JP2008051790A - Method for trace mass analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for mass analysis that reduces the quantity of the molecules to be analyzed and a reagent, that is easy to prepare the samples for high-sensitivity mass analysis/measurements, obtaining reliable information regarding chemical structure, and by improving the ionization efficiency and the measurement reproducibility, in particular, when it is applied to a trace amount of molecule, such as glycoprotein, that is derived from a living body sample or is located within the biological sample, and obtaining information useful for clarifying the functions and clinical conditions. <P>SOLUTION: The mass analysis method of molecules has a process (1) for placing the analysis molecules and a derivatization agent on a sample support member used for mass analysis method; a process (2) for making the analysis molecule and the derivatization agent react on the sample support member; a process (3) for supplying the derivative of the analyzed molecule, generated by a reaction on the sample support member, to a mass spectrometer; and a process (4) for ionizing the derivative of the analysis molecule. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to molecular mass spectrometry.

  “Mass spectrometry” is a method for obtaining chemical structure information by ionizing a sample containing molecules, separating the ionized molecules according to mass / charge (m / z), and detecting them.

Molecules such as sugar chains, proteins (including peptides), glycoproteins (including glycopeptides), nucleic acids, glycolipids, etc. can be used for matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry ( Structural information is obtained and identified by ESI-MS). In these molecules, whether or not generation of molecular ions ([M + Na] ⁺ , [M + H] ⁺ , [M−H] ^−, etc.) efficiently occurs greatly affects the detection sensitivity. In particular, since there are a plurality of isomers having the same molecular weight and composition in the sugar chain, MS ⁿ analysis that repeatedly selects and fragments molecular ions as precursor ions and further selects and fragments the generated ions as precursor ions is performed. It is essential.

  In general, the signal intensity of ions obtained by MS / MS measurement is reduced to 1/10 of the previous stage ions. Therefore, by performing multi-step MS measurement, the structure information becomes extremely large, but the detection sensitivity decreases, so it is necessary that the initial molecular ion generation amount is sufficiently large.

  However, since the molecules derived from biological samples as described above are not easily ionized as they are, and molecules are easily destroyed, it is difficult to obtain a sufficient amount of molecular ions. For example, if an attempt is made to analyze a sugar chain by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) as it is, 10 pmol or more is required. To solve this problem, a method of derivatizing a sugar chain using 4-aminobenzoic acid ethyl ester, 2-aminopyridine, 1-pyrenebutanoic acid hydrazide (PBH), or the like has been proposed. (For example, refer nonpatent literature 1).

However, these derivatizations are performed prior to mass spectrometry, but include many steps of reaction operations and excessive reagent removal, which takes time and labor, and in addition, molecules derived from ultra-small biological samples. It was difficult to apply to the current situation.
Sugahara, D. et al., Anal. Sci., 19, 167-169 (2003)

  The present invention has been made in view of the above-mentioned background art, and its problem is that the amount of molecules to be analyzed and the amount of reaction reagents can be reduced, and a sample for high-sensitivity mass spectrometry measurement can be easily prepared. It is to provide a mass spectrometry method capable of obtaining information on a highly reliable chemical structure by improving the reproducibility of measurement, and in particular, a molecule derived from a trace biological sample such as glycoprotein, or An object of the present invention is to provide a mass spectrometry method that can be applied to molecules in a biological sample to obtain information useful for elucidating the function and pathophysiology.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor conducted a reaction on a sample support member used for direct mass spectrometry, so that the sample can be easily and efficiently stabilized without reducing the amount of the sample. The present inventors have completed the present invention by creating a derivative, realizing improved ionization efficiency of molecules that have been difficult to ionize and stabilizing the generated ions, and improving the sensitivity of mass spectrometry. .

That is, the present invention is a molecular mass spectrometry method, and includes at least steps (1), (2), (3) and (4).
(1) placing the molecule to be analyzed and the derivatizing agent on a sample support member used for mass spectrometry;
(2) reacting the molecule to be analyzed and the derivatizing agent on the sample support member;
(3) a step of subjecting a derivative of a molecule to be analyzed generated on reaction on a sample support member to a mass spectrometer;
(4) To provide a mass spectrometric method characterized by having a step of ionizing a derivative of a molecule to be analyzed.

  Moreover, this invention provides the derivatizing agent characterized by using for the said mass spectrometry.

  According to the present invention, by reacting a reaction reagent with a molecule on the sample support member, the amount of the molecule to be used and the amount of the reaction reagent can be reduced (high yield can be achieved), and the treatment process can be performed under mild conditions. Thus, a sufficient amount of molecular ions can be generated without reducing the amount of measurement molecules, and a sample for high-sensitivity mass spectrometry can be easily prepared. As a result, in a normal processing step, mass spectrometry can be performed even when only a very small amount of sample that can be analyzed or less is obtained. As a result, ionization efficiency can be improved, cutting ionization position can be controlled, and product ions can be stabilized, thereby improving measurement reproducibility and obtaining highly reliable chemical structure information. be able to. In addition, since the present invention can be applied to a small amount of molecules derived from biological samples or molecules in biological samples, for example, sugar chains, proteins (including peptides), glycoproteins (including glycopeptides), nucleic acids, glycolipids, etc. It is possible to provide a method for obtaining useful information for elucidation of molecular functions and pathological conditions.

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified and implemented.

  In the present invention, a molecule to be analyzed and a reaction reagent such as a derivatizing agent are directly reacted on a sample support member used for a mass spectrometer, and the molecule generated by the reaction is analyzed by mass spectrometry.

1. Molecules to be analyzed (1) Types of molecules to be analyzed “Moleculars to be analyzed” are not particularly limited, but are sugars, sugar chains, proteins, nucleic acids, complex carbohydrates (glycoproteins, glycolipids, etc.), etc. Is preferable because the effects of the present invention can be exhibited. “Molecules to be analyzed” include those prepared from natural products, those prepared by partially modifying natural products chemically or enzymatically, and those prepared chemically or enzymatically. preferable. Moreover, what has the partial structure of the molecule | numerator contained in the biological body, and the thing produced by imitating the molecule | numerator contained in the biological body are also preferable. In addition, the sample placed on the sample support member used for mass spectrometry, that is, the object containing “molecules to be analyzed” may be only “molecules to be analyzed” itself or those containing “molecules to be analyzed”. For example, living tissue, cells, body fluids and secretions (for example, blood, serum, urine, semen, saliva, tears, sweat, feces, etc.) may be used. That is, a biological sample may be used directly. Alternatively, a molecule to be analyzed may be prepared by placing a sample on a sample support member and performing an enzyme treatment or the like. The term “protein” in the present invention includes a peptide, and “glycoprotein” includes a glycopeptide.

  In many cases, the above-mentioned molecules are subjected to a small amount of sample to be analyzed, and in particular, sugars, sugar chains, complex carbohydrates such as glycoproteins and glycolipids, or the like are chemically or enzymatically released from them. Since there are a plurality of isomers having the same molecular weight and composition, the mass spectrometry of the present invention is suitable for analyzing the chemical structure of these molecules. In particular, it is preferable because the above-described effects are exhibited.

(2) Functional group possessed by the molecule to be analyzed In the present invention, the molecule to be analyzed is reacted with a derivatizing agent on a sample support member to form a compound suitable for mass spectrometry and used in a mass spectrometer. . The functional group possessed by the molecule to be analyzed is not particularly limited as long as it reacts easily with the derivatizing agent and the reaction product does not adversely affect the mass spectrometry, but the aldehyde group, carboxyl A group, amino group, mercapto group or hydroxyl group is preferred. That is, the molecule analyzed in the present invention preferably has one or more functional groups selected from the group consisting of an aldehyde group, a carboxyl group, an amino group, a mercapto group, and a hydroxyl group.

Hereinafter, the “molecule to be analyzed” in the present invention will be described in detail for each of the preferred functional groups.
(A) "Analyzed molecule" having an aldehyde group
A sugar chain having an adjacent diol such as a non-reducing end or a hexose having a substituent only at the C ₂ , C _4, or C ₆ position produces an aldehyde group by periodate oxidation. As preferred. In particular, a sugar chain containing sialic acid can selectively oxidize only the C _7-9 position of sialic acid with periodic acid to generate an aldehyde group. The selective oxidation of the C _7-9 position of sialic acid is performed, for example, by reacting in 5 mM NaIO ₄ aqueous solution at 0 ° C. for 10 to 20 minutes. Such “molecules to be analyzed” having an aldehyde group derived from sialic acid react with a derivatizing agent having an amino group, a hydrazide group, etc., as described later, to be derivatized for mass spectrometry. Since the obtained molecule can be easily obtained by reacting on the sample support member, it is preferable as the molecule analyzed by the mass spectrometry of the present invention.

A sugar chain containing galactose at the non-reducing end is also preferred. This is because the galactose at the non-reducing end can be oxidized specifically at the C ₆ position with galactose oxidase to generate an aldehyde group. The enzyme reaction can be performed, for example, in a neutral buffer solution at room temperature for 2 hours. Using a derivatizing agent having an amino group, a hydrazide group, etc., the aldehyde group generated by the above enzymatic oxidation and the amino group or hydrazide group are subjected to a condensation reaction on the sample support member, and then reduced if necessary. By this, the derivatized molecule | numerator used for mass spectrometry can be made to react suitably on a sample support member, and can be obtained suitably.

Although the reduction performed as necessary is not particularly limited, it is preferable to add a reducing agent such as NaCNBH ₃ , NaBH ₄ , NaBH (OCOCH ₃ ) ₃ and perform the reaction at warming or room temperature. It is also possible to have a reducing agent coexist in the reaction solution of the “molecule to be analyzed” and the derivatizing agent from the beginning. Reduction has the effect of stabilizing the derivatized molecule.

For example, a chemical reaction in which a “molecule to be analyzed” having an aldehyde group is reacted with, for example, a derivatizing agent having a hydrazide group is represented by the following formula (1).
R ¹ CONHNH ₂ + R ² CHO → R ¹ CONHN═CHR ² (1)
[In formula, R < ¹ >, R < ² > shows arbitrary organic groups mutually independently. ]

In the reduction, for example, in the above case, the chemical reaction is represented by the following formula (2).
R ¹ CONHN═CHR ² → R ¹ CONHNH—CH ₂ R ² (2)
[In formula, R < ¹ >, R < ² > shows arbitrary organic groups mutually independently. ]

  The above method of labeling sialic acid or galactose can also be applied to sialic acid or galactose on a glycoprotein molecule. In that case, since the glycoprotein molecule can be labeled, the ionization efficiency of the molecule becomes high, and mass spectrometry becomes particularly sensitive, which is preferable. In addition, since the present invention labels sugar chains on the molecule without releasing the sugar chains, tandem mass spectrometry spectrum analysis of proteins containing sugar chains can be performed, and the presence or absence of sugar chain structures on peptide chains of proteins and The sugar chain binding position can be specified.

  In elucidating the function of a glycoprotein, it is extremely important to clarify what position and structure the sugar chain is added to on the peptide chain. In many cases, since a plurality of sugar chains having different structures are bonded to a plurality of binding positions of the peptide chain, it is necessary to clarify the correspondence between the position of the sugar chain and the structure. The present invention makes it possible to obtain sugar chain structure information of a labeled sugar chain moiety. Therefore, glycoproteins are particularly preferred as the “molecule to be analyzed” in the present invention.

(B) “Analyzed molecule” having a carboxyl group, amino group, mercapto group, etc.
For example, proteins and glycoproteins can be reacted with a derivatizing agent using a carboxyl group, amino group, mercapto group or the like contained therein. When reacting using the carboxyl group contained in the “molecule to be analyzed”, reacting them with a derivatizing agent having an amino group, a hydrazide group, a diazomethyl group, etc. on the sample support member A compound to be subjected to mass spectrometry can be suitably obtained. Also, the carboxyl group in the “molecule to be analyzed” reacts with methyl iodide or trimethylsilyldiazomethane, which are derivatizing agents, to become a methyl ester, which easily supports the derivatized molecule for mass spectrometry. It can be obtained by reaction on the member. If necessary, a dehydrating condensing agent can be added and reacted. In the case of a molecule having sialic acid, the carboxyl group of sialic acid may be used.

  In addition, when the amino group contained in the “molecule to be analyzed” is used for the reaction, it is used for mass spectrometry by reacting a derivatizing agent having a succinimidyl ester group, a sulfonyl chloride group or the like. The derivatized molecule can be easily and suitably obtained by reaction on the sample support member.

  In addition, for example, when a reaction is performed using a mercapto group such as a cysteine residue contained in a protein or glycoprotein, it is subjected to mass spectrometry by reacting a derivatizing agent having an iodo group (-I) or the like. The derivatized molecule can be suitably obtained on the sample support member.

2. Derivatizing agent The “molecule to be analyzed” having the above functional group obtains a molecule to be subjected to mass spectrometry by reacting the derivatizing agent. The derivatizing agent is not particularly limited, but is preferably one that enhances the ionization efficiency of the derivatized molecule, that is, the molecule subjected to mass spectrometry. The ionization efficiency may be increased in the laser desorption ionization method, or the ionization efficiency may be increased in the electrospray ionization method. A compound having an effect as a matrix molecule in mass spectrometry, or a compound further having a reactive functional group or a spacer portion described later is also preferable.

  The chemical structure of such a derivatizing agent is not particularly limited as long as it has the above effects, but a condensed polycyclic derivative compound having a condensed polycycle such as naphthalene, anthracene, and pyrene in the molecule preferably has the above effects. This is particularly preferable. Here, the “fused polycyclic derivative compound” is capable of binding to the “analyzed molecule” with a condensed polycyclic moiety that may partially contain a heterocyclic ring containing nitrogen, sulfur or oxygen molecules. The compound which has a certain reactive functional group and the spacer part which connects this condensed polycyclic part and this reactive functional group as needed. In particular, a compound having an aromatic ring is more preferable.

  The derivatizing agent preferably reacts with the molecule to be analyzed so that the ionized cleavage position of the derivatized molecule, ie the molecule subjected to mass spectrometry, can be controlled.

  As the derivatizing agent, those having the functional group described in the section “Analyzed molecule” are preferably used. That is, a derivatizing agent having an amino group, a hydrazide group, a diazomethyl group, a succinimidyl ester group, a sulfonyl chloride group, an iodo group (—I) or the like is preferable. As a particularly preferred derivatizing agent, specifically, a condensed polycyclic derivative compound in which the above group is bonded to a condensed polycycle such as a naphthalene ring, an anthracene ring, or a pyrene ring directly or through another group (spacer portion) Methyl iodide; diazomethane; trimethylsilyldiazomethane and the like.

  Among these, a pyrene derivative compound is particularly preferable in terms of increasing ionization efficiency of a derivatized molecule, that is, a molecule subjected to mass spectrometry, and controlling an ionization cleavage position. Here, the “pyrene derivative compound” means a pyrene ring, a reactive functional group capable of binding to the “molecule to be analyzed”, and, if necessary, the pyrene ring and the reactive functional group. A compound having a spacer portion.

  Specifically, 1-pyrenebutanoic acid, hydrazide (hereinafter abbreviated as “PBH”), 1-pyreneacetic acid, hydrazide, 1-pyrenepropionic acid hydrazide ( 1-pyrenepropionic acid, hydrazide), 1-pyreneacetic acid, succinimidyl ester, 1-pyrenepropionic acid, succinimidyl ester, 1-pyrenepropionic acid, succinimidyl ester Sinimidyl ester (1-pyrenebutanoic acid, succinimidyl ester), N- (1-pyrenebutanoyl) cysteic acid succinimidyl ester (N- (1-pyrenebutanoyl) cysteic acid, succinimidyl ester), N- (1-pyrene) iodoacetamide (N- (1-pyrene) iodoacetamide), N- (1-pyrene) iodomaleimide (N- (1-pyrene) maleimide), N- (1-pyrenemethyl) Iodoacetamide (N- (1-pyrenemethyl) iodoacetamide), 1-pyrenemethyl iodoacetate, aminopyrene, 1-pyrenemethylamine, 1-pyrenepropylamine (3 -(1-pyrenyl) propylamine), 1-pyrenebutylamine (4- (1-pyrenyl) butylamine), 1-pyrenesulfonyl chloride, 1-pyrenyldiazomethane (hereinafter, Preferred examples include 1-pyrenecarbaldehyde hydrazone, 1-pyrenylthiocyanate, 1-pyrenylisothiocyanate and the like. . Of these, PBH or PDAM is most preferred.

  Preferred examples of the derivatizing agent include those obtained by replacing the pyrene ring with a naphthalene ring or an anthracene ring in the above specific compound. Also preferred are methyl iodide, diazomethane or trimethylsilyldiazomethane.

3. Combination of molecule to be analyzed and derivatizing agent As a preferable combination of “molecule to be analyzed” and derivatizing agent, “molecule to be analyzed” is a molecule having a sugar chain containing an aldehyde group. May have an amino group or a hydrazide group. Further, as a preferable combination, the “molecule to be analyzed” is a protein or glycoprotein having a carboxyl group, an amino group or a mercapto group, and the derivatizing agent has an amino group, a hydrazide group, a diazomethyl group or the like. In some cases, the “molecule to be analyzed” is a protein or glycoprotein having a carboxyl group, and the derivatizing agent is methyl iodide or trimethylsilyldiazomethane. These combinations make it possible for the “molecules to be analyzed” and the derivatizing agent to easily react on the sample support member, to not inhibit ionization, to be selective in reaction, and generally to be analyzed in trace amounts. This is preferable because the above effect is easily achieved.

4). Step The present invention includes at least steps (1), (2), (3) and (4).
(1) placing the molecule to be analyzed and the derivatizing agent on a sample support member used for mass spectrometry;
(2) reacting the molecule to be analyzed and the derivatizing agent on the sample support member;
(3) A step of subjecting a derivative of a molecule to be analyzed generated by reaction on a sample support member to a mass spectrometer;
(4) A molecular mass spectrometry method comprising a step of ionizing a derivative of a molecule to be analyzed. Below, it describes about each process.

(1) Step of placing molecule to be analyzed and derivatizing agent on sample support member used for mass spectrometry It is preferable to place “molecule to be analyzed” in a state dissolved or dispersed in a solvent. Particularly preferably, it is better to dissolve it so that it can be placed uniformly. The amount of “molecules to be analyzed” placed on the sample support member is not particularly limited, but 1 amol to 100 pmol is preferable for achieving the effect of the present invention, and 1 fmol to 1 pmol is particularly preferable. The amount of the “analyzed molecule” solution placed on the sample support member is not particularly limited, but is preferably 10 pL to 50 μL as the whole solution or the whole dispersion, and particularly preferably 10 nL to 500 nL. The solvent is not particularly limited, and examples thereof include water, ethanol, dimethyl sulfoxide (hereinafter abbreviated as “DMSO”), and a mixed solution thereof. When the solvent in which the “molecule to be analyzed” is dissolved or dispersed is different from the solvent in which the derivatizing agent is dissolved, it is preferable that the derivatizing agent is loaded after drying. In addition, those containing “molecules to be analyzed” such as biological samples such as biological tissues, cells, body fluids and secretions (blood, urine, saliva, tears, etc.) are used for “mass spectrometry. It can also be placed directly on the `` sample support member '', but in that case, the tissue is made into ultrathin sections, the cells are placed in a dispersed state, the body fluid and the secretion fluid are applied, and dried as necessary It is preferable to fix it with methanol or ethanol.

  The derivatizing agent is placed in a state dissolved or dispersed in a solvent. Preferably, it is better to dissolve in a solvent in that the minimum amount can be uniformly loaded. The amount of the derivatizing agent placed on the sample support member is not particularly limited, but is preferably 1 fmol to 10 nmol, particularly preferably 10 pmol to 1 nmol. The amount of the liquid is not particularly limited, but 1 nL to 5 μL is preferable as the whole solution or the whole dispersion, and 10 nL to 500 nL is particularly preferable. Although there is no limitation in particular as a solvent, DMSO, isopropanol, n-butanol etc. are preferable at the point of the solubility and volatility of a derivatizing agent and the molecule | numerator to be analyzed. Particularly preferred is DMSO.

  The derivatizing agent may be placed on the sample support member first, and the derivatizing agent is dissolved in a highly volatile solvent such as methanol or ethanol, and after the sample is placed on the sample support member, the solvent is volatilized and then DMSO etc. It is preferred to load the molecule to be analyzed dissolved in the. In addition, when the molecule to be analyzed and the derivatizing agent are dissolved in a solvent such as DMSO, either may be placed on the sample support member first, and may be dried and / or reacted each time it is placed. Alternatively, the following may be placed without substantial reaction, followed by drying and / or reaction. Further, when the molecule to be analyzed and the derivatizing agent are dissolved in DMSO or the like, a mixture of both may be placed on the sample support member. 1 type may be used for a derivatizing agent and it may use 2 or more types. Further, when two or more derivatizing agents are used, for example, after the molecules to be analyzed are placed on the sample support member and dried, the derivatizing agent A is placed and dried and / or reacted, and then the derivatizing agent is used. It is also preferable that B is loaded and dried and / or reacted.

  For example, conventionally, the chemical reaction represented by the chemical reaction formula (1) is usually in an organic solvent such as methanol and DMSO (10 μL to 100 μL as a solution), and in the presence or absence of acetic acid as a catalyst. Below, for example, the sugar chain and a large excess of the reaction reagent were heated (for example, 80 ° C.). However, in the present invention, the reaction is performed on the sample support member in DMSO (50 nL to 500 nL). For example, it was possible to easily realize a very small amount of reaction system in which a sugar chain (1 fmol to 500 fmol) and a derivatizing agent (1 pmol to 100 pmol) were reacted to obtain a high yield. In the parentheses, the most preferred amounts of use when the sugar chain and the derivatizing agent are reacted using the chemical reaction formula (1) are shown.

  In addition, if necessary, after reacting only the “molecule to be analyzed” and the derivatizing agent, a reducing agent or the like is further placed on the sample support member in a state dissolved or dispersed in a solvent. It is also preferable to mix the reducing agent with the derivatizing agent and place it on the sample support member because the operation is completed in one step. The amount of the reducing agent or the like placed on the sample support member is not particularly limited, but 1 pmol to 1 nmol is preferable, and 10 pmol to 500 pmol is particularly preferable in that ionization is not inhibited. The amount of the liquid is not particularly limited, but 10 pL to 50 μL is preferable as the whole solution or the whole dispersion, and 10 nL to 500 nL is particularly preferable.

(2) Step of reacting molecule to be analyzed and derivatizing agent on sample support member The reaction is preferably performed by maintaining the sample support member at -30 ° C to 100 ° C. The reaction temperature is particularly preferably 20 ° C to 80 ° C, more preferably 40 ° C to 60 ° C. The reaction time may be selected in an optimum range for each reaction type and / or for each solvent type, but the time for the solvent to dry is preferred. Usually, it is 1 minute to 24 hours, preferably 2 minutes to 20 minutes.

  If the solvent remains after completion of the reaction, the solvent is removed. When using a vacuum desiccator or the like, it is preferably maintained at room temperature for about 10 minutes to 24 hours.

  When the laser desorption ionization method is used, matrix molecules may coexist. After completion of the reaction, the solvent on the sample support member is removed, and then a matrix molecule or a solution of the matrix molecule is placed and MALDI-MS measurement is performed. The matrix molecule is not particularly limited, and known ones can be used. 2,5-dihydroxybenzoic acid (hereinafter abbreviated as “DHBA”), 1,5-diaminonaphthalene, nor-Harman (9H-pyrido [3,4-b] indole) and the like.

  In addition, the derivatizing agent is also preferable as the matrix molecule. Furthermore, a by-product obtained by reacting the derivatizing agent with a molecule other than the “molecule to be analyzed” in the step of reacting the derivatizing agent on a sample support member is also preferable. Examples of such by-products include PBH by-products when PBH is used as a derivatizing agent; and PDAM by-products such as 1-pyrenylmethyl alcohol when PDAM is used as a derivatizing agent.

  As the matrix molecule, a pyrene derivative or the like is also preferable. Of the pyrene derivatives, PBH, PDAM, 1-pyrenylmethyl alcohol and the like are particularly preferable. When PBH, PDAM or the like is used as the derivatizing agent, only unreacted PBH, PDAM or the like may be used, but a derivatizing agent such as PBH or PDAM may be newly added after the reaction. The matrix molecule may be used alone or two or more kinds of compounds may be used. For “two or more types”, for example, a combination of a pyrene derivative (as an unreacted product, an added product after reaction and / or a byproduct) and a known matrix molecule such as DHBA, nor-Harman is also preferable.

  The amount of the matrix molecule placed on the sample support member is not particularly limited, but the matrix molecule is preferably 0.1 μg to 20 μg, particularly preferably 0.2 μg to 1 μg. The solvent is not particularly limited, but ethanol-water, acetonitrile-water mixtures and the like are preferable because the molecules to be analyzed and the matrix are well mixed. Particularly preferred is a mixed solution of acetonitrile and water.

  The “sample support member” is not particularly limited as long as it has a surface that provides a field where molecules can react and can substantially be subjected to mass spectrometry, but is preferably a target plate formed of stainless steel or the like. These are sample tubes dedicated to supplying analytical samples such as sample plates and autosampler sample tubes to the analyzer, microwell plates, nitrocellulose membranes, polyvinylidene fluoride (PVDF) membranes, and the like. Particularly preferred is a sample plate for mass spectrometry. The sample plate may be coated with gold or may be chemically or physically treated.

  In the present invention, the expression “put on the sample support member” also means to put on the inner wall of the sample tube, for example. That is, the “sample support member” includes not only plates and dishes, but also containers, and “put on top” is placed in a container, that is, placed in a container. Is also included.

  When the present invention is not carried out, extraction with an organic solvent-water mixture or separation operation with a column such as C18 is necessary to remove excess derivatizing agent, etc. after the reaction. When these operations are performed, the amount may be reduced for each process, and it may be difficult to finally detect. However, according to the present invention, there is no loss of the sample by performing the reaction directly on the sample support member, and it is not necessary to use an excessive amount of the reaction reagent because the reaction solution is the minimum amount. Therefore, in mass spectrometry of sugar chains and the like that are difficult to ionize as they are, a large number of molecular ions can be generated easily and efficiently, and as a result, sufficient sensitivity can be obtained.

  It can be imagined that the reaction occurring in the test tube or flask also occurs on the sample support member. However, in the past, it was considered that the reaction was inadequate due to the inability to stir or drying without a lid, and therefore it was not usually considered to react on the sample support member. In addition, since the unreacted derivatizing agent may inhibit ionization, it was thought that it was necessary to increase the reaction yield or to perform purification, and thus it is considered that the present invention could not be conceived.

  The present invention includes (b) reacting a derivatizing agent with (a) analyzing a trace substance (for example, analyzing a sugar chain derived from a trace amount of biological sample or a sugar chain in a trace amount of biological sample), (c ) The reaction is allowed to proceed on the sample support member, but by combining them, the sample used for the reaction can be used as a molecule to be analyzed substantially, and unreacted derivatization Since the agent and its by-products do not inhibit ionization, a synergistic effect is recognized only when (a), (b) and (c) are combined.

  Furthermore, unreacted derivatizing agents and by-products formed by the reaction of derivatizing agents with molecules other than “molecules to be analyzed” not only inhibit ionization but also promote (act as a matrix). For this reason, the present invention is considered to have achieved the above effect. In the present invention, step (2) is an indispensable step, but the present invention can be used only when the reaction product generated by the reaction of the derivatizing agent and the “molecule to be analyzed” promotes ionization. It is not limited.

(3) Step of providing a derivative of a molecule to be analyzed generated by reaction on a sample support member to a mass spectrometer Any known mass spectrometer can be used for the mass spectrometry of the present invention. When measuring by laser desorption ionization, the sample support member from which the solvent has been removed can be directly used in a mass spectrometer after completion of the reaction. In the case of measuring by the MALDI method, after completion of the reaction, the matrix can be placed on the sample support member from which the solvent has been removed, and then used as it is for a mass spectrometer. When measuring by the ESI method, the molecule to be analyzed on the sample support member is dissolved in an ammonium acetate aqueous solution-acetonitrile mixed solution or the like, and injected into the liquid chromatograph mass spectrometer through flow injection or a column. In this case, the sample support member is preferably a sample tube used for an autosampler.

(4) Step of ionizing molecular derivative to be analyzed There is no particular limitation on the ionization method, and electrospray ionization (ESI) method including nanoelectrospray ionization (nanoESI) method, atmospheric pressure ionization (APCI) method, electron FAB method in which neutral ion beam generated by applying argon or xenon ionized by impact to neutral atom is applied to the sample, photo spray method in which ultraviolet rays are applied to the sprayed sample solution, ionization by heating and jet spraying Examples thereof include a thermospray method using charging, a cold spray method in which spraying is performed without heating, a laser desorption ionization (LDI) method, and a matrix-assisted laser desorption ionization (MALDI) method.

  Among these, in the present invention, the electrospray ionization (ESI) method including the nanoelectrospray ionization (nanoESI) method is preferable in that the molecules to be directly analyzed are sucked and injected into the mass spectrometer. Laser desorption ionization (LDI) is a derivative produced by the reaction of a molecule to be analyzed and a derivatizing agent on a sample support member, possibly with an unreacted derivatizing agent or by-product of the derivatizing agent, This is preferable in that the ionization efficiency can be improved, the cutting ionization position can be controlled, and the product ions can be stabilized. It is difficult to ionize macromolecules derived from biological samples such as sugar chains, proteins, glycoproteins, nucleic acids, glycolipids, etc., but the molecules to be analyzed and the derivatizing agent react on the sample support member to generate derivatives. This can further double the ionization efficiency of the matrix, possibly with unreacted derivatizing agents and by-products of the derivatizing agent, and substantially all the molecules to be analyzed placed on the sample support member in the mass spectrometer. The MALDI method is particularly preferable because it can be ionized and the polymer can be suitably ionized monovalently.

  Lasers used in laser desorption / ionization (LDI) and matrix-assisted laser desorption / ionization (MALDI) include nitrogen laser (337 nm), YAG laser triple wave (355 nm), NdYAG laser (256 nm), carbon dioxide gas laser (2940 nm) and the like, and a nitrogen laser is preferable. Further, when a condensed polycyclic derivative compound such as a pyrene derivative compound is used as a derivatizing agent, a nitrogen laser is particularly preferable. In the present invention, the molecule to be analyzed and the derivatizing agent react on the sample support member to become a derivative, or the unreacted derivatizing agent and the by-product of the derivatizing agent coexist, thereby reducing the laser intensity. It can be reduced to 20% to 80%. The reduction in laser intensity has the effect of improving the resolution.

  Next, ionization was performed using a double focusing method, a quadrupole focusing method (quadrupole (Q) filter method), a tandem quadrupole (QQ) method, an ion trap method, a time of flight (TOF) method, or the like. Molecules are separated and detected according to mass / charge ratio (m / z). QIT-TOF is preferable.

Since molecules such as sugar chains, proteins, glycoproteins, nucleic acids, and glycolipids contain many isomers having the same molecular weight and composition, derivatization is performed to improve ion generation efficiency, and molecular fragmentation is repeated n times ( The MS ⁿ ) method is preferred. In the present invention, a derivatizing agent is reacted and labeled on a sample support member, and an ion containing the selected label is analyzed by MS ⁿ , whereby a binding position or the like in the molecule can be determined. In the present invention, MS ² (MS / MS), MS ³ or MS ⁴ is preferably performed. Hereinafter, “MS ² ” is represented as “MS / MS”.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples as long as the gist thereof is not exceeded. In particular, the present invention will be specifically described in Examples using PBH which is a derivatizing agent having a hydrazide group. However, the present invention is not limited to the derivatizing agent, and other derivatizing agents may be used. However, it is possible to react on the sample support member using the method substantially the same as that and to use for the mass spectrometer.

Example 1
0.5 μL of 0.5 pmol of Lacto-N-fucohexose-I (Compound 1) represented by the formula (3) was spotted on a stainless steel target plate and dried. A DMSO solution of 0.25 μL of PBH 125 pmol and NaBH ₃ CN 300 pmol was layered thereon and heated at 60 ° C. for 10 minutes. Next, the plate was placed in a desiccator, and DMSO was volatilized by pulling with a vacuum pump for 10 minutes.

  As a matrix, 0.5 μL of a solution of DHBA in “acetonitrile / water = 50/50 (volume ratio)” (concentration: 10 mg / mL) was placed, dried at room temperature, and MALDI-QIT-TOFMS apparatus AXIMA-QIT (Shimadzu / Kratos) ).

As shown in FIG. 1 (a), m / z 1527, which is [M + Na] ⁺ ion, and m / z 1503, which is [M−H] ⁻ ion, as shown in FIG. 1 (b). A signal was obtained. Compared to the conventional method shown in Comparative Example 1, the amount of sample used for the reaction was less than 1/2000, and the operation was completed within 30 minutes.

Comparative Example 1
Compound 1 aqueous solution (compound 1 containing 1 nmol) was added to a glass reaction tube with a screw lid and dried. 500 nmol of PBH was dissolved in 20 μL of methanol and added to the reaction tube, and further 2 μL of acetic acid diluted with methanol (the ratio of acetic acid to methanol was 1: 8 by volume) was added to completely close the lid. After stirring well, the mixture was heated at 80 ° C. for 20 minutes and neutralized by adding 1M NaOH aqueous solution. 30 μL of 1.7 M NaBH ₄ solution was added and reacted at 40 ° C. for 30 minutes, and further 10 μL of 1.7 M NaBH ₄ solution was added and reacted at 40 ° C. for 30 minutes.

Subsequently, the following operation was performed in order to remove excess reagents. After adding 400 μL of pure water and 400 μL of chloroform and shaking well, the mixture was allowed to stand. The lower chloroform layer was discarded, and 400 μL of fresh chloroform was added, followed by extraction once more. The upper layer was taken to dryness. The Sep-pak C ₁₈ cartridge was washed with methanol and then with pure water, and the dried reaction product was dissolved in pure water and passed through the cartridge. The cartridge was washed with pure water, and then eluted with acetonitrile-pure water (volume ratio 6: 4) to obtain a sugar labeled and reduced with PBH. The solvent was distilled off and stored at −30 ° C. protected from light.

  The time required for the preparation of the measurement sample so far is 9 hours, which is much longer than that of Example 1 which was 30 minutes. The amount of compound 1 required was about 2000 times that of Example 1. Furthermore, the resulting reduced labeled sugar had to be stored at −30 ° C. protected from light until measurement.

Example 2
Unsaturated chondro disaccharide ΔDi-4S (compound 2) shown in formula (4) and ΔDi-6S (compound 3) aqueous solution (solute / solution = 100 fmol / 10 nL) 10 nL shown in formula (5), respectively, The sample was spotted on a stainless steel target plate with a micro dispenser and dried. PBH (60 pmol) DMSO solution 0.1 μL was layered thereon and heated at 50 ° C. for 7 minutes. The plate was put in a desiccator and pulled with a vacuum pump for 30 minutes to volatilize DMSO. As a matrix, 0.5 μL of a 50 vol% acetonitrile solution (concentration 1 mg / mL) of nor-Harman (9H-pyrido [3,4-b] indole) was placed and dried at room temperature. The same apparatus as in Example 1 Measured with

The derivatizing agent obtained by reacting the compound 2 from the "△ Di-4S pyrene derivative", [M-H] ^- obtained signals m / z742 is ion was found to PBH bound (Fig. 2a).

  MS / MS measurement was performed by selecting m / z 742 as the precursor ion. As a result, fragment ion signals of m / z 566, m / z 584, and m / z 626 decreased in this order from “ΔDi-6S pyrene derivative” obtained by reacting compound 3 with a derivatizing agent ( FIG. 3a).

  On the other hand, from the “ΔDi-4S pyrene derivative” obtained by reacting compound 2 with a derivatizing agent, the same fragment ions were detected in a decreasing order (FIG. 3b).

Further, MS ³ measurement was performed using m / z 626 as a precursor ion, and different fragment ion patterns were obtained between the two isomers (FIGS. 4a and b). Thus, MS / MS or MS ³ measurement was possible even with a small amount. This indicates that the fragment ions necessary to identify the isomeric structure were obtained.

In addition, the compound 2 or compound 3 isolated from a biological sample or the like usually has a very small amount of the sample as in this example, and is a method of reacting with a derivatizing agent other than on the sample support member. Then, MS / MS and MS ⁿ (n is an integer of 3 or more) cannot be measured.

  Even if 0.5 μL of DHBA (concentration 1 mg / mL) or 0.5 μL of 1,5-diaminonaphthalene (concentration 1 mg / mL) is used as the matrix instead of nor-Harman, the same result as above can be obtained. It was.

Comparative Example 2
An unsaturated chondro disaccharide ΔDi-4S (compound 2) aqueous solution (solute / solution = 100 fmol / 10 nL) was spotted on a stainless steel target plate with a microdispenser. Without using a derivatizing agent, 0.5 μL of nor-Harman (concentration 1 mg / mL) was placed and dried at room temperature, and the measurement was performed using the same apparatus as in Example 1.

[M-H] ^- an ion was detected m / z 458 (FIG. 2b) is the same amount loaded on the target plate, in Example 2 was pyrene derivative with PBH as derivatizing agent (Fig. 2a) In comparison, the signal was very low, and MS / MS measurement was difficult. For this reason, it was impossible to identify the structure of Compound 2, such as where the sulfate group was bonded.

Example 3
Using an unsaturated chondro disaccharide ΔDi-4S represented by formula (4) ΔDi-4S (compound 2) and an aqueous solution of ΔDi-6S (compound 3) represented by formula (5) (solute / solution = 5 fmol / 10 nL) 10 nL The same operation as in Example 2 was performed, and the measurement was performed using the same apparatus as in Example 1. As shown in FIG. 5, [M-H] ^- was detected from any the signal of m / z742 is an ion.

Example 4
A synthetic glycopeptide (Compound I) represented by the following formula (6), 100 nmol and 500 mM of iodoacetamide aqueous solution 10 μmol were reacted in 50 μL of 20 mM aqueous ammonium bicarbonate solution at room temperature for 1 hour under light shielding, and the alkyl of cysteine residues. Made.

The reaction mixture was applied to a Sep-Pak C18 light, washed with an aqueous solution of 0.1% by volume trifluoroacetic acid (hereinafter abbreviated as “TFA”), and with an aqueous solution of “60% by volume acetonitrile-0.1% by volume TFA”. Eluted. In 20 μL of “50 mM HEPES buffer (pH 7) containing 20 mM CaCl ₂ , 20 mM MnCl ₂ ” added with 20 nmol of this alkylated glycopeptide, 4 mU of β1,4 galactosyltransferase, and 200 nmol of UDP-galactose at 37 ° C. The reaction was performed for 21 hours.

  After the reaction, the glycopeptide was purified by Sep-Pak C18 light. When positive-ion mode MS measurement of this glycopeptide was performed, an increase of 162 Da corresponding to one galactose residue was confirmed.

Alkylated glycopeptide 20 nmol, α2,6-sialyltransferase or α2,3-sialyltransferase 2U, and 200 nmol of CMP-N-acetylneuraminic acid, 20 μL of “20 mM CaCl ₂ , 20 mM MnCl ₂ In a 100 mM HEPES buffer (pH 7) ", the reaction was performed at 37 ° C for 21 hours, and this glycopeptide was purified by Sep-Pak C18 light.

  According to the MS measurement, 291 Da corresponding to one residue of sialic acid is increased, and the alkylated compound (II) represented by the following formula (7) or the alkylated compound (III) represented by the following formula (8) is obtained. It was confirmed that it was generated.

10 μL of 1 mM NaIO ₄ was added to 2 nmol each of the alkylated compound (II) or (III), and the reaction was carried out on ice under light shielding for 20 minutes. Next, 0.5 μL of 100-fold diluted ethylene glycol was added, and the mixture was allowed to stand at room temperature for 20 minutes under light shielding. The reaction mixture was applied to a Sep-Pak C18 light, washed with 0.1 vol% TFA aqueous solution, and eluted with “60 vol% acetonitrile-0.1 vol% TFA” aqueous solution.

  From the MS measurement, it was confirmed that the periodate oxidation reaction decreased 62 Da, the carbon-carbon bond was cleaved between the sialic acid residues C7 to C8, and one aldehyde group was generated.

  The glycopeptide thus obtained was reacted with PBH on a stainless steel target plate and dried in the same manner as in Example 2. The amounts used are also the same as in Example 2. As a matrix, 0.5 μL of an “acetonitrile / water = 50/50 (volume ratio)” solution (concentration 5 mg / mL) of 1,5-diaminonaphthalene was placed and dried at room temperature, and MALDI-QIT-TOFMS apparatus AXIMA- It was measured by QIT (manufactured by Shimadzu / Kratos).

From those used alkylating compound (II), as shown in FIG. ^{6a, [M-H] -} m / z3383 are ions are detected, PBH that bound was confirmed. In addition, it was confirmed from the thing using the alkylation compound (III) that PBH couple | bonded similarly.

In addition, MS / MS analysis was performed using m / z2681 in which 6 amino acid residues were released as a precursor ion. As a result, as shown in 6b, the ions PBH to ring opening ^{0, 2} A fragment of ring opening ^{3, 5} A and N- acetylglucosamine galactose was introduced, m / z585 and m / z794 is detected It was.

  On the other hand, when PBH was introduced into the alkylated compound (III), m / z 585 was not detected, and only m / z 794 was detected.

  As in the case of the isolated alkylated compounds (I) to (III), the glycopeptides prepared and isolated from biological samples or the like usually have a very small amount of the sample. In the process, MS / MS analysis cannot be performed.

  By carrying out the step of reacting the molecule to be analyzed with the derivatizing agent on the sample support member provided directly to the analyzer, it was possible to simplify the preparation of the sample for high sensitivity mass spectrometry measurement and increase the yield. This improves detection sensitivity while keeping the amount of sample used small, achieves ionization acceleration and product ion stabilization, thereby generating a sufficient amount of molecular ions even at a much smaller sample level. Analysis was possible.

Example 5
In proteomics, proteins are identified by mass spectrometry of peptide fragments obtained by in-gel digestion of proteins separated by electrophoresis, but micrograms (μg) are required to identify sugar chains. . By applying the present invention, it was possible to identify a sugar chain recovered from a gel after electrophoresis of a very small amount of glycoprotein.

[Electrophoresis and in-gel digestion]
50 μg of prostate specific antigen (hereinafter abbreviated as “PSA”) purified from human semen (Chemicon) in 20 μL of sample buffer (0.125 M Tris-HCl, pH 6.8, 10% by mass mercaptoethanol) It melt | dissolved in the mass% SDS, 10 mass% sucrose (0.004 mass% bromophenol blue), and it heated at 100 degreeC for 3 minutes, Then, it left still in ice and cooled. After electrophoretic separation on a 9% by mass acrylamide gel, the gel was lightly washed with pure water and subjected to CBB staining.

  The gel of the stained protein portion was cut out, transferred to a 1.5 mL tube, and washed with pure water, a 50% acetonitrile aqueous solution, and finally acetonitrile. After the gel was dried, a reducing solution (10 mM DTT, 25 mM ammonium bicarbonate, pH 8.0, aqueous solution) was added, and the mixture was allowed to react with shaking at 56 ° C. for 1 hour. The solution was removed, an alkylated solution (55 mM iodoacetamide aqueous solution) was added, and the mixture was allowed to react with shaking at room temperature for 45 minutes. After washing with aqueous ammonium bicarbonate (25 mM, pH 8.0) and acetonitrile, the gel was dried.

  Lysyl Endopeptidase solution (250 ng: Wako Mass Spectrometry Grade, 25 mM ammonium bicarbonate pH 8.0 aqueous solution) was added to the dried gel, and the mixture was left to swell for 45 minutes in ice and then gently stirred at 37 ° C. for 18 hours. Reacted. An extract (75% acetonitrile, 0.1% aqueous trifluoroacetic acid solution) was added, and the solution was recovered after shaking for 20 minutes.

  After drying with a centrifugal concentrator, PNGase F was added in 1 unit (1 unit / μL, 1 μg AEBSF: Roche, 50 mM ammonium bicarbonate pH 8.0 aqueous solution), and reacted at 37 ° C. with shaking for 18 hours. 1 μL of a 1% trifluoroacetic acid aqueous solution was added to the reaction mixture, and the sample was repeatedly sucked and discharged onto a C18 chip to adsorb the peptide, and separated from the sugar chains in the solution. After loading and washing 30 mg of carbon graphite (GL Science) in a microspin column, the sugar chain fraction that did not bind to the C18 chip was added to the column, and 5% acetonitrile 0.1% trifluoroacetic acid aqueous solution was added, followed by centrifugation. (300 × g, 1 minute to 2 minutes). A 50% acetonitrile 0.1% trifluoroacetic acid aqueous solution was added, and sugar chains were eluted by centrifugation (300 × g, 1 minute to 2 minutes). The recovered sugar chain eluate was dried by a centrifugal concentrator and dissolved in 2 μL of pure water to obtain a mass spectrometry sample.

[Signs]
On the target plate, 0.5 μL of the sample obtained above (containing about 10 ng of glycoprotein-derived sugar chain) was air-dried, and then 0.25 μL of PBH (500 pmol) in DMSO was overlaid thereon, Heated for about 15 minutes to dry at ° C. In addition, after 0.5 μL of the sample is placed on another part of the target plate and air-dried, 0.25 μL of a DMSO solution of 1-pyrenyldiazomethane (PDAM) (500 pmol) is layered thereon and dried at 40 ° C. Until about 25 minutes. In addition, after 0.5 μL of the sample is placed on another target plate and air-dried, 0.25 μL of a DMAM solution of PDAM (500 pmol) is layered thereon and heated at 40 ° C. for about 25 minutes, Further, 0.25 μL of PBH (500 pmol) in DMSO was layered thereon and heated at 50 ° C. for about 15 minutes until dried. As a control, 0.5 μL of the sample obtained above was placed on another portion of the target plate and air-dried. The target plate was suction dried overnight in a desiccator. The measurement was performed with a mass spectrometer in the same manner as in Example 1.

  FIG. 7 shows the MS spectrum of the sample. FIG. 7A shows an MS spectrum in which a sugar chain derived from about 10 ng of PSA was placed on a plate, a derivatizing agent was not added, and negative ions were measured. FIG. 7B was the same amount of sugar chain placed on a plate and PBH was added. FIG. 7C shows the MS spectrum measured after the reaction, and the MS spectrum measured after adding the same amount of sugar chain to the plate and adding PDAM to react. FIG. 7D shows the MS spectrum measured after the reaction. The MS spectrum measured after adding PDAM on a plate and reacting, followed by adding PBH and reacting is shown.

  In FIG. 7, the full scale of the vertical axis is shown as 20 mV. White arrows indicate unlabeled sugar chain ions, white stars indicate sugar chain ions labeled with PBH, gray stars indicate sugar chain ions labeled with PDAM, and sugar chain ions with white and gray stars indicate It shows that it labeled with both derivatizing agents of PDAM and PBH.

As shown in FIG. 7A, no signal was detected in the sample to which the derivatizing agent was not added (FIG. 7, A).
When PBH was added and reacted on the plate, sugar chain b ion m / z 2362 labeled with PBH was detected (FIG. 7, B).
When another derivatizing agent PDAM was added to the same sample and reacted, ions labeled with the following three sugar chains a, b and c, m / z 2144, m / z 2290, and m / z 2581 were detected ( FIG. 7, C).
Furthermore, after reacting PDAM with the sample and subsequently reacting with PBH, in addition to the sugar chain ions individually labeled, the following sugar chain c ions labeled with both derivatizing agents, m / z 2867 Was detected (FIG. 7, D). After reacting PBH with the sample, PDAM was subsequently reacted, and the same mass spectrum was obtained even if the order of derivatization was reversed. In addition, the laser intensity could be measured at the usual 80% during these measurements.

  Since protein recovery from the gel after electrophoresis is said to be about 50% at the maximum, it seems that PSA was actually equivalent to 5 ng. Therefore, only about 160 fmol of sugar chain is on the plate at the maximum. There will be no. It should be noted that this sugar chain amount is too small to be measured when no derivatizing agent is added (in the case of non-labeling), as shown in FIG. 7A.

  When both a derivatizing agent such as PBH and PDAM and a sugar chain are placed on the sample support member, there is an effect of improving not only the sugar chain labeled by the reaction of the derivatizing agent but also the ionization of the unlabeled sugar chain. . The reason for this was thought to be that the ionization of the coherent peptide fragments was suppressed, and that these derivatizing agents coexisted to produce an effect similar to a matrix. In particular, the matrix effect of PDAM was considered to be a synergistic effect of 1-pyrenylmethyl alcohol produced by the side reaction together with this. That is, in the present invention, the above step “(2) the step of reacting the molecule to be analyzed and the derivatizing agent on the sample support member” is an indispensable step, but the present invention includes only the reactants of the present invention. However, the present invention is not limited only to the case where the effect of

  FIG. 8A is an MS spectrum (vertical axis 300 mV) in which about 10 ng of PSA-derived glycan was placed on a plate and negative ions were measured without adding a derivatizing agent. FIG. 8B was the same amount of glycan placed on a plate. It is the MS spectrum (vertical axis 120mV) measured after adding PDAM and making it react. Black arrows indicate peptide ions, and white arrows indicate sugar chain ions.

PDAM has a strong ability to produce sugar chain ions. As shown in FIG. 8, when the derivatizing agent is not added (FIG. 8, A), for example, many ions of the peptide on the low molecular side indicated by the black arrow are generated, and most of the sugar chain ions are detected. There wasn't. However, after PDAM was added and reacted, for example, peptide ions indicated by black arrows were suppressed to 1/10, and instead, sugar chain ions (white arrows) that could hardly be detected were formed predominantly (Fig. 8, B). Further, the labeled sugar chain c ion (gray star) could be subjected to MS ³ analysis.

Example 6
After the following molecules and derivatizing agent are reacted on the sample support member, mass spectrometry can be performed.
(1) A molecule having an aldehyde group (for example, a molecule that is a sugar or a molecule that has an aldehyde group by oxidizing sugar or glycoprotein) and the following first group of compounds as a derivatizing agent.
(2) A molecule having an amino group or a carboxyl group (for example, a molecule that is a sugar, a protein, or a glycoprotein) and the following second group of compounds as a derivatizing agent.
(3) A molecule having an SH group (for example, a molecule that is a sugar, protein, or glycoprotein) and the following third group of compounds as a derivatizing agent.
(4) A molecule having an OH group (for example, a molecule that is a sugar, protein, or glycoprotein) and a compound of the following fourth group as a derivatizing agent.

Group 1: 1-pyrenebutanoic acid hydrazide, 1-pyreneacetic acid hydrazide, 1-pyrenepropionic hydrazide, 1-pyrenecarbaldehyde hydrazone, aminopyrene, 1-pyrenemethylamine, 1-pyrenepropylamine, 1-pyrenebutylamine and similar aromatic compounds.
Group 2: Methyl iodide, trimethylsilyldiazomethane, 1-pyreneacetic acid succinimidyl ester, 1-pyrenepropionic acid succinimidyl ester, 1-pyrenebutanoic acid succinimidyl ester, N- (1-pyrenebutanoyl) cysteic acid succini Midyl ester, 1-pyrenesulfonic acid chloride, 1-pyrenyldiazomethane, 1-pyrenyl thiocyanate, 1-pyrenyl isothiocyanate, and similar aromatic compounds.
Group 3: N- (1-pyrene) iodoacetamide, N- (1-pyrene) iodomaleimide, N- (1-pyrenemethyl) iodoacetamide, 1-pyrenemethyliodoacetate, and similar aromatic compounds Group 4: Methyl iodide, trimethylsilyldiazomethane, 1-pyrenyldiazomethane, and similar aromatic compounds

  The mass spectrometry method of the present invention can obtain information on a highly reliable chemical structure by reducing the amount of sample and reaction reagent to be analyzed and improving ionization efficiency and reproducibility of measurement. It is widely used for elucidating its function and pathological condition by applying it to a small amount of biological sample such as glycoprotein or molecules in the biological sample.

In Example 1, it is a MALDI-QIT-TOF mass spectrometry (MS) spectrum of what was obtained by making compound 1 react with PBH on a plate. (A) Shows the peak of [M + Na] ⁺ ion. (B) shows the peak of [M−H] ⁻ ion. (A) in Example 2, but obtained by reacting PBH on plates Compound 2 [M-H] ^- a spectrum of ions. (B) in Comparative Example 2, the compound 2 not by reacting PBH [M-H] ^- a spectrum of ions. It is a MS / MS spectrum measured by selecting m / z 742 as a precursor ion (Example 2). (A) MS / MS spectrum obtained by reacting compound 3 with PBH on a plate. (B) MS / MS spectrum obtained by reacting compound 2 with PBH on a plate. (Example 2) which is an MS ³ spectrum measured by selecting m / z 626 as a precursor ion. (A) MS ³ spectrum obtained by reacting compound 3 with PBH on a plate. (B) MS ³ spectrum obtained by reacting compound 2 with PBH on a plate. Is ^- (spectrum of the ion [M-H]) in Example 3, but obtained by reacting PBH on plates MALDI-QIT-TOF mass spectrometry (MS) spectrum. (A) 5 fmol of compound 3 is used. (B) 5 fmol of compound 2 is used. In Example 4, it is a MALDI-QIT-TOF mass spectrometry (MS) spectrum of what was obtained by making the alkylated compound (II) and PBH react on a plate. (A) MS spectrum. (B) MS / MS spectrum using m / z 2681 as a precursor ion. 6 is an MS spectrum measured in Example 5. A: MS spectrum in which about 10 ng of PSA sugar chain was placed on a plate and negative ions were measured without adding a derivatizing agent. B: MS spectrum measured after placing the same amount of sugar chain on a plate and adding PBH to react. C: MS spectrum measured after PDAM was added and reacted with the same amount of sugar chain on a plate. D: MS spectrum measured after placing the same amount of sugar chains on a plate and adding PDAM to react, followed by PBH and reacting. 6 is an MS spectrum measured in Example 5. A: MS spectrum obtained by placing about 10 ng of PSA sugar chain on a plate and measuring negative ions without adding a derivatizing agent. B: An MS spectrum measured after placing the same amount of sugar chain on a plate and adding PDAM for reaction.

Claims (16)

Molecular mass spectrometry, comprising at least steps (1), (2), (3) and (4)
(1) placing the molecule to be analyzed and the derivatizing agent on a sample support member used for mass spectrometry;
(2) reacting the molecule to be analyzed and the derivatizing agent on the sample support member;
(3) A step of subjecting a derivative of a molecule to be analyzed generated by reaction on a sample support member to a mass spectrometer;
(4) A mass spectrometry method comprising ionizing a derivative of a molecule to be analyzed.   The mass spectrometric method according to claim 1, wherein the derivative of the molecule to be analyzed is ionized by laser desorption ionization.   The mass spectrometric method according to claim 2, wherein a matrix molecule coexists with a derivative of a molecule to be analyzed on the sample support member after the reaction and is used for a mass spectrometer.   The mass spectrometric method according to claim 1, wherein the derivative of the molecule to be analyzed is ionized by electrospray ionization.   The mass spectrometric method according to any one of claims 1 to 4, wherein the molecule to be analyzed is a sugar, a protein or a glycoprotein.   The molecule according to any one of claims 1 to 5, wherein the molecule to be analyzed has one or more functional groups selected from the group consisting of an aldehyde group, a carboxyl group, an amino group, a mercapto group, and a hydroxyl group. Mass spectrometry described in 1.   The mass spectrometric method according to any one of claims 1 to 6, wherein the molecule to be analyzed is a molecule obtained by oxidizing a sugar or glycoprotein containing sialic acid or galactose to introduce an aldehyde group.   The mass spectrometry method according to any one of claims 1 to 7, wherein the derivatizing agent increases ionization efficiency by reacting with a molecule to be analyzed.   The mass spectrometric method according to any one of claims 1 to 8, wherein the derivatizing agent controls a cleavage ionization position by reacting with a molecule to be analyzed.   The derivatizing agent has at least one functional group selected from the group consisting of an amino group, a hydrazide group, a carboxyl group, a diazomethyl group, a succinimidyl ester group and a sulfonyl chloride group. The mass spectrometry method according to claim 9.   The mass spectrometric method according to any one of claims 1 to 10, wherein the derivatizing agent is methyl iodide or trimethylsilyldiazomethane.   The mass spectrometric method according to any one of claims 1 to 11, wherein the derivatizing agent is a condensed polycyclic derivative compound having a condensed polycycle in its molecule.   The molecule according to any one of claims 1 to 10, wherein the molecule to be analyzed is a molecule having a sugar chain containing an aldehyde group, and the derivatizing agent has an amino group or a hydrazide group. Mass spectrometry described in 1.   The analyzed molecule is a protein or glycoprotein having a carboxyl group, an amino group or a mercapto group, and the derivatizing agent is an amino group, a hydrazide group or a diazomethyl group, or methyl iodide or trimethylsilyldiazomethane. The mass spectrometry method according to any one of claims 1 to 6 or any one of claims 8 to 12.   The mass spectrometry method according to any one of claims 1 to 14, wherein the molecule to be analyzed is 1 picomolar (pmol) or less.   A derivatizing agent used for mass spectrometry according to any one of claims 1 to 15.