relative to the acidic molecular components such as DHB, preferably 0.5 to 20 mol%, and more preferably 1 to 15 mol%. In the present invention, the addition of aromatic basic molecules promotes the ionization of glycan fragment components, but if alkali metal ions are not added, ionization of components other than glycan fragments also occurs, and multiple aglycon-derived fragment peaks are generated along with multiple metal ion adduct peaks. When the amount of alkali metal ions added is 1 mol%, the aglycon-derived fragment peaks are significantly reduced, and when the amount is 2 mol% or more, no further suppression effect is exerted, and when the amount exceeds 25 mol%, the peak intensity begins to decrease. In addition, when DAN is added together with aromatic basic molecules, alkali metal ions are not essential, but 0.1 to 20 mol% is preferable, and 1 to 15 mol% is more preferable.

In a second aspect of the present invention, there is provided a MALDI-TOF mass spectrometry method that realizes selective analysis of glycan fragments of high molecular weight glycoconjugates.
The MALDI-TOF mass spectrometry method according to the present invention uses the solid matrix system according to the present invention. More specifically, an aqueous solution of a solid matrix composition prepared by adding an alkali metal ion source such as, but not limited to, sodium bicarbonate to a mixture of an aromatic basic molecule in small excess relative to an acidic matrix molecule commonly used in MALDI methods such as DHB is spotted on a target plate and dried to form a matrix layer on at least a portion of the target plate. An aqueous solution of the analyte is deposited on the matrix layer and dried to prepare a measurement sample of the analyte.
In the present invention, a plate having a liquid-repellent coating formed on its surface having one or more hydrophilic patches (anchors) is useful (e.g., AnchorChip ^™ (Bruker Daltonics, Bremen, Germany)). "Liquid-repellent" refers to low wettability not only with water, but also with most organic solvents including alcohol, acetonitrile, and even acetone.

The obtained measurement sample is subjected to MALDI-TOF mass spectrometry (e.g., Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics GmbH; Bremen, Germany)) to perform MALDI-TOFMS targeting glycan fragment ions selectively generated by in-source decay (ISD) or post-source decay (PSD). The method of the present invention is particularly useful for measurements using the reflectron mode, which enables more precise analysis.

In a third aspect of the present invention, there is provided a MALDI-TOF/TOF mass spectrometry method utilizing the second aspect and further targeting glycan fragment ions selectively generated by in-source decay (ISD) of high molecular weight glycoconjugates.
The MALDI-TOF/TOF mass spectrometry according to the present invention uses the matrix system according to the present invention. A glycan fragment ion to be analyzed is selected as a parent ion by a timed ion gate, and LIFT-TOF/TOF analysis is performed.

The compounds used to prepare the matrix compositions are shown in Figures 2 to 5. As acidic molecules commonly used as matrices in conventional MALDI methods, 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and CHCA derivatives 4-chloro-α-cyanocinnamic acid (Cl-CCA), 4-bromo-α-cyanocinnamic acid (Br-CCA), α-cyano-4-hydroxycinnamic acid methyl ester (CNME), 2,4,6-trihydroxyacetophenone (THAP), and picolinic acid (PA) were used. As arylamine-type basic substances, aniline, aniline derivatives N-methylaniline (NMA) and N,N-dimethylaniline (DMA), aminopyridine derivative 3-aminoquinoline (3AQ), and aminooxy group-containing aromatic derivative O-benzylhydroxylamine were used. In addition, 3-amino-4-hydroxybenzoic acid (AHBA) was used as an aromatic amphoteric molecule having a carboxyl group, a hydroxyl group, and an amino group in one molecule, and for comparison, 3,5-diaminobenzoic acid (DABA) was used as an aromatic amphoteric molecule having a carboxyl group and two amino groups in one molecule but no hydroxyl group. Sodium bicarbonate was used as a monovalent metal ion source. Furthermore, 1,5-diaminonaphthalene (DAN) was used as a glycan fragmentation promoter.

The subject of analysis was ribonuclease B (RNase B), an enzyme that catalyzes the reaction of decomposing ribonucleic acid into oligonucleotides or mononucleotides. The glycan that binds to RNase B is composed of N-acetylglucosamine (GlcNAc) and mannose (Fig. 6g-1). In addition, quail egg white, a protein mixture, was used (Fig. 6g-2). Furthermore, lipopolysaccharides derived from Escherichia coli O157 and O6, which are gram-negative bacteria, were used as high molecular weight glycolipids (Fig. 6g-3).

RNase B was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Quail egg white was separated from eggs produced at the Muroran Quail Farm, purchased from a mass retailer. Lipopolysaccharide derived from Escherichia coli O157 (ultracentrifuged product) was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. 2,4,6-trihydroxyacetophenone (THAP) and 3-aminoquinoline (3AQ) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2,5-Dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), 4-chloro-α-cyanocinnamic acid (Cl-CCA), 4-bromo-α-cyanocinnamic acid (Br-CCA), α-cyano-4-hydroxycinnamic acid methyl ester (CAME), picolinic acid (PA), and 1,5-diaminonaphthalene (DAN) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Acetonitrile, aniline, and sodium bicarbonate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). O-benzylhydroxylamine (BOA) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Standard strains of E. coli O6 (ATCC 25922, serotype O6:K1) and E. coli O157 (ATCC 43888, serotype O157:H7) were purchased from Microbiologics.

[MALDI-MS analysis]
An Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics GmbH; Bremen, Germany) equipped with a 200 Hz smart beam Nd:YAG UV laser (355 nm) with a maximum power of 30 mW was used in reflectron mode. The spot positions on the target plate were calibrated using the autoteaching function. Mass spectra of all samples were acquired in the range of m/z 700–5000 for reflectron mode with 500 laser shots in random walk mode with a shooting position pattern at a laser frequency of 200 Hz. Bruker peptide calibration standard II was used as an external standard for calibration. Otherwise, the sample positions and laser powers were taken with the default settings prepared using FlexControl 3.5 software.

In MALDI-TOF mass spectrometry, as the laser intensity increases, fragment ions are generated by cleavage of the internal bonds of the generated molecular ions. This leads to backbone fragmentation, called in-source decay (ISD) and/or post-source decay (PSD), even when using the "cool" DHB matrix, which is less likely to decompose than CHCA and allows for soft ionization.

In-source decay (ISD) is a decomposition phenomenon in which the analyte fragments in the ionization chamber simultaneously with or immediately after ionization, while post-source decay (PSD) is a decomposition phenomenon that occurs in the drift space after ionization due to excitation caused by the internal energy of the ion itself or collisions with residual gas.
Cleavage of the analyte due to ISD or PSD occurs at multiple locations within the molecule, resulting in the generation of various fragment ions. Since the internal energy of the ion depends on the power of the irradiated laser, it is known that the degree of increase in the amount of fragments generated varies for each cleavage location in response to an increase in laser power. Therefore, when measurements are performed on the same analyte using multiple laser powers, the resulting mass spectrum changes. By analyzing the relative intensity ratio of the other spectra using a peak that is commonly observed from low power to high power among multiple mass spectra as a standard, it is possible to analyze the structure of the analyte.

[Example 1]
The spectral pattern resulting from the degradation of the analyte RNase B was evaluated in a reflectron mode MALDI-TOFMS using matrix composition 1 (DHBNa-aniline).
First, Na+ ion-added matrix composition 1 was prepared by increasing the volume of 0.5 M DHB ( ¹⁰⁰ μL) in _CH3CN / _H2O (90:10, v/v), 1.0 M aniline solution (60 μL) in _CH3CN , and 0.1 M sodium bicarbonate in water (50 μL) to 1 mL with _CH3CN / _H2O (50:50, v/v).
When the glycoprotein RNase B was analyzed using matrix composition 1 at a laser intensity of 80%, a glycan fragmentation pattern consisting of ^2,4 A ₇ -type cross-ring cleavage of the reducing end GlcNAc residue was observed, without the generation of peptide fragments.
It was shown that by using the matrix composition according to the present invention, not only glycopeptides but also glycoproteins having larger molecular weights and complex structures can be directly analyzed without cleavage or modification of the glycans.

[Example 1-2]
The TOF/TOF degradation pattern from the analyte RNase B was evaluated in the LIFT-TOF/TOF MS mode using matrix composition 1 (DHBNa-aniline).
To focus on the PSD process of the glycan fragment peak consisting of ^2,4 A ₇ -type cross-ring cleavage of the reducing end GlcNAc residue of the RNase B glycan, a molecular weight of 1096 was selected as the parent ion peak and further ion acceleration was performed in the LIFT cell (fragment mode). The fragmentation pattern of the reducing end GlcNAc residue was mainly observed.
It has been shown that by using the matrix composition according to the present invention, it is possible to directly analyze the glycan components of not only glycopeptides but also glycoproteins with larger molecular weights and complex structures without excising or modifying the glycans.

[Example 2]
The degradation pattern of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MSF using matrix composition 2 (DHBNa-aniline+DAN).
First, Na+ ion-added matrix composition 2 was prepared by increasing the volume of 0.5 M DHB (100 μL) in _CH3CN / _H2O (90:10, v/v), 1.0 M aniline solution (50 μL) in _CH3CN , 1.0 M 1,5-diaminonaphthalene (10 μL) in _CH3CN , and 0.1 M sodium bicarbonate in water (50 μL) to 1 mL with _CH3CN ^/ _H2O (50:50, v/v).
Matrix composition 2 is prepared by adding 1,5-diaminonaphthalene (DAN) to matrix composition 1 (DHBNa-aniline). When a matrix composition containing DAN was used, the sensitivity and resolution of direct glycan analysis were improved. This is thought to be because DAN promotes the fragmentation of glycans without impairing the sugar-selective ionization ability.

[Example 2-2]
The TOF/TOF degradation pattern from the analyte RNase B was evaluated in the LIFT-TOF/TOF MS mode using matrix composition 2 (DHBNa-aniline+DAN).
To focus on the PSD process of the glycan fragment peak consisting of ^2,4 A ₇ -type cross-ring cleavage of the reducing end GlcNAc residue of the RNase B glycan, a molecular weight of 1096 was selected as the parent ion peak and further ion acceleration was performed in the LIFT cell (fragment mode). The fragmentation pattern of the reducing end GlcNAc residue was mainly observed.
It was shown that by using the matrix composition according to the present invention, it is possible to directly analyze the glycan components of glycoproteins having large molecular weights and complex structures without excising or modifying the glycans.

[Example 3]
The degradation pattern of the analyte quail egg white (a glycoprotein mixture) was evaluated in reflectron mode MALDI-TOF MS using matrix composition 2 (DHBNa-aniline+DAN).
By using the matrix composition containing DNA according to the present invention, direct glycan analysis was achieved with high sensitivity and resolution.
It has been shown that by using the matrix composition according to the present invention, it is possible to directly analyze glycan components not only of purified glycoproteins but also of mixtures of glycoproteins, which are complex compositions, without excision or modification of the glycans.

[Example 4]
The degradation pattern of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using matrix composition 3 (CHANa-3AQ).
First, matrix composition 3 was prepared by increasing the volume of 0.5 M DHB (100 μL) in _CH3CN / _H2O (90:10, v/v), 1.0 M 3-aminoquinoline solution (60 μL) in _CH3CN , and 0.1 M sodium bicarbonate in water (50 μL) to 1 mL with _CH3CN / _H2O (50:50, v/v).
This matrix composition selectively ionized the glycan-derived fragments of the glycoprotein RNase B, enabling direct analysis of glycan components.

[Example 5]
The degradation pattern of the O157-derived lipopolysaccharide to be analyzed was evaluated in reflectron mode MALDI-TOF MS using matrix composition 1 (DHBNa-aniline).
When O157-derived lipopolysaccharide was analyzed using matrix composition 1 at a laser intensity of 80%, a glycan fragment cycle derived from the O-antigen tetrasaccharide, which is characteristic of O157, was observed, and no fragments derived from Lipid A or impurity peptides were generated.
It was shown that by using the matrix composition of the present invention, it is possible to directly analyze the repeating structures of glycan components not only of glycoproteins but also of lipopolysaccharides, a type of glycolipid whose glycan components have a large molecular weight and a complex structure, without excising or modifying the glycans.

[Example 5-2]
The internal structural pattern of the O-antigen repeating glycan sequence in the analyzed O157-derived lipopolysaccharide was evaluated in LIFT-TOF/TOF mode using matrix composition 1 (DHBNa-aniline). To focus on the PSD process of the O-antigen repeating glycan sequence unit in O157-derived lipopolysaccharide, a molecular weight of 721, which corresponds to the sodium adduct ion of the O157 antigen glycan sequence unit, was selected as the parent ion peak and further ion acceleration was performed in the LIFT cell (fragment mode), and the fragment pattern of the 4 sugar units that constitute the O157 antigen glycan was observed.

[Example 6]
The degradation pattern of the target O157-derived lipopolysaccharide was evaluated in reflectron mode MALDI-TOF MS using matrix composition 2 (DHBNa-aniline+DAN).
When O157-derived lipopolysaccharide was analyzed using matrix composition 2 at a laser intensity of 80%, a glycan fragment cycle derived from the O-antigen tetrasaccharide, which is characteristic of O157, was observed, and no fragments derived from Lipid A or impurity peptides were generated.
Matrix composition 2 is prepared by adding 1,5-diaminonaphthalene (DAN) to matrix composition 1 (DHBNa-aniline). When a matrix composition containing DAN was used, the sensitivity and resolution of direct glycan analysis were improved. This is thought to be because DAN promotes the fragmentation of glycans without impairing the sugar-selective ionization ability.

[Example 6-2]
The internal structural pattern of the O-antigen repeating glycan sequence in the analyzed O157-derived lipopolysaccharide was evaluated in the LIFT-TOF/TOF mode using matrix composition 2 (DHBNa-aniline + DAN).
To focus on the PSD process of the O-antigen repeating glycan sequence unit in O157-derived lipopolysaccharide, a molecular weight of 721, which corresponds to the sodium adduct ion of the O157 antigen glycan sequence unit, was selected as the parent ion peak and subjected to further ion acceleration in the LIFT cell (fragment mode) and analyzed, and the fragment pattern of the 4 sugar units that constitute the O157 antigen glycan was observed.

[Example 7]
The degradation pattern of the O157-derived lipopolysaccharide to be analyzed was evaluated in reflectron mode MALDI-TOF MS using matrix composition 4 (DHBNa-BOA).
When O157-derived lipopolysaccharide was analyzed using matrix composition 4 at a laser intensity of 80%, a glycan fragment cycle derived from the O-antigen tetrasaccharide, which is characteristic of O157, was observed, and no fragments derived from Lipid A or impurity peptides were generated.
Matrix composition 4 is a matrix composition in which O-benzylhydroxylamine (BOA) was added instead of aniline in matrix composition 1 (DHBNa-aniline). When a matrix composition containing BOA was used, the sensitivity and resolution of direct glycan analysis were improved. On the other hand, fragment patterns with relatively large molecular weights were observed, indicating that the fragmentation-promoting ability was reduced compared to aniline derivatives and DAN.

[Example 7-2]
The internal structural pattern of the O-antigen repeating glycan sequence in the analyzed O157-derived lipopolysaccharide was evaluated in the LIFT-TOF/TOF mode using matrix composition 4 (DHBNa-BOA).
To focus on the PSD process of the O-antigen repeating glycan sequence unit in O157-derived lipopolysaccharide, a molecular weight of 721, which corresponds to the sodium adduct ion of the O157 antigen glycan sequence unit, was selected as the parent ion peak and subjected to further ion acceleration in the LIFT cell (fragment mode) and analyzed, and the fragment pattern of the 4 sugar units that constitute the O157 antigen glycan was observed.

[Example 8]
In Examples 5 to 7, it was confirmed that the method of the present invention can detect lipopolysaccharide containing bacterial O antigens. Next, the internal structural pattern of the O-antigen repeating glycan sequence in the lipopolysaccharide on the bacterial surface of colonies of Escherichia coli, a gram-negative bacterium, was evaluated.
First, two different serotypes of E. coli strains, O6 (ATCC25922, serotype O6:K1) and a non-verotoxin-producing strain O157 (ATCC43888, serotype O157:H7), were cultured on agar media. The resulting colonies were collected into Eppendorf tubes using a platinum loop. The tubes containing the collected bacterial cells were washed by adding water, stirring, and centrifuging at 10,000G twice. A portion of the washed bacterial cells was suspended in 250 mM hydrochloric acid, left to stand at 90°C for 30 minutes, and then centrifuged at 20,000G. After drying 1 μL of the supernatant, matrix composition 5 (DHBNa-DAN) was added and measurement was performed using reflectron mode MALDI-TOF MS. The supernatant from the treatment of O6 (ATCC25922) cells showed the O6 antigen, and the supernatant from the treatment of O157 (ATCC43888) cells showed a fragment pattern indicating the O157 antigen.
According to the present invention, it is possible to easily cleave the KDO (2-keto-3-deoxyoctanoic acid) sequence present between Lipid A and O antigen of gram-negative bacteria by acid treatment and identify the sequence pattern of the excised O antigen. Based on the library of fragment patterns of O antigen for each bacterial cell, it is now possible to identify the type of bacterial cell and even its strain.

[Example 9]
The effect of the amount of added sodium on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using matrix composition 1 (DHBNa-aniline).
First, different volumes of 0.1 M aqueous sodium bicarbonate (0–50 μL) were added to a mixture of 0.5 M DHB (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v) and 1.0 M aniline solution (60 μL) in CH ₃ CN, and then the volume was increased to 1 mL to prepare matrix solutions related to matrix composition 1 with different amounts of Na ⁺ ions added.
When the glycoprotein RNase B was analyzed at 80% laser intensity using matrix solutions related to matrix composition 1 with different amounts of added Na ⁺ ions, the ion peaks derived from peptide fragments were suppressed with increasing amounts of added sodium, and were no longer detectable at 3% or more. On the other hand, the peak sensitivity derived from glycan components increased with increasing amounts of added sodium, and no change occurred in the peak pattern at sodium concentrations of 4% or more relative to DHB.

[Example 10]
The effect of the amount of added sodium on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using matrix composition 2 (DHBNa-aniline+DAN).
First, different volumes of 0.1 M aqueous sodium bicarbonate solution (0 to 50 μL) were added to a mixture of 0.5 M DHB (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), 1.0 M aniline solution (50 μL) in CH ₃ CN, and 1.0 M 1,5-diaminonaphthalene (10 μL) in CH ₃ CN, and then the volume was increased to 1 mL to prepare matrix solutions related to matrix composition 2 with different amounts of Na ⁺ ions added.
When the glycoprotein RNase B was analyzed using matrix solutions related to matrix composition 2 with different amounts of added Na ⁺ ions at a laser intensity of 80%, the glycan fragment pattern was clearly detected even in the case of no added sodium, and the peaks derived from peptide fragments were suppressed. When the amount of added sodium was 1% or more, only the glycan fragment pattern was observed, and no peptide fragments were generated. Furthermore, the sensitivity of the glycan fragment peak increased with increasing sodium addition, and no change in the peak pattern occurred at sodium concentrations of 2% or more relative to DHB.

[Example 11]
In reflectron mode MALDI-TOF MS using matrix composition 5 (DHBNa-DAN), the effect of the amount of DAN added on the spectral pattern derived from the degradation of the analyte RNase B was evaluated.
First, 0.1 M aqueous sodium bicarbonate (50 μL) and different volumes of 1.0 M 1,5-diaminonaphthalene (0 to 60 μL) in CH ₃ CN were added to 0.5 M DHB (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), and then the volume was increased to 1 mL to prepare matrix solutions related to matrix composition 5 with different amounts of DAN added.
When the glycoprotein RNase B was analyzed using matrix solutions related to matrix composition 5 with different amounts of DAN added at a laser intensity of 80%, only the glycan fragment pattern was observed with 20% DAN added, and no peptide fragments were generated. Furthermore, no change in the peak pattern occurred at DAN concentrations of 60% or less relative to DHB, but very weak peptide fragments were generated along with the glycan fragment pattern with 80% DAN added.

[Example 12]
The effect of the amount of added sodium on the spectral pattern resulting from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using matrix composition 6 (DHBNa) that does not contain an aromatic base component.
First, different volumes of 0.1 M aqueous sodium bicarbonate (0–50 μL) were added to 0.5 M DHB (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), and then the volume was increased to 1 mL to prepare matrix solutions related to matrix composition 6 with different amounts of Na ⁺ ions added.
When the glycoprotein RNase B was analyzed using matrix solutions related to matrix composition 6 with different amounts of Na ⁺ ions at a laser intensity of 80%, a glycan fragment pattern was generated along with very weak peptide fragments when sodium was added at 2%. When sodium was added at 4% or more, only the glycan fragment pattern was observed, and no peptide fragments were generated. Furthermore, the glycan fragment peak sensitivity increased with increasing sodium addition, and no change in the peak pattern occurred at sodium concentrations of 4% or more relative to DHB.

[Reference Example]
The spectral patterns derived from the degradation of the analyte RNase B were evaluated using 1,5-diaminonaphthalene (DAN) in reflectron mode MALDI-TOF MS.
The DAN matrix solution was prepared by adding different volumes of 0.1 M aqueous sodium bicarbonate (0 or 50 μL) to 1.0 M 1,5-diaminonaphthalene (50 μL) in CH ₃ CN, and then increasing the volume to 1 mL with CH ₃ CN/H ₂ O (50:50, v/v).
When the glycoprotein RNase B was analyzed using this DAN matrix solution at a laser intensity of 80%, no glycan fragment pattern was observed, and only peptide fragment patterns were generated. In particular, peptide fragments showing sequence patterns beyond cysteine, which could not be measured using DHB alone, were also observed due to the disulfide bond cleavage effect of DAN. Furthermore, the selective ionization ability of glycan fragments with the addition of Na, which was observed with the acidic solid matrix, was not observed with DAN alone.

[Example 13]
The effect of adding α-cyano-4-hydroxycinnamic acid (DAN) on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using a matrix composition containing α-cyano-4-hydroxycinnamic acid (CHCA), aniline, sodium, and DAN.
First, different volumes of 1.0 M 1,5-diaminonaphthalene solution in CH ₃ CN (0 or 10 μL) were added to a mixture of 0.5 M CHCA solution (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), 1.0 M aniline solution (50 μL) in CH ₃ CN, and 0.1 M aqueous sodium bicarbonate solution (50 μL), and then the volume was increased to 1 mL to prepare CHCA-containing matrix solutions with the combination of DAN addition.
When the glycoprotein RNase B was analyzed using each matrix solution at a laser intensity of 80%, only a weak glycan fragment pattern was observed without the addition of DAN, and no peptide fragments were generated, whereas only a strong glycan fragment pattern was observed with the addition of DAN.

[Example 14]
The effect of adding 2,4,6-trihydroxyacetophenone (THAP), aniline, sodium, and DAN on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using a matrix composition containing DAN.
First, different volumes of 1.0 M 1,5-diaminonaphthalene solution in CH ₃ CN (0 or 10 μL) were added to a mixture of 0.5 M THAP solution (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), 1.0 M aniline solution (50 μL) in CH ₃ CN, and 0.1 M aqueous sodium bicarbonate solution (50 μL), and then the volume was increased to 1 mL to prepare THAP-containing matrix solutions with the combination of DAN addition.
When the glycoprotein RNase B was analyzed using each matrix solution at a laser intensity of 80%, a strong glycan fragment pattern was generated along with weak peptide fragments in the absence of DAN, whereas only a strong glycan fragment pattern was observed in the presence of DAN.

[Example 15]
The effect of adding DAN on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using a matrix composition containing picolinic acid (PA), aniline, sodium, and DAN.
First, different volumes of 1.0 M 1,5-diaminonaphthalene in CH ₃ CN (0 or 10 μL) were added to a mixture of 0.5 M PA solution (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), 1.0 M aniline solution (50 μL) in CH ₃ CN, and 0.1 M aqueous sodium bicarbonate solution (50 μL), and then the volume was increased to 1 mL to prepare PA-containing matrix solutions with the combination of DAN addition.
When the glycoprotein RNase B was analyzed using each matrix solution at a laser intensity of 80%, only a weak glycan fragment pattern was observed without the addition of DAN, and no peptide fragments were generated, whereas with the addition of DAN, only a glycan fragment pattern was observed.

[Example 16]
The effect of adding methyl α-cyano-4-hydroxycinnamate (CNME), aniline, sodium, and DAN on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using a matrix composition containing DAN.
First, different volumes of 1.0 M 1,5-diaminonaphthalene in CH ₃ CN (0 or 10 μL) were added to a mixture of 0.5 M CNME solution (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), 1.0 M aniline solution (50 μL) in CH ₃ CN, and 0.1 M aqueous sodium bicarbonate solution (50 μL), and then the volume was increased to 1 mL to prepare CNME-containing matrix solutions with the combination of DAN addition.
When the glycoprotein RNase B was analyzed using each matrix solution at a laser intensity of 80%, only a weak glycan fragment pattern was observed without the addition of DAN, and no peptide fragments were generated, whereas with the addition of DAN, only a glycan fragment pattern was observed.

[Example 17]
The effect of adding DAN on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using a matrix composition containing 4-chloro-α-cyanocinnamic acid (Cl-CCA), aniline, sodium, and DAN.
First, different volumes of 1.0 M 1,5-diaminonaphthalene in CH ₃ CN (0 or 10 μL) were added to a mixture of 0.5 M Cl-CCA solution (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), 1.0 M aniline solution (50 μL) in CH ₃ CN, and 0.1 M aqueous sodium bicarbonate solution (50 μL), and then the volume was increased to 1 mL to prepare Cl-CCA-containing matrix solutions with the combination of DAN addition.
When the glycoprotein RNase B was analyzed using each matrix solution at a laser intensity of 80%, only a glycan fragment pattern was observed without the addition of DAN, and no peptide fragments were generated, whereas only a strong glycan fragment pattern was observed with the addition of DAN.

[Example 18]
The effect of adding DAN on the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using a matrix composition containing 4-bromo-α-cyanocinnamic acid (Br-CCA), aniline, sodium, and DAN.
First, different volumes of 1.0 M 1,5-diaminonaphthalene in CH ₃ CN (0 or 10 μL) were added to a mixture of 0.5 M Br-CCA solution (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v), 1.0 M aniline solution (50 μL) in CH ₃ CN, and 0.1 M aqueous sodium bicarbonate solution (50 μL), and then the volume was increased to 1 mL to prepare Br-CCA-containing matrix solutions with the combination of DAN addition.
When the glycoprotein RNase B was analyzed using each matrix solution at a laser intensity of 80%, only a glycan fragment pattern was observed without the addition of DAN, and no peptide fragments were generated, whereas only a strong glycan fragment pattern was observed with the addition of DAN.

[Example 19]
<Direct analysis of N-linked glycans>
The possibility of direct analysis of N-linked glycans in terms of the spectral pattern derived from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using matrix composition 7 containing 3-amino-4-hydroxybenzoic acid (AHBA) having a carboxyl group, a hydroxyl group, and an amino group in one molecule and sodium.
First, a Na + ion-added matrix composition was prepared by increasing the volume of 0.5 M AHBA (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v) and 0.1 M sodium bicarbonate in water (50 μL) to 1 mL with CH ₃ CN ^/ H ₂ O (50:50, v/v).
When the above matrix composition was used to analyze the glycoprotein RNase B at a laser intensity of 80%, a glycan fragment pattern consisting of ^2,4 A-type and ^0,2 A-type cross-ring cleavage of the reducing end GlcNAc residue was observed, and no peptide fragments were generated. The AHBA-Na matrix tended to have a lower glycan fragmentation ability than the alkali metal-added aniline derivative/DHB matrix and sodium-added DHB/DAN. Although AHBA has a low glycan fragmentation ability in the analysis of N-linked glycans on glycoproteins, it has a higher sensitivity in detecting N-linked glycan fragments than sodium-added DHB/aniline, which also has a low glycan fragmentation ability. This is expected to be due to the formation of intramolecular ion pairs (zwitterions) and improved crystallization ability, which gives it more stable solid ion matrix properties. In addition, as described in [Non-Patent Document 15], the occurrence of saturation peaks and peak widths were suppressed even at high laser intensity, and it is expected that noise in the low molecular weight region is less likely to occur, and ions of the analyte are efficiently detected.
It was confirmed that the use of matrix composition 7 according to the present invention enables direct analysis of N-linked glycans.

[Comparative Examples 1 to 4]
The effect of adding 3,5-diaminobenzoic acid (DABA), which has a carboxyl group and an amino group but no hydroxyl group in one molecule, on the spectral pattern resulting from the degradation of the analyte RNase B was evaluated in reflectron mode MALDI-TOF MS using a matrix composition containing DABA.
(a) Analysis was not possible in the system containing DABA alone. (b) In the system containing sodium, not only the spectrum of the glycan but also the spectrum of the peptide was observed. (c) In the system containing sodium and DAN, not only the spectrum of the glycan but also the spectrum of the peptide was observed. (d) In the system containing sodium and DHB, only the spectrum of the glycan was observed.

[Example 20]
<Direct analysis of O-linked glycans>
The possibility of direct analysis of O-linked glycans in the decomposition-derived spectral pattern of the target porcine stomach mucin (Mucin from porcine stomach, type II, Sigma-Aldrich M2378) was evaluated using reflectron mode MALDI-TOF MS with matrix composition 7 containing 3-amino-4-hydroxybenzoic acid (AHBA) with carboxyl, hydroxyl and amino groups in one molecule and sodium. O-linked glycans have a structure in which glycans are bound to the side chain hydroxyl groups of serine/threonine residues (Ser/Thr). In addition, since there is no enzyme that uniformly cleaves the binding site with peptides under mild conditions like N-linked glycans, the analysis of O-linked glycans generally involves cleaving the glycans by a β-elimination reaction in the Ser/Thr residues by base treatment, and then separating and analyzing the peptide fragments and glycan fragments. However, because the common core structure of O-linked glycans is smaller than that of N-linked glycans, it is generally difficult to detect small glycan fragments, and analysis of O-linked glycans has been more difficult than that of N-linked glycans. In fact, when porcine gastric mucin, a glycoprotein that is rich in O-linked glycans, was analyzed using DHB, the noise was high in the low molecular weight region, and it was difficult to systematically search for peaks characteristic of glycans even in the high molecular weight region. Therefore, we decided to analyze porcine gastric mucin using sodium-added AHBA.
First, a Na + ion-added matrix composition was prepared by increasing the volume of 0.5 M AHBA (100 μL) in CH ₃ CN/H ₂ O (90:10, v/v) and 0.1 M sodium bicarbonate in water (50 μL) to 1 mL with CH ₃ CN ^/ H ₂ O (50:50, v/v).
When the above matrix composition was used to analyze the glycoprotein porcine gastric mucin at a laser intensity of 80%, the noise in the low molecular weight region was reduced, and a glycan pattern with a GalNAc residue at the reducing end, which was generated through β-elimination from the serine or threonine residue to which the glycan is bound, was observed together with its dehydrated product, and no peptide fragments were generated. In addition, by comparing the signal corresponding to the observed glycan pattern with the background signal obtained from the spot to which only sodium-added AHBA was added, it was confirmed that the signal corresponding to the observed glycan pattern was derived from porcine gastric mucin.
It was confirmed that by using matrix composition 7 according to the present invention, it is possible to directly analyze O-linked glycans not only from glycoproteins having N-linked glycans such as those from RNase B, but also from mucin, which is composed of glycoproteins with molecular weights of several million, without the need for cleavage or modification of the glycans.
The fragment pattern observed here was in good agreement with the reported O-linked glycan pattern from porcine gastric mucin prepared by cleavage of the glycans with base treatment, labeling, and column purification [Non-patent Document 19].

[Discussion]
The generalities can be summarised as follows:
(1) In MALDI-TOF MS, increasing the intensity of the irradiated laser promotes the production of fragment ions.
(2) The addition of sodium or aromatic base molecules to the acidic solid matrix further promotes the production of fragment ions.
(3) DAN promotes fragmentation of the target sample.
(4) The addition of an acidic solid matrix plus sodium or an acidic solid matrix plus an aromatic base molecule increases the ionization efficiency of glycan fragments and decreases the ionization efficiency of peptide fragments. This effect is enhanced with an increase in the amount of sodium or aromatic base molecules added.
(5) The combination of an acidic solid matrix, sodium, and aromatic base molecules further improves the ionization efficiency of glycan fragments, reducing peptide fragments to undetectable levels.
(6) N-linked and O-linked glycans can be directly analyzed using an aromatic amphoteric molecule containing an acidic group, a hydroxyl group, and a basic group in one molecule plus sodium.

The matrix composition of the present invention is a matrix containing an acidic or weakly acidic solid matrix, a basic molecule selected from aromatic basic molecules and hydroxylamine-type basic molecules, the conjugate acid of which has a pKa of 4.0 to 5.0, and, if desired, a monovalent metal ion, preferably a fragmentation promoter (1,5-diaminonaphthalene), added thereto. This solid matrix composition is a mass spectrometry method characterized by selective ionization of glycan components in a fragment ion measurement method for high molecular weight analytes such as glycoproteins and glycolipids using a MALDI-TOF mass spectrometer, and has the main advantage of being able to analyze with high sensitivity and good reproducibility a large number of high molecular weight analytes (glycoproteins, lipid-bound polysaccharides, etc.) for which direct observation of glycan structures has been difficult in the past.
Laser-power-dependent in-source or post-source decay patterns of analytes also allow pseudo- ^MS3 strategies for detailed sequencing studies of the analytes.
That is, according to the present invention, the laser power-dependent pseudo- ^MS2 and ^MS3 strategies have achieved direct structural analysis of glycoconjugates with higher molecular weights and more complex structures, such as glycoproteins and lipid-bound glycans, without any modification, enabling the search for disease biomarkers. In addition, direct analysis of O-antigen glycans, which are used to classify Gram-negative bacteria such as E. coli, has been achieved, enabling the rapid diagnosis of infectious diseases.

Claims (17)

An amphoteric solid matrix having an aromatic hydroxyl group and a basic residue whose conjugate acid exhibits a pKa of 4.0 to 5.0 in one molecule; and a monovalent metal ion, wherein the amphoteric solid matrix is represented by the formula (1):

(wherein R ¹ to R ⁵ are each independently a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, a carboxyl group and/or an amino group, and any two of R ¹ to R ⁵ are each a hydroxyl group and an amino group), and the amount of the monovalent metal ion added is 1 to 50 mol % relative to 100 mol % of the amphoteric solid matrix. The solid matrix composition according to claim 1, wherein the monovalent metal ion is selected from the group consisting of lithium ions, sodium ions, potassium ions, rubidium ions, and cesium ions. The aminohydroxybenzoic acid has the following formula:

2. The solid matrix composition of claim 1, wherein the aminohydroxybenzoic acid is selected from the group consisting of compounds represented by the formula: The solid matrix composition according to claim 1, wherein the monovalent metal ion is selected from the group consisting of lithium ions, sodium ions, potassium ions, rubidium ions, and cesium ions. The present invention comprises an acidic solid matrix; a basic compound selected from an aromatic basic molecule and a hydroxylamine-type basic molecule, the conjugate acid of which exhibits a pKa of 4.0 to 5.0; and a monovalent metal ion,
The amount of the monovalent metal ion added is 1 to 50 mol % relative to 100 mol % of the acidic solid matrix.
Solid matrix compositions. The solid matrix composition according to claim 5, wherein the acidic solid matrix contains a carboxylic acid and exhibits a pKa of 1.0 to 6.0. The solid matrix composition according to claim 5, wherein the acidic solid matrix contains an aromatic hydroxyl group and exhibits a pKa of 6.0 to 10.0. The solid matrix composition according to claim 5, wherein the acidic solid matrix is selected from the group consisting of 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), CHCA derivatives 4-chloro-α-cyanocinnamic acid (Cl-CCA), 4-bromo-α-cyanocinnamic acid (Br-CCA), α-cyano-4-hydroxycinnamic acid methyl ester (CNME), 2,4,6-trihydroxyacetophenone (THAP), and picolinic acid (PA). The solid matrix composition according to claim 5, wherein the monovalent metal ion is selected from the group consisting of lithium ions, sodium ions, potassium ions, rubidium ions, and cesium ions. The solid matrix composition according to claim 5, wherein the basic compound whose conjugate acid exhibits a pKa of 4.0 to 5.0 is selected from the group consisting of aniline, N-methylaniline (NMA), N,N-dimethylaniline (DMA) and O-benzylhydroxylamine (BOA). The solid matrix composition according to claim 5, wherein the amount of the basic compound whose conjugate acid has a pKa of 4.0 to 5.0 is 10 to 150 mol % relative to 100 mol % of the acidic solid matrix. The solid matrix composition according to claim 5 further comprises 1 to 100 mol % of 1,5-diaminonaphthalene as a fragmentation promoter relative to 100 mol % of the acidic solid matrix. A matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry method for glycoconjugates, characterized by selective ionization of glycan components of glycoproteins or glycolipids, using a solid matrix composition according to any one of claims 1 to 12. The mass spectrometry method according to claim 13, in which in-source decay and post-source decay products are measured in reflectron mode. The mass spectrometry method according to claim 14, which utilizes the selective ionization ability of glycan-derived fragments. The mass spectrometry method according to claim 13, which utilizes the laser output-dependent fragmentation tendency and the ability to selectively ionize glycan-derived fragments in MALDI-TOF/TOFMS analysis to perform pseudo-MS/MS/MS analysis. A measurement plate for matrix-assisted laser desorption/ionization mass spectrometry, at least a portion of which is formed with a matrix layer made of the solid matrix composition according to any one of claims 1 to 12.

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