JPWO2008133148A1 - Method for localizing target molecule on plate and mass spectrometry using the same - Google Patents

Abstract

A sufficient amount of the target molecule can be secured on the plate without washing it off, and it can be used for mass analysis of unknown target molecules, and also used for mass analysis of multiple target molecules at the same time. A method for localizing a molecule to be measured on a plate is provided. (A) preparing a plate to which at least one compound that localizes the molecule to be measured is bound; and (B) placing a biological sample containing at least one molecule to be measured on the plate. And (C) dropping and drying a solution containing a matrix on the biological sample to localize the molecule to be measured on the plate.

Description

  The present invention relates to a method for localizing a molecule to be measured on a plate. More specifically, the present invention relates to a method capable of selectively localizing only a measurement object from a biological sample on a plate. The present invention relates to a mass spectrometry method that uses such a method to obtain a high signal intensity of a molecule to be measured.

  In recent years, high-sensitivity mass spectrometry methods for proteins and / or sugar chains, which are biopolymers, have been actively developed, and the analysis methods are rapidly spreading in the fields of biochemistry and medicine. Such mass spectrometry is used, for example, for analysis of biomolecule expression and its function, biomarker search, and the like.

  In a biological sample containing a biopolymer, many contaminating molecules are mixed in addition to the molecule to be measured. For this reason, when a biological sample containing a very small amount of a molecule to be measured is highly purified, the mass analysis sensitivity of the molecule to be measured may be less than the detection sensitivity, and thus the molecule may be missed in mass spectrometry. In addition, when a contaminating molecule has a higher ionization efficiency than the measurement target molecule, it is difficult to detect the measurement target molecule.

  Considering such circumstances, for example, the following is disclosed as a technique related to ionization of a biological sample as a previous stage of mass spectrometry.

  Non-Patent Document 1 discloses that surface-enhanced laser desorption / ionization (SELDI) is used to immobilize functional groups and / or antibody molecules on the chip surface, capture only specific molecules in a biological sample, wash them, and perform laser irradiation. Discloses a technique for performing ionization.

  Patent Document 1 discloses a technique for monitoring the purification of a target molecule, including, for example, creating a surface enhanced laser desorption ionization (SELDI) mass spectral profile of biological components in a cell culture medium or a purification fraction of such medium. Has been.

JPO homepage, sample room, standard technology collection, general, mass spectrometry (mass spectrometry), 2-1-1-1-1 surface enhanced laser desorption ionization (SELDI) (http: //www.jpo.go (See .jp / shiryou / index.htm) JP 2005-509173 A

  Prior to mass spectrometry, a technique (SELDI) disclosed in Non-Patent Document 1 and Patent Document 1 that leaves only a molecule to be measured by a predetermined washing with respect to a biological sample on a plate is adopted. In some cases, some of the molecules to be measured are washed away when the biological sample is washed. For this reason, when the amount of the molecule to be measured is very small and / or the binding is weak, the amount of the molecule to be measured on the plate is extremely small, and the analysis sensitivity is much higher than the desired detection sensitivity in mass spectrometry. May fall below.

  In addition, mass spectrometry after adopting SELDI is effective when the molecule to be measured is known, but is not effective when the molecule to be measured is unknown, because the molecule may be completely removed from the plate. .

  Furthermore, in mass spectrometry after adopting SELDI, by selecting a predetermined chip and performing a predetermined washing, it is possible to limit to a specific type of measurement target molecule having the maximum content or the strongest binding. However, in actual mass spectrometry, it may be desirable to simultaneously analyze a plurality of molecules to be measured with the amount of biological sample used for one SELDI measurement.

  The object of the present invention is to (a) ensure a sufficient remaining amount on the plate without washing away the measurement target molecule, (b) can be used for mass spectrometry of unknown measurement target molecules, and (c) multiple Another object of the present invention is to provide a method for localizing a molecule to be measured on a plate, which can be used when mass-analyzing the molecules to be measured simultaneously. Another object of the present invention is to provide a mass spectrometry method that can obtain a high signal intensity of a molecule to be measured using such a method.

  The present invention includes (A) a step of preparing a plate to which at least one compound for localizing a molecule to be measured is bound, and (B) a biological sample containing at least one molecule to be measured on the plate. And (C) dropping and drying a solution containing a matrix on the biological sample and localizing the molecule to be measured on the plate, the molecule to be measured on the plate Relates to a method for localizing. Here, the biological sample placed on the plate always contains at least one kind of measurement target molecule. In other words, the case where the biological sample is composed of only contaminating molecules is excluded.

  In such a method for localizing a molecule to be measured on a plate, the compound preferably contains an aryl group, a dihydroxyboryl group, an alkyl group, an amino group, or a hydroxyl group. Among these, the aryl group can be a phenyl group, a thienyl group, a pyrrolyl group, a pyridyl group, a naphthyl group, an anthryl group, a pyrenyl group, a quinolyl group, an indolyl group, an acrylidinyl group, or a benzothiazolyl group. The alkyl group may be a C4 to C18 alkyl group.

  The localization method can localize a single molecule to be measured on the plate, and can also localize a plurality of molecules to be measured on the plate. That is, when the biological sample contains at least two kinds of molecules to be measured, different compounds are bound to at least two regions of the plate in the step (A), and at least two regions in the step (C). It is possible to localize different molecules to be measured.

  The localization method can perform not only localization of a molecule to be measured but also localization of a contaminating molecule. That is, when the biological sample further contains at least one kind of contaminant molecule, in the step (A), a different compound is bound to at least two regions of the plate, and in the step (C), The molecule to be measured can be localized in one region and the contaminating molecule can be localized in another region.

  In such a method for localizing a molecule to be measured on a plate, it is desirable to further include (B ′) a step of derivatizing the biological sample between steps (B) and (C). .

  The measurement target molecule can be, for example, a sugar chain (free sugar chain, glycopeptide, glycoprotein, etc.).

The present invention is a method for mass spectrometry of a molecule to be measured,
(1) a step of localizing a molecule to be measured in a biological sample on a plate by any one of the above methods, and (2) a molecule to be measured localized in the step (1) above, It includes a step of performing MS ⁿ measurement, wherein n is an integer of 1 or more.

The present invention includes (A1) binding a first compound that localizes the molecule to be measured on the first plate, and (A2) localizing the molecule to be measured on the second plate. A step of binding a second compound different from the compound, and (B1) sandwiching a biological sample containing at least one molecule to be measured between the first plate and the second plate, and drying (B1) B2) separating the first plate and the second plate; and (C) dropping and drying a solution containing a matrix on the first plate and the second plate, so that the first plate and the second plate are separated. And a method of localizing the molecule to be measured on the plate, comprising the step of localizing the molecule to be measured on at least one of the plates. Here, the biological sample placed on the plate always contains at least one kind of measurement target molecule. In other words, the case where the biological sample is composed of only contaminating molecules is excluded.

  In such a method for localizing a molecule to be measured on a plate, the compound preferably contains an aryl group, a dihydroxyboryl group, an alkyl group, an amino group, or a hydroxyl group. Among these, the aryl group can be a phenyl group, a thienyl group, a pyrrolyl group, a pyridyl group, a naphthyl group, an anthryl group, a pyrenyl group, a quinolyl group, an indolyl group, an acrylidinyl group, or a benzothiazolyl group. The alkyl group may be a C4 to C18 alkyl group.

  The localization method can localize different measurement target molecules on two plates. That is, when the biological sample includes at least two kinds of molecules to be measured, in the step (C), a molecule to be measured different from the molecule to be measured on the first plate can be localized on the second plate. it can.

  The localization method not only can localize different measurement target molecules in each of the two plates, but also localizes the measurement target molecules and the contaminant molecules in each of the two plates. Can do. That is, when the biological sample further contains at least one kind of contaminating molecule, in the step (C), at least one kind of contaminating molecule can be localized on the second plate.

  In such a method for localizing a molecule to be measured on a plate, it is desirable to further include (B ′) a step of derivatizing the biological sample between steps (B2) and (C). .

  The measurement target molecule can be, for example, a sugar chain (free sugar chain, glycopeptide, glycoprotein, etc.).

A method for mass spectrometry of a molecule to be measured,
(1) Localizing at least one of the measurement target molecule in the biological sample on the first plate and the measurement target molecule in the biological sample on the second plate by any one of the methods described above, and (2) Mass spectrometry including a step of performing MS ⁿ measurement on the measurement target molecule localized in the step (1), wherein n is an integer of 1 or more.

  In the localization method of a molecule to be measured on the plate of the present invention, the affinity between the molecule to be measured and the plate is obtained by performing a predetermined surface treatment (binding of a predetermined compound) in advance on the plate on which the biological sample is placed. The affinity can be increased more than the affinity between other contaminant molecules and the plate. For this reason, the measurement target molecule can be selectively and sufficiently left on the plate simply by drying the biological sample containing the measurement target molecule on the plate.

  In addition, since the localization method of the present invention employs a technique of drying on the plate without washing away the molecule to be measured like SELDI, naturally the unknown molecule to be measured remains on the plate. For this reason, this method can be applied even if the target molecule to be subjected to mass spectrometry is unknown.

  Furthermore, since the localization method of the present invention does not wash away the measurement target molecule unlike SELDI, a plurality of measurement target molecules can remain in the dried biological sample. For this reason, this method can be applied even if there are a plurality of molecules to be measured.

  In addition, according to the mass spectrometry using the localization method of the present invention that exhibits the effects as described above, it is possible to obtain a sufficient signal intensity of the molecule to be measured, and to deal with an unknown molecule to be measured, In addition, a plurality of molecules to be measured can be analyzed simultaneously.

FIG. 1 is a photograph of the measurement sample obtained in Example 1. FIG. 2 is a graph showing the distribution of unlabeled sugar chain 1-derived deprotonated ions (m / z = 2077) in Example 1. FIG. 3 is a graph showing the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) in Example 1. FIG. 4 is a diagram showing a mass spectrum at point A in FIGS. FIG. 5 is a diagram showing a mass spectrum at a point B in FIGS. FIG. 6 is a diagram showing the distribution of unlabeled sugar chain 1-derived deprotonated ions (m / z = 2077) with respect to Comparative Example 1. FIG. 7 is a view showing the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) in Comparative Example 1. FIG. 8 is a diagram showing a mass spectrum at a point C in FIGS. FIG. 9 is a graph showing the distribution of peptide ions (m / z = 1075) in Example 2. FIG. 10 is a graph showing the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) in Example 2. FIG. 11 is a diagram showing a mass spectrum at a point D in FIGS. FIG. 12 is a diagram showing a mass spectrum at a point E in FIGS. FIG. 13 is a diagram showing an MS spectrum of negative ions (average of all measurement points) when pyrene labeling is not performed in Example 3. FIG. 14 is a diagram showing an MS spectrum (average of all measurement points) of negative ions when pyrene labeling is performed in Example 3. FIG. 15 is a diagram showing an MS spectrum of negative ions in a specific region when pyrene labeling is performed in Example 3. FIG. 16 is a diagram showing an MS spectrum of negative ions of a phenyl plate after separation when pyrene labeling is not performed in Example 4. FIG. 17 is a diagram showing an MS spectrum of negative ions when pyrene labeling is performed on the phenyl plate after separation in Example 4. FIG. 18 is a diagram showing an MS spectrum of negative ions of the C18 plate after separation in Example 4 when no pyrene labeling is performed. FIG. 19 is a diagram showing the MS spectrum of negative ions when pyrene labeling is performed on the C18 plate after separation in Example 4. FIG. 20 is a diagram showing an MS spectrum of negative ions of the amino plate after separation in Example 5 when no pyrene labeling was performed. FIG. 21 is a diagram showing a negative ion MS spectrum of pyrene-labeled amino plates after separation in Example 5. FIG. 22 is a diagram showing an MS spectrum of negative ions of Example 5 when no pyrene labeling is performed in Example 5. 23 is a diagram showing an MS spectrum of negative ions when pyrene labeling is performed on an amino plate in Example 5. FIG.

  Embodiments of the present invention will be described below, but the present invention is not limited to these ranges, and can be appropriately changed within the range of ordinary creation ability of those skilled in the art.

<Localization method of molecule to be measured on plate (Type 1)>
The method (type 1) for localizing a molecule to be measured on the plate of the present invention comprises (A) preparing a plate to which at least one compound that localizes the molecule to be measured is bound; B) A step of placing a biological sample containing at least one molecule to be measured on the plate; and (C) dropping and drying a solution containing a matrix on the biological sample, and then measuring the measurement target on the plate. And localizing the molecule. Here, the molecule to be measured means a molecule to be measured in mass spectrometry performed after performing the localization method.

[Process for preparing plates]
(plate)
The plate is not particularly limited as long as it has a functional group capable of capturing at least one of a molecule to be measured and a contaminating molecule in a biological sample, and is either a metal plate or a non-metal film. You can also. Moreover, the surface of the plate can have any shape such as a flat shape or an uneven shape, and can also be made porous. Furthermore, a predetermined functional group may not be directly bonded to the plate, for example, a spacer portion or a polymer between the plate and the functional group may be interposed.

  Examples of capturing a molecule to be measured on a plate (type 1-1) include a plate to which an aryl group is bonded, a plate to which a dihydroxyboryl group is bonded, a plate to which an amino group is bonded, or a plate to which a hydroxy group is bonded. An example of using a different plate is given. These plates are an example of a plate that has higher affinity for the molecule to be measured than affinity for other contaminant molecules. In particular, when pyrene labeling is performed on a measurement target molecule, it is possible to achieve a high affinity between the labeled measurement target molecule and the plate by using a plate to which an aryl group is bonded, and to achieve good localization. It is preferable at the point achieved. In addition, if the molecule to be measured contains a sugar chain, it is possible to use a plate with a dihydroxyboryl group, a plate with an amino group, a plate with a hydroxy group, etc. This is preferable in that a high affinity between the target molecule and the plate is realized, and the localization is satisfactorily achieved.

  In contrast, a functional group having an affinity for a contaminating molecule (for example, C4, C8, C18, etc.) is bound to a part of the plate, thereby capturing the optionally derivatized contaminating molecule on the plate. In addition, the molecule to be measured and the contaminating molecule can be separated on the plate to localize the molecule to be measured (type 1-2).

  Furthermore, as a combination of the above types 1 and 2, both the measurement target molecule and the contamination molecule are separately captured on the plate, and the measurement target molecule and the contamination molecule are separated on the plate, and the measurement target molecule is localized. (Type 1-3). In such a case, for example, a predetermined functional group corresponding to the derivatization of the molecule to be measured and the derivatization of the contaminating molecule is appropriately selected, and the functional group having affinity for each derivatized molecule is separated from the plate. The localized region of each molecule can be separated.

  In any of the types 1-1 to 1-3, a plurality of types of molecules to be measured and / or a plurality of types of contaminant molecules are captured on the plate, and a predetermined plurality of molecules to be measured are localized. You can also plan. In such a case, different derivatization is performed according to a predetermined molecule to be measured and / or a contaminating molecule, and each functional group having an affinity for each molecule is separately bound to the plate and localized. The regions may be separated. Alternatively, the plates can be sandwiched in order using plates having functional groups that have affinity for each, and each molecule can be captured and separated on each plate.

(Plate creation method)
Below, the preparation method of the plate which combined the aryl group as an example of such a plate of types 1-1 to 1-3 is demonstrated.

  The method for producing the plate to which the aryl group is bonded is not particularly limited, but for example, it can be produced as follows. That is, the aryl group can be introduced into the plate according to, for example, the Micro Contact Contact Printing method (Harvard Whitesides et al. Langmuir Vol. 10.1498-1511 (1994). Specifically, it is made of polydimethylsiloxane. Then, the plate is dipped in an ethanol diluted solution of benzyl mercaptan [α-toluenethiol] for a predetermined time and air-dried, and then this plate is stamped on a separately prepared Au substrate for 3 seconds. Mercapto groups react with the gold surface to form Au-S bonds to form self-assembled monolayers (SAMs) Finally, the Au substrate was washed with ethanol and dried to bond the phenyl groups. Plates can be obtained: Aryl group applied to Au substrate It can not limited to the above stamp, spin coating, roll coating, also possible to employ various coating methods such as screen printing.

  As the metal material of the substrate used here, gold, silver, platinum, palladium, rhodium, ruthenium, or an alloy thereof capable of adsorbing thiol can be used. Among these, gold that can be stably produced and that does not undergo surface oxidation in the atmosphere is preferred in practice.

  When the aryl group is uniformly bonded to the entire surface of the plate, it is important to prepare a plate larger than the Au substrate because the aryl group is uniformly introduced to the end of the Au substrate. It is.

  In the introduction of the aryl group into such a plate, the aryl group can be bonded nonuniformly as well as uniformly bonded to the entire surface of the plate. For example, an aryl group can be bonded on the plate in a dot shape, or an aryl group can be bonded in a donut shape.

The aryl group in the present invention may be a monocyclic group such as phenyl group, thienyl group, pyrrolyl group, pyridyl group, or naphthyl group, anthryl group, pyrenyl group, quinolyl group, indolyl group, acrylidinyl group. Or a condensed polycyclic group such as a benzothiazolyl group. Moreover, not only one type of aryl group but also a plurality of types of aryl groups can be introduced into the plate. In such a case, for example, a plurality of aryl groups may be mixed and introduced, or the introduction region on the plate may be divided and unevenly distributed depending on the type of the aryl group. Note that a functional group such as COOH, SO ₃ H, NH ₂ , NO ₂ , OH, COOCH ₃ , and CN may be bonded to the aryl group.

  Furthermore, an aryl group and another compound such as boronic acid can be mixed and exist on the same plate, and these may be introduced separately in different regions.

The functional group introduced into the plate may be an alkyl group (for example, C4, C8, C18, etc.). Furthermore, functional groups such as COOH, SO ₃ H, NH ₂ , NO ₂ , OH, COOCH ₃ , and CN may be bonded to these alkyl groups.

  Further, a compound in which two or more single functional groups or a plurality of functional groups are bonded to the same molecule may be introduced.

[Process for placing biological sample containing target molecule]
(Measuring molecule)
“Moleculars to be measured” refers to sugars, sugar chains, proteins, nucleic acids, complex carbohydrates (glycoproteins, glycolipids, etc.) that are prepared from nature or chemically or enzymatically prepared. Including. The term “protein” in the present invention includes a peptide, and the term “glycoprotein” in the present invention includes a glycopeptide. Proteins may be modified including sugar chains, phosphates and the like. Moreover, what has the partial structure of the molecule | numerator contained in the biological body, or the thing produced by imitating the molecule | numerator contained in the biological body is included.

  The molecules to be measured may be used as they are, but by partially derivatizing them, the distribution of the derivatized and non-labeled substances to be measured at each point on the plate is compared. Mass confirmation can be made possible by confirmation of localization. Derivatization may be performed before placing on the plate, or may be performed after placing the molecule to be measured on the plate and drying. The derivatization may be labeling using a labeling compound. Hereinafter, derivatization will be described in detail.

A sugar chain (hereinafter referred to as a sugar chain) obtained by chemical or enzymatic release from a sugar, sugar chain, or complex carbohydrate is an aldehyde group of a sugar at the reducing end, an amino group or a hydrazide group (- A labeled compound which is a condensed polycyclic hydrocarbon derivative having CONHNH ₂ ) or the like is subjected to a condensation reaction to form a labeled intermediate, and then the labeled intermediate is reduced to give a reduction-labeled molecule. The condensation reaction is usually performed in an organic solvent such as methanol or DMSO by heating the sugar chain and the labeled compound in the presence or absence of acetic acid as a catalyst. Thereafter, a reducing agent such as NaCNBH ₃ or NaBH ₄ is added, and the reduction reaction is performed at warming or room temperature. Alternatively, a reducing agent may coexist from the beginning in the condensation reaction solution. In the method of labeling by condensing the aldehyde group of the sugar at the reducing end and PBH, the stability of the parent ion such as [M + Na] ⁺ or [M−H] ⁻ is low and the sensitivity is poor. For this reason, many parent ions can be produced | generated by performing a reductive reaction further.

  For sugars, sugar chains or complex carbohydrates containing sialic acid, a condensed polycyclic hydrocarbon derivative having an amino group, a hydrazide group, a diazomethyl group or the like is used as a labeling compound, and the carboxy group of sialic acid It can be labeled by reacting with an amino group, a hydrazide group, a diazomethyl group or the like of the labeling compound. This reaction is performed, for example, in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide.

Furthermore, saccharides, sugar chains, or complex carbohydrates containing sialic acid can selectively oxidize only the _C7-9 position of sialic acid to produce an aldehyde group. Selective oxidation of the C _7-9 position of sialic acid can be carried out by reacting in 5 mM NaIO ₄ aqueous solution at 0 ° C. for 10-20 minutes. Using a labeled compound that is a condensed polycyclic hydrocarbon derivative having an amino group or hydrazide group, the aldehyde group produced by the selective oxidation and the amino group or hydrazide group of the labeled compound are subjected to a condensation reaction as described above. By forming a labeled intermediate and then reducing the labeled intermediate, a reduction-labeled molecule can be obtained.

Alternatively, many sugars, sugar chains, or glycoconjugates contain galactose at the non-reducing end. The non-reducing terminal galactose can be specifically cause oxidation aldehyde groups of C ₆ position by galactose oxidase. The enzyme reaction can be performed, for example, in a neutral buffer solution at room temperature for 2 hours. Using a labeled compound which is a condensed polycyclic hydrocarbon derivative having an amino group or a hydrazide group, the aldehyde group generated by the above enzymatic oxidation and the amino group or hydrazide group of the labeled compound are subjected to a condensation reaction as described above. By forming a labeled intermediate and then reducing the labeled intermediate, a reduction-labeled molecule can be obtained.

  These methods of labeling galactose can be applied to sialic acid, galactose, etc. on glycoprotein (including glycopeptide) molecules. In that case, since the glycoprotein molecule can be labeled, the ionization efficiency of the molecule is increased.

  Proteins (including peptides) and glycoproteins (including glycopeptides) can be labeled with a carboxy group, amino group, or SH group contained therein. When labeling with a carboxy group, a condensed polycyclic hydrocarbon derivative having an amino group, a hydrazide group, a diazomethyl group, etc. is used as a labeling compound, and these groups react with a carboxy group in a protein or glycoprotein. To obtain a labeled molecule. On the other hand, when labeling with an amino group, a condensed polycyclic hydrocarbon derivative having a succinimidyl ester group, a sulfonyl chloride group or the like is used as a labeling compound, and these groups are combined with an amino group in a protein or glycoprotein. To give a labeled molecule. Furthermore, when labeling using the SH group of a cysteine residue contained in a protein or glycoprotein, a condensed polycyclic hydrocarbon derivative having an iodo group (-I) or the like is used as a labeling compound, Labeled molecules can be obtained by reacting with SH groups in proteins or glycoproteins.

  The labeling compound may be an aromatic compound or a condensed polycyclic hydrocarbon derivative. “Aromatic compound” means an aromatic ring part, a reactive functional group capable of binding to a molecule to be analyzed, and a spacer part that connects the aromatic ring part and the reactive functional group. The compound which has. Here, the aromatic ring moiety is a monocyclic aromatic ring such as a benzene ring, thiophene ring, pyrrole ring, pyridine ring, or naphthalene ring, anthracene ring, pyrene ring, quinoline ring, indole ring, acridine ring, benzothiazole. It may be a condensed aromatic ring such as a ring. “Condensed polycyclic hydrocarbon derivative” means a condensed polycyclic hydrocarbon moiety such as naphthalene, anthracene, pyrene, a reactive functional group capable of binding to a molecule to be analyzed, and the condensed polycyclic hydrocarbon And a spacer moiety that links the reactive functional group.

  The labeling compound used in the present invention is preferably a pyrene derivative compound. The “pyrene derivative compound” is a compound having a pyrene ring, a reactive functional group capable of binding to a molecule to be analyzed, and a spacer portion that connects the pyrene ring and the reactive functional group. Say. Specifically, 1-pyrenyldiazomethane, 1-pyrenebutanoic acid, hydrazide, 1-pyreneacetic acid, hydrazide, 1-pyrenepropionic acid hydrazide (1-pyrenepropionic acid, hydrazide), 1-pyreneacetic acid, succinimidyl ester, 1-pyrenepropionic acid, succinimidyl ester, 1-pyrenebutanoic acid Succinimidyl ester (1-pyrenebutanoic acid, succinimidyl ester), N- (1-pyrenebutanoyl) cysteic acid succinimidyl ester (N- (1-pyrenebutanoyl) cysteic acid, succinimidyl ester), N- (1-pyrene) iodo Acetamide (N- (1-pyrene) iodoacetamide), N- (1-pyrene) iodomaleimide (N- (1-pyrene) maleimide), N- (1-pyrene) Lenmethyl) 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 and the like. Among these, it is preferable to use 1-pyrenyldiazomethane in terms of high reactivity.

  Further, as an example of derivatization, alkylation that does not include an aromatic group may be performed.

(Biological sample placement method)
In the biological sample mounting step, the derivatized measurement molecule or the non-derivatized measurement molecule is dissolved in an appropriate solvent and dropped onto the plate with a dropping device such as a pipette or an automatic spotter. Drying may be left at room temperature or warmed if the molecule does not change. If necessary, a derivatization reaction is subsequently performed.

[Step of localizing target molecule on the plate]
(matrix)
The role of the matrix in the subsequent mass spectrometry is to absorb the light energy of the irradiated laser and cause the matrix itself to ionize and transfer protons or electrons to and from the molecule to be measured. Since the matrix has an inherent light energy absorption band depending on its type, the matrix used differs depending on the laser to be irradiated. In addition to the absorption of light energy, the matrix also has a role as a dispersing agent for uniformly dispersing the molecule to be measured at the molecular level. For this reason, it is important to select a matrix appropriately according to the difference in the molecule to be measured (protein, nucleic acid, saccharide, lipid, etc.).

  Examples of such a matrix include α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHBA), norharman, 6-aza-2-thiothymine ( 6-ATT), 2,4,6-trihydroxyacetophenone, etc. can be used. In order to achieve high localization, it is preferable to appropriately select the matrix concentration. Specifically, the matrix concentration needs to be selected depending on the measurement sample. The matrix concentration is particularly preferably from 0.1 to 20 mg / ml, for example, and particularly preferably from 2 to 10 mg / ml, since the crystal form can be controlled.

(solvent)
As the solvent for dissolving the matrix, acetone, methanol, ethanol, acetonitrile and the like, and a mixture of these with water can be used. In particular, the use of a mixed solution of acetonitrile and water is preferable in that it can be well mixed with the sample and the matrix to realize the subsequent localization. In order to achieve high localization, it is preferable to select a solvent concentration as appropriate. Specifically, the solvent concentration needs to be selected depending on the measurement sample. The solvent concentration, for example, acetonitrile concentration is preferably 0 to 100%, and particularly preferably 40 to 90%, from the viewpoint that the drying time and / or the drying process can be controlled.

(Localization method of target molecule)
Mixing of the matrix and the solvent is not particularly limited, and any known technique can be used. For example, 5 mg of DHBA (high purity matrix reagent, Shimadzu GL) is dissolved in 0.5 ml of water containing 40% acetonitrile, and a final concentration of 10 mg / ml solution is prepared at the time of use. At this time, the centrifuged supernatant is used if necessary.
In the step of localizing the molecule to be measured on the plate, first, the matrix solution is dropped onto the plate with a dropping device such as a pipette or an automatic spotter so as to cover the site where the measurement sample is placed. At this stage, the molecule to be measured is redissolved to form a mixed solution with the matrix.

  Next, by drying the mixed solution, the measurement target molecule is re-migrated to realize localization. Specifically, even if it is allowed to stand at room temperature, it may be heated if the molecule does not change. The drying is performed at a time when localization sufficiently occurs, depending on the measurement molecule. The drying time is preferably 2 minutes to 24 hours. Furthermore, from the viewpoint of shortening the analysis time and not changing the sample and crystal form, it is more preferable that the time is 5 minutes to 30 minutes.

  According to such a localization method (type 1) of a molecule to be measured on a plate, when the biological sample includes at least two kinds of molecules to be measured, in step (A), at least two of the plates In the step (C), different compounds to be measured can be localized in at least two regions by binding different compounds to the regions. Further, when the biological sample contains at least one kind of contaminating molecule, in step (A), different compounds are bound to at least two regions of the plate, and in step (C), molecules to be measured are detected in one region of the plate. In other areas, contaminating molecules can be localized.

<Localization method of molecule to be measured on plate (type 2)>
[Process for preparing plates]
In Type 2, the step of preparing a plate includes (A1) a step of binding a first compound that localizes a molecule to be measured on the first plate, and (A2) a molecule to be measured on the second plate. Binding a second compound that is localized and different from the first compound.

  The type of plate used in this step is the same as type 1 described above. However, different compounds are bound to the first plate and the second plate. Thereby, as will be described later, different kinds of molecules to be measured can be localized on different plates.

[Process for placing biological sample containing target molecule]
In Type 2, the step of placing the biological sample containing the molecule to be measured includes (B1) a step of sandwiching and drying the biological sample containing at least one type of molecule to be measured between the first plate and the second plate. And (B2) separating the first plate and the second plate.

  The kind of molecule to be measured used in this step is the same as that of Type 1 described above. When a biological sample containing a molecule to be measured is sandwiched between the first plate and the second plate, it is preferable to place a weight or the like so that the biological sample is placed in close contact with each other because the localization can be performed with high efficiency. In addition, when the biological sample is dried, it is preferable that the biological sample is dried over an appropriate time and further completely dried from the viewpoint that localization can be achieved with high efficiency. For example, it is preferable to dry at room temperature to 45 ° C. for 30 minutes to 24 hours.

[Step of localizing target molecule on the plate]
In Type 2, the step of localizing the molecule to be measured on the plate is as follows. (C) The solution containing the matrix is dropped on the first plate and the second plate and dried to localize the molecule to be measured on the plate. It is a process to convert.

  About the matrix used in this process, and the kind of solvent, it is the same as that of the above-mentioned type 1.

  According to such a localization method (type 2) of a molecule to be measured on the plate, when the biological sample includes at least two kinds of molecules to be measured, in step (C), the measurement target of the first plate A molecule to be measured different from the molecule can be localized on the second plate. Further, when the biological sample contains at least one kind of contaminating molecule, in the step (C), at least one kind of contaminating molecule can be localized on the second plate.

<Mass Spectrometry (Type 1)>
The method for mass spectrometry of the molecule to be measured of the present invention is as follows.
(1) a method of localizing a molecule to be measured on the plate by a method (type 1) for localizing the molecule to be measured on the plate, and (2) localized in the above step (1) The method includes the step of performing MS ⁿ measurement on a molecule to be measured, wherein n is an integer of 1 or more.

(Process (1))
Step 1 is a step of performing the above-described localization method of the molecule to be measured on the plate. This process is as described above.

(Process (2))
Step 2 is a step of performing MS ⁿ measurement on the molecule to be measured localized in Step 1, and n is a step in which n is an integer of 1 or more.

  “Mass spectrometry” means matrix-assisted laser desorption ionization (MALDI) method, laser desorption (LD) method, fast electron impact (FAB) method, electrospray ionization (ESI) method, atmospheric pressure chemistry (APCI) method Samples containing molecules are ionized by ionization methods such as time-of-flight method (TOF method), double-focusing method, quadrupole focusing method, etc. It is a method of separating and detecting according to the ratio (m / z).

  In the present invention, the ionization method is not limited, but is preferably the LD method, FAB method, or MALDI method, and more preferably the MALDI method.

  In the MALDI method, the ionization efficiency is increased because the methylated or condensed polycyclic hydrocarbon is bonded to the molecule.

Since molecules such as sugars, sugar chains, proteins, sugar-modified proteins, nucleic acids, glycolipids, etc. have structural isomers having the same molecular weight and composition, precursor ions are labeled after labeling with condensed polycyclic hydrocarbon derivatives. The structure information can be obtained by performing MS ⁿ (n> 1) analysis and generating ions specific to the isomer. When MS ⁿ (n> 1) analysis is performed on a molecule labeled on a sugar chain part of a glycopeptide or glycoprotein, a sugar chain binding position in the molecule can be determined by selecting a product ion containing the label.

  In the type 1 mass spectrometry method, any method usually used in the technical field can be used.

<Mass Spectrometry (Type 2)>
The method for mass spectrometry of the molecule to be measured of the present invention is as follows.
(1) Localizing the molecule to be measured in the biological sample on the plate by a method (type 2) for localizing the molecule to be measured on the plate; This is a method including the step of performing MS ⁿ measurement on the activated molecule to be measured, wherein n is an integer of 1 or more.

  (Step (1)), (Step (2)), the term “mass spectrometry” and the like are the same as the above-described method (type 1) for localizing a molecule to be measured on a plate.

  In the type 2 mass spectrometry method, any method usually used in the technical field can be used.

  Below, the effect of this invention is demonstrated based on an Example.

Example 1
1-1. Preparation of Phenyl Plate A phenyl group was introduced into the plate according to the Micro / Contact / Printing method. Specifically, a plate material made of polydimethylsiloxane was prepared, and dipped in an ethanol diluted solution (2 mM) of benzyl mercaptan [α-toluenethiol] for 10 minutes and air-dried. Next, stamping was performed for 3 seconds on a separately prepared Au substrate. Thereafter, the substrate was washed with ethanol and dried to obtain a phenyl plate. In this example, since the phenyl group was entirely coated on the plate, a plate larger than the Au substrate was used as the plate material.

1-2. Localization and Mass Spectrometry of Measurement Object Molecule Sugar chain 1 (500 fmol) was placed on the plate prepared in advance as described above and dried. On top of that, 0.25 μL of DMSO solution containing 500 pmol of 1-pyrenyldiazomethane was added dropwise and heated to 40 ° C. for 25 minutes to dry, thereby obtaining labeled sugar chain 1 partially labeled with pyrene. As a matrix, 0.5 μL of DHBA (10 mg / ml solution, 40% acetonitrile) was added dropwise so as to cover the sample and dried at room temperature to prepare a labeled sugar chain-containing measurement sample 1 shown in FIG.

  The obtained measurement sample 1 was divided into a total of 441 regions of X × Y = 21 × 21, and negative ions in each region were measured by a raster scan of a mass spectrometer (AXIMA-QIT, Shimadzu Corporation). The information of the measurement file of each region obtained by the raster scan was converted into text including measurement position coordinates (x, y), m / z, and signal intensity. Based on the text-converted data, the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) and the distribution of deprotonated ions derived from unlabeled sugar chain 1 (m / z = 2077) are disclosed. Displayed two-dimensionally using Soft Graph-R.

  FIG. 2 shows the signal intensity of the deprotonated ion derived from the labeled sugar chain 1, and FIG. 3 shows the signal intensity of the deprotonated ion derived from the unlabeled sugar chain 1. FIG. 4 shows a mass spectrum at point A in FIGS. FIG. 5 shows a mass spectrum at a point B in FIGS. 4 and 5 are shown such that the maximum values are the same.

  2 and 3, it was found that the regions where the labeled sugar chain ion and the unlabeled sugar chain ion are generated are different, in other words, the localization of the labeled sugar chain can be realized. Also, according to FIGS. 4 and 5, from the results of FIGS. 2 and 3, a predetermined region, that is, a region in which the point of FIG. 2 is darker among the points of FIGS. Only when () was selected, it was found that a high signal intensity compared with other ions of the desired ion (labeled sugar chain 1-derived deprotonated ion) was selectively obtained.

  The above effect was achieved by using a plate in which the plate was subjected to predetermined processing and bonded with an aryl group (phenyl group), thereby demonstrating the effect of the present application.

(Comparative Example 1)
Scanning was performed in the same manner as in Example 1 using a generally used stainless steel plate instead of the phenyl plate. The results are shown below.

  FIG. 6 shows the signal intensity of the deprotonated ion derived from the labeled sugar chain 1, and FIG. 7 shows the signal intensity of the deprotonated ion derived from the unlabeled sugar chain 1. FIG. 8 shows only the mass spectrum at point C in FIGS.

  According to FIGS. 6 and 7, it was found that the regions where the labeled sugar chain ion and the unlabeled sugar chain ion are generated are the same, in other words, the localization of the labeled sugar chain has not been realized. In FIG. 8, only the mass spectrum at the point C in FIGS. 6 and 7 is displayed. However, since there is no difference in the color intensity of each point in FIGS. It is expected that the result of FIG. 8 will be obtained.

  The above is a result obtained by using a conventional stainless steel plate without performing predetermined processing on the plate.

(Example 2)
A commercially available glycoprotein prostate specific antigen was electrophoresed and then digested in the gel with lysyl endoprotease C, digested with peptide N glycosidase F, and a C18 chip flow-through fraction was obtained. This fraction was placed as a sample on the phenyl plate prepared in Example 1, and scanning was performed in the same manner as in Example 1 below. The results are shown below.

  FIG. 9 shows the signal intensity of peptide ions (m / z = 1075), and FIG. 10 shows the signal intensity of labeled sugar chain 1-derived deprotonated ions (m / z = 2582). FIG. 11 shows a mass spectrum at a point D in FIGS. FIG. 12 shows a mass spectrum at point E in FIGS. 11 and 12 are shown such that the maximum values are the same.

  9 and 10, it was found that the regions where peptide ions and labeled sugar chain ions are generated are different, in other words, localization of peptide ions and labeled sugar chains can be realized. Further, according to FIGS. 11 and 12, a predetermined region based on the results of FIGS. 9 and 10, that is, a region with a darker color (for example, a point E in the point of FIG. Only when () was selected, it was found that a high signal intensity compared with other ions of the desired ion (labeled sugar chain 1-derived deprotonated ion) was selectively obtained.

  The above effect was achieved by using a plate in which the plate was subjected to predetermined processing and bonded with an aromatic compound, thereby demonstrating the effect of the present application.

  Furthermore, conventionally, when a peptide is mixed with a sugar chain in a measurement sample, since the peptide is easily ionized, sugar chain ions were hardly detected in mass spectrometry. However, in Example 2, separation of both substances by localization in a microregion is realized even if a peptide is mixed with a sugar chain, and even a trace sugar chain derived from a biological sample can be identified by mass spectrometry. It is expected that.

(Example 3)
Example 3 is an example in which a glycopeptide and a peptide are localized using a phenyl plate. First, purification of the PSA-ACT-derived glycopeptide was performed as follows. That is, 95 μL of 50 mM ammonium bicarbonate aqueous solution containing 0.5 μg of PSA-ACT and 5 μL of 10 U / μL thermolysin aqueous solution were added to a 0.6 mL tube, and the mixed solution was stirred and incubated at 56.5 ° C. overnight. This mixed solution is dried with a centrifugal evaporator to obtain a dried product, and 150 μL of 0.8% trifluoroacetic acid (TFA) is added to the dried product, and the mixture is reacted at 80 ° C. for 40 seconds using a heat block. Obtained samples were obtained.

Next, H ₂ O was added to the sample and dried repeatedly. Specifically, 20 μL of H ₂ O, 20 μL of ethanol, and 80 μL of butanol were added to the sample and stirred well, and 10 μL of washed and equilibrated Sepharose CL-4B gel suspension (1: 1) was added and stirred well. Thereafter, it was shaken for 1 hour. The Sepharose CL-4B gel was washed and equilibrated three times with 50% ethanol and three times with a mixed solution of butanol / ethanol / water (4: 1: 1). After shaking, the supernatant was discarded by centrifugation, and this was repeated for washing. 100 μL of 50% ethanol was added and shaken for 30 seconds. After centrifugation, the supernatant was collected and dried with a centrifugal evaporator.

  Finally, localization and MS measurement using a phenyl plate were performed in the same manner as in Example 1. That is, 1 μL of 10 mg / mL DHBA dissolved in 60% acetonitrile was dropped, air-dried, and measured by AXIMA-QIT. As a result, negative ion MS spectra are shown in FIGS. 13 shows the result of MS measurement without pyrene labeling, and FIG. 14 shows the result of MS measurement with pyrene labeling.

  According to FIGS. 13 and 14, only peptide signal a was detected when pyrene labeling was not performed. In contrast, when pyrene labeling was performed, it was revealed that the signals b, c, and d of the glycopeptide were specifically detected. Similar results were obtained in the positive ion MS spectrum.

  Furthermore, FIG. 15 shows an example of the MS spectrum of a specific region of the measurement sample. Compared with the average spectrum of all measurement points shown in FIGS. 13 and 14, it was shown that there are clearly measurement points where the signal intensity of only the glycopeptide is relatively high, that is, localized. .

Example 4
Example 4 is an example in which localization was performed by sandwiching a glycopeptide and a peptide between a C18 plate and a phenyl plate. First, the C18 plate and the phenyl plate were produced as follows. That is, the phenyl plate was prepared in the same manner as in Example 1. In order to hold the measurement sample solution, a sheet made of PDMS (polydimethylsiloxane) with a hole having a diameter of 3 mm (thickness: 100 μm, outer shape is substantially the same as that of the phenyl plate) was prepared and attached. Adhesion is not used because of the self-adsorption of the PDMS sheet. A C18 plate was prepared in the same manner as the phenyl plate. A plate material made of polydimethylsiloxane was prepared and immersed in an ethanol dilution (2 mM) of [1-octadecanethiol] for 10 minutes and air-dried. Next, stamping was performed for 3 seconds on a separately prepared Au substrate. Thereafter, the substrate was washed with ethanol and dried. In this example, since C18 was coated on the entire surface of the plate, a material larger than the Au substrate was used as the plate material.
The glycopeptide was prepared in the same manner as in Example 3.

  Further, localization and MS measurement using C18 plate and phenyl plate were performed as follows. That is, 12.5 μL of 5% acetonitrile was added to the dried PSA-ACT-derived glycopeptide and dissolved. An acetonitrile solution containing 5% of 4 pmol / μL adrenocorticotropic hormone fragment (ACTH) fragment (Adrenocorticotropic Hormone Fragment 18-39 human, Sigma) was mixed with PSA-ACT-derived glycopeptide as a peptide to prepare a measurement sample. A sample solution of 0.5 μL was dropped onto a phenyl plate to which a PDMS sheet with a hole was attached, and a C18 plate was placed on top, and a weight was placed on the plate to bring it into close contact. The adhesion body was placed on a heat block and left in this state at 40 ° C. for 2.5 hours to evaporate the solvent. After peeling off the phenyl plate and the C18 plate, the PDMS sheet was peeled off, and a 0.25 μL dimethyl sulfoxide (DMSO) solution of 500 pmol of 1-pyrenyldiazomethane (PDAM) was dropped for pyrene labeling and left at 40 ° C. And reacted. After drying, it was further dried in a vacuum desiccator overnight.

  To this dried product, 1 μL of 10 mg / mL 2,5-dihydroxybenzoic acid (DHBA) dissolved in 60% acetonitrile was dropped, air-dried, and measured with AXIMA-QIT. As a result, a negative ion MS spectrum was shown in FIG. , 17 (for phenyl plates) and 18, 19 (for C18 plates). 16 and 18 show the results of MS measurement without pyrene labeling, and FIGS. 17 and 19 show the results of MS measurement with pyrene labeling.

  According to FIGS. 16-19, only the peptide signal a was detected in any plate, when pyrene labeling was not performed. In addition, according to FIG. 16, it turns out that the signal of a peptide has fallen to 1/2 of Example 3 (FIG. 13).

  On the other hand, when pyrene labeling is performed, it is understood that the signals b, c and d of the glycopeptide are not detected on the C18 plate, but are specifically detected on the phenyl plate. Similar results were obtained in the positive ion MS spectrum.

  From these results (Examples 3 and 4), in Example 4, the peptide, which is a contaminant molecule in the measurement sample on the phenyl plate, was captured by the C18 plate superimposed thereon, and the signal intensity decreased. It can be seen that the glycopeptide remains on the phenyl plate and is localized and does not reduce the signal. Further, in Example 4, it was shown that the signal / noise ratio was improved as compared with Example 3. That is, it can be said that Example 4 achieves more effective localization than Example 3. In addition, it is thought that this effect is acquired also in what arranged the C18 coupling | bond part and the phenyl group coupling | bond part on the same plate.

(Example 5)
The localization was performed by mounting the glycopeptide and peptide on the amino plate or sandwiching the glycopeptide and peptide between the C8 plate and amino plate. In these examples, the same operation as in Examples 3 and 4 was performed except for the type of plate. In Example 5, the results relating to the amino plate after localization, with the glycopeptide sandwiched between the C8 plate and the amino plate, and then separating these plates are shown in FIGS. In contrast, in Example 5, a glycopeptide or the like was placed on the amino plate for localization, and then the results relating to the amino plate are shown in FIGS. In these figures, FIGS. 20 and 22 show the results of MS measurement without pyrene labeling, and FIGS. 21 and 23 show the results of MS measurement with pyrene labeling.

  According to the results of measurement without pyrene labeling (FIGS. 20 and 22), only contaminating peptides were observed. On the other hand, according to the results of measurement using pyrene labeling (FIGS. 21 and 23), when the C8 plate was overlapped (FIG. 23), compared to the case of the amino plate localized without overlapping C8 (FIG. 23) FIG. 21) shows that the signal intensity of the glycopeptide is excellent as in Example 4. As described above, it was found that even when the C8 plate and the amino plate were used, the affinity between the glycopeptide and the amino plate was high, and the signal of the glycopeptide was observed even in the presence of the contaminating peptide.

  The localization method of the molecule to be measured on the plate of the present invention can secure a sufficient remaining amount on the plate without washing the molecule to be measured, and can be used for mass analysis of unknown molecules to be measured. In addition, it can also be used when a plurality of molecules to be measured are subjected to mass spectrometry at the same time. Therefore, the method is promising in that it can be suitably applied to mass spectrometry of various molecules, which are expected to be used more frequently in the fields of biochemistry and medicine.

  The present invention relates to a method for localizing a molecule to be measured on a plate. More specifically, the present invention relates to a method capable of selectively localizing only a measurement object from a biological sample on a plate. The present invention relates to a mass spectrometry method that uses such a method to obtain a high signal intensity of a molecule to be measured.

  In recent years, high-sensitivity mass spectrometry methods for proteins and / or sugar chains, which are biopolymers, have been actively developed, and the analysis methods are rapidly spreading in the fields of biochemistry and medicine. Such mass spectrometry is used, for example, for analysis of biomolecule expression and its function, biomarker search, and the like.

  In a biological sample containing a biopolymer, many contaminating molecules are mixed in addition to the molecule to be measured. For this reason, when a biological sample containing a very small amount of a molecule to be measured is highly purified, the mass analysis sensitivity of the molecule to be measured may be less than the detection sensitivity, and thus the molecule may be missed in mass spectrometry. In addition, when a contaminating molecule has a higher ionization efficiency than the measurement target molecule, it is difficult to detect the measurement target molecule.

  Considering such circumstances, for example, the following is disclosed as a technique related to ionization of a biological sample as a previous stage of mass spectrometry.

  Non-Patent Document 1 discloses that surface-enhanced laser desorption / ionization (SELDI) is used to immobilize functional groups and / or antibody molecules on the chip surface, capture only specific molecules in a biological sample, wash them, and perform laser irradiation. Discloses a technique for performing ionization.

  Patent Document 1 discloses a technique for monitoring the purification of a target molecule, including, for example, creating a surface enhanced laser desorption ionization (SELDI) mass spectral profile of biological components in a cell culture medium or a purification fraction of such medium. Has been.

JPO homepage, sample room, standard technology collection, general, mass spectrometry (mass spectrometry), 2-1-1-1-1 surface enhanced laser desorption ionization (SELDI) (http: //www.jpo.go (See .jp / shiryou / index.htm) JP 2005-509173 A

  Prior to mass spectrometry, a technique (SELDI) disclosed in Non-Patent Document 1 and Patent Document 1 that leaves only a molecule to be measured by a predetermined washing with respect to a biological sample on a plate is adopted. In some cases, some of the molecules to be measured are washed away when the biological sample is washed. For this reason, when the amount of the molecule to be measured is very small and / or the binding is weak, the amount of the molecule to be measured on the plate is extremely small, and the analysis sensitivity is much higher than the desired detection sensitivity in mass spectrometry. May fall below.

  In addition, mass spectrometry after adopting SELDI is effective when the molecule to be measured is known, but is not effective when the molecule to be measured is unknown, because the molecule may be completely removed from the plate. .

  Furthermore, in mass spectrometry after adopting SELDI, by selecting a predetermined chip and performing a predetermined washing, it is possible to limit to a specific type of measurement target molecule having the maximum content or the strongest binding. However, in actual mass spectrometry, it may be desirable to simultaneously analyze a plurality of molecules to be measured with the amount of biological sample used for one SELDI measurement.

  The object of the present invention is to (a) ensure a sufficient remaining amount on the plate without washing away the measurement target molecule, (b) can be used for mass spectrometry of unknown measurement target molecules, and (c) multiple Another object of the present invention is to provide a method for localizing a molecule to be measured on a plate, which can be used when mass-analyzing the molecules to be measured simultaneously. Another object of the present invention is to provide a mass spectrometry method that can obtain a high signal intensity of a molecule to be measured using such a method.

  The present invention includes (A) a step of preparing a plate to which at least one compound for localizing a molecule to be measured is bound, and (B) a biological sample containing at least one molecule to be measured on the plate. And (C) dropping and drying a solution containing a matrix on the biological sample and localizing the molecule to be measured on the plate, the molecule to be measured on the plate Relates to a method for localizing. Here, the biological sample placed on the plate always contains at least one kind of measurement target molecule. In other words, the case where the biological sample is composed of only contaminating molecules is excluded.

  In such a method for localizing a molecule to be measured on a plate, the compound preferably contains an aryl group, a dihydroxyboryl group, an alkyl group, an amino group, or a hydroxyl group. Among these, the aryl group can be a phenyl group, a thienyl group, a pyrrolyl group, a pyridyl group, a naphthyl group, an anthryl group, a pyrenyl group, a quinolyl group, an indolyl group, an acrylidinyl group, or a benzothiazolyl group. The alkyl group may be a C4 to C18 alkyl group.

  The localization method can localize a single molecule to be measured on the plate, and can also localize a plurality of molecules to be measured on the plate. That is, when the biological sample contains at least two kinds of molecules to be measured, different compounds are bound to at least two regions of the plate in the step (A), and at least two regions in the step (C). It is possible to localize different molecules to be measured.

  The localization method can perform not only localization of a molecule to be measured but also localization of a contaminating molecule. That is, when the biological sample further contains at least one kind of contaminant molecule, in the step (A), a different compound is bound to at least two regions of the plate, and in the step (C), The molecule to be measured can be localized in one region and the contaminating molecule can be localized in another region.

  In such a method for localizing a molecule to be measured on a plate, it is desirable to further include (B ′) a step of derivatizing the biological sample between steps (B) and (C). .

  The measurement target molecule can be, for example, a sugar chain (free sugar chain, glycopeptide, glycoprotein, etc.).

The present invention is a method for mass spectrometry of a molecule to be measured,
(1) a step of localizing a molecule to be measured in a biological sample on a plate by any one of the above methods, and (2) a molecule to be measured localized in the step (1) above, It includes a mass spectrometry method including a step of performing MS ⁿ measurement, wherein n is an integer of 1 or more.

  The present invention includes (A1) binding a first compound that localizes the molecule to be measured on the first plate, and (A2) localizing the molecule to be measured on the second plate. A step of binding a second compound different from the compound, and (B1) sandwiching a biological sample containing at least one molecule to be measured between the first plate and the second plate, and drying (B1) B2) separating the first plate and the second plate; and (C) dropping and drying a solution containing a matrix on the first plate and the second plate, so that the first plate and the second plate are separated. And a method of localizing the molecule to be measured on the plate, comprising the step of localizing the molecule to be measured on at least one of the plates. Here, the biological sample placed on the plate always contains at least one kind of measurement target molecule. In other words, the case where the biological sample is composed of only contaminating molecules is excluded.

  In such a method for localizing a molecule to be measured on a plate, the compound preferably contains an aryl group, a dihydroxyboryl group, an alkyl group, an amino group, or a hydroxyl group. Among these, the aryl group can be a phenyl group, a thienyl group, a pyrrolyl group, a pyridyl group, a naphthyl group, an anthryl group, a pyrenyl group, a quinolyl group, an indolyl group, an acrylidinyl group, or a benzothiazolyl group. The alkyl group may be a C4 to C18 alkyl group.

  The localization method can localize different measurement target molecules on two plates. That is, when the biological sample includes at least two kinds of molecules to be measured, in the step (C), a molecule to be measured different from the molecule to be measured on the first plate can be localized on the second plate. it can.

  The localization method not only can localize different measurement target molecules in each of the two plates, but also localizes the measurement target molecules and the contaminant molecules in each of the two plates. Can do. That is, when the biological sample further contains at least one kind of contaminating molecule, in the step (C), at least one kind of contaminating molecule can be localized on the second plate.

  In such a method for localizing a molecule to be measured on a plate, it is desirable to further include (B ′) a step of derivatizing the biological sample between steps (B2) and (C). .

  The measurement target molecule can be, for example, a sugar chain (free sugar chain, glycopeptide, glycoprotein, etc.).

A method for mass spectrometry of a molecule to be measured,
(1) Localizing at least one of the measurement target molecule in the biological sample on the first plate and the measurement target molecule in the biological sample on the second plate by any one of the methods described above, and (2) Mass spectrometry including a step of performing MS ⁿ measurement on the measurement target molecule localized in the step (1), wherein n is an integer of 1 or more.

  In the localization method of a molecule to be measured on the plate of the present invention, the affinity between the molecule to be measured and the plate is obtained by performing a predetermined surface treatment (binding of a predetermined compound) in advance on the plate on which the biological sample is placed. The affinity can be increased more than the affinity between other contaminant molecules and the plate. For this reason, the measurement target molecule can be selectively and sufficiently left on the plate simply by drying the biological sample containing the measurement target molecule on the plate.

  In addition, since the localization method of the present invention employs a technique of drying on the plate without washing away the molecule to be measured like SELDI, naturally the unknown molecule to be measured remains on the plate. For this reason, this method can be applied even if the target molecule to be subjected to mass spectrometry is unknown.

  Furthermore, since the localization method of the present invention does not wash away the measurement target molecule unlike SELDI, a plurality of measurement target molecules can remain in the dried biological sample. For this reason, this method can be applied even if there are a plurality of molecules to be measured.

  In addition, according to the mass spectrometry using the localization method of the present invention that exhibits the effects as described above, it is possible to obtain a sufficient signal intensity of the molecule to be measured, and to deal with an unknown molecule to be measured, In addition, a plurality of molecules to be measured can be analyzed simultaneously.

FIG. 1 is a photograph of the measurement sample obtained in Example 1. FIG. 2 is a graph showing the distribution of unlabeled sugar chain 1-derived deprotonated ions (m / z = 2077) in Example 1. FIG. 3 is a graph showing the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) in Example 1. FIG. 4 is a diagram showing a mass spectrum at point A in FIGS. FIG. 5 is a diagram showing a mass spectrum at a point B in FIGS. FIG. 6 is a diagram showing the distribution of unlabeled sugar chain 1-derived deprotonated ions (m / z = 2077) with respect to Comparative Example 1. FIG. 7 is a view showing the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) in Comparative Example 1. FIG. 8 is a diagram showing a mass spectrum at a point C in FIGS. FIG. 9 is a graph showing the distribution of peptide ions (m / z = 1075) in Example 2. FIG. 10 is a graph showing the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) in Example 2. FIG. 11 is a diagram showing a mass spectrum at a point D in FIGS. FIG. 12 is a diagram showing a mass spectrum at a point E in FIGS. FIG. 13 is a diagram showing an MS spectrum of negative ions (average of all measurement points) when pyrene labeling is not performed in Example 3. FIG. 14 is a diagram showing an MS spectrum (average of all measurement points) of negative ions when pyrene labeling is performed in Example 3. FIG. 15 is a diagram showing an MS spectrum of negative ions in a specific region when pyrene labeling is performed in Example 3. FIG. 16 is a diagram showing an MS spectrum of negative ions of a phenyl plate after separation when pyrene labeling is not performed in Example 4. FIG. 17 is a diagram showing an MS spectrum of negative ions when pyrene labeling is performed on the phenyl plate after separation in Example 4. FIG. 18 is a diagram showing an MS spectrum of negative ions of the C18 plate after separation in Example 4 when no pyrene labeling is performed. FIG. 19 is a diagram showing the MS spectrum of negative ions when pyrene labeling is performed on the C18 plate after separation in Example 4. FIG. 20 is a diagram showing an MS spectrum of negative ions of the amino plate after separation in Example 5 when no pyrene labeling was performed. FIG. 21 is a diagram showing a negative ion MS spectrum of pyrene-labeled amino plates after separation in Example 5. FIG. 22 is a diagram showing an MS spectrum of negative ions of Example 5 when no pyrene labeling is performed in Example 5. 23 is a diagram showing an MS spectrum of negative ions when pyrene labeling is performed on an amino plate in Example 5. FIG.

  Embodiments of the present invention will be described below, but the present invention is not limited to these ranges, and can be appropriately changed within the range of ordinary creation ability of those skilled in the art.

<Localization method of molecule to be measured on plate (Type 1)>
The method (type 1) for localizing a molecule to be measured on the plate of the present invention comprises (A) preparing a plate to which at least one compound that localizes the molecule to be measured is bound; B) A step of placing a biological sample containing at least one molecule to be measured on the plate; and (C) dropping and drying a solution containing a matrix on the biological sample, and then measuring the measurement target on the plate. And localizing the molecule. Here, the molecule to be measured means a molecule to be measured in mass spectrometry performed after performing the localization method.

[Process for preparing plates]
(plate)
The plate is not particularly limited as long as it has a functional group capable of capturing at least one of a molecule to be measured and a contaminating molecule in a biological sample, and is either a metal plate or a non-metal film. You can also. Moreover, the surface of the plate can have any shape such as a flat shape or an uneven shape, and can also be made porous. Furthermore, a predetermined functional group may not be directly bonded to the plate, for example, a spacer portion or a polymer between the plate and the functional group may be interposed.

  Examples of capturing a molecule to be measured on a plate (type 1-1) include a plate to which an aryl group is bonded, a plate to which a dihydroxyboryl group is bonded, a plate to which an amino group is bonded, or a plate to which a hydroxy group is bonded. An example of using a different plate is given. These plates are an example of a plate that has higher affinity for the molecule to be measured than affinity for other contaminant molecules. In particular, when pyrene labeling is performed on a measurement target molecule, it is possible to achieve a high affinity between the labeled measurement target molecule and the plate by using a plate to which an aryl group is bonded, and to achieve good localization. It is preferable at the point achieved. In addition, if the molecule to be measured contains a sugar chain, it is possible to use a plate with a dihydroxyboryl group, a plate with an amino group, a plate with a hydroxy group, etc. This is preferable in that a high affinity between the target molecule and the plate is realized, and the localization is satisfactorily achieved.

  In contrast, a functional group having an affinity for a contaminating molecule (for example, C4, C8, C18, etc.) is bound to a part of the plate, thereby capturing the optionally derivatized contaminating molecule on the plate. In addition, the molecule to be measured and the contaminating molecule can be separated on the plate to localize the molecule to be measured (type 1-2).

  Furthermore, as a combination of the above types 1 and 2, both the measurement target molecule and the contamination molecule are separately captured on the plate, and the measurement target molecule and the contamination molecule are separated on the plate, and the measurement target molecule is localized. (Type 1-3). In such a case, for example, a predetermined functional group corresponding to the derivatization of the molecule to be measured and the derivatization of the contaminating molecule is appropriately selected, and the functional group having affinity for each derivatized molecule is separated from the plate. The localized region of each molecule can be separated.

  In any of the types 1-1 to 1-3, a plurality of types of molecules to be measured and / or a plurality of types of contaminant molecules are captured on the plate, and a predetermined plurality of molecules to be measured are localized. You can also plan. In such a case, different derivatization is performed according to a predetermined molecule to be measured and / or a contaminating molecule, and each functional group having an affinity for each molecule is separately bound to the plate and localized. The regions may be separated. Alternatively, the plates can be sandwiched in order using plates having functional groups that have affinity for each, and each molecule can be captured and separated on each plate.

(Plate creation method)
Below, the preparation method of the plate which combined the aryl group as an example of such a plate of types 1-1 to 1-3 is demonstrated.

  The method for producing the plate to which the aryl group is bonded is not particularly limited, but for example, it can be produced as follows. That is, the aryl group can be introduced into the plate according to, for example, the Micro Contact Contact Printing method (Harvard Whitesides et al. Langmuir Vol. 10.1498-1511 (1994). Specifically, it is made of polydimethylsiloxane. Then, the plate is dipped in an ethanol diluted solution of benzyl mercaptan [α-toluenethiol] for a predetermined time and air-dried, and then this plate is stamped on a separately prepared Au substrate for 3 seconds. Mercapto groups react with the gold surface to form Au-S bonds to form self-assembled monolayers (SAMs) Finally, the Au substrate was washed with ethanol and dried to bond the phenyl groups. Plates can be obtained: Aryl group applied to Au substrate It can not limited to the above stamp, spin coating, roll coating, also possible to employ various coating methods such as screen printing.

  As the metal material of the substrate used here, gold, silver, platinum, palladium, rhodium, ruthenium, or an alloy thereof capable of adsorbing thiol can be used. Among these, gold that can be stably produced and that does not undergo surface oxidation in the atmosphere is preferred in practice.

  When the aryl group is uniformly bonded to the entire surface of the plate, it is important to prepare a plate larger than the Au substrate because the aryl group is uniformly introduced to the end of the Au substrate. It is.

  In the introduction of the aryl group into such a plate, the aryl group can be bonded nonuniformly as well as uniformly bonded to the entire surface of the plate. For example, an aryl group can be bonded on the plate in a dot shape, or an aryl group can be bonded in a donut shape.

The aryl group in the present invention may be a monocyclic group such as phenyl group, thienyl group, pyrrolyl group, pyridyl group, or naphthyl group, anthryl group, pyrenyl group, quinolyl group, indolyl group, acrylidinyl group. Or a condensed polycyclic group such as a benzothiazolyl group. Moreover, not only one type of aryl group but also a plurality of types of aryl groups can be introduced into the plate. In such a case, for example, a plurality of aryl groups may be mixed and introduced, or the introduction region on the plate may be divided and unevenly distributed depending on the type of the aryl group. Note that a functional group such as COOH, SO ₃ H, NH ₂ , NO ₂ , OH, COOCH ₃ , and CN may be bonded to the aryl group.

  Furthermore, an aryl group and another compound such as boronic acid can be mixed and exist on the same plate, and these may be introduced separately in different regions.

The functional group introduced into the plate may be an alkyl group (for example, C4, C8, C18, etc.). Furthermore, functional groups such as COOH, SO ₃ H, NH ₂ , NO ₂ , OH, COOCH ₃ , and CN may be bonded to these alkyl groups.

  Further, a compound in which two or more single functional groups or a plurality of functional groups are bonded to the same molecule may be introduced.

[Process for placing biological sample containing target molecule]
(Measuring molecule)
“Moleculars to be measured” refers to sugars, sugar chains, proteins, nucleic acids, complex carbohydrates (glycoproteins, glycolipids, etc.) that are prepared from nature or chemically or enzymatically prepared. Including. The term “protein” in the present invention includes a peptide, and the term “glycoprotein” in the present invention includes a glycopeptide. Proteins may be modified including sugar chains, phosphates and the like. Moreover, what has the partial structure of the molecule | numerator contained in the biological body, or the thing produced by imitating the molecule | numerator contained in the biological body is included.

  The molecules to be measured may be used as they are, but by partially derivatizing them, the distribution of the derivatized and non-labeled substances to be measured at each point on the plate is compared. Mass confirmation can be made possible by confirmation of localization. Derivatization may be performed before placing on the plate, or may be performed after placing the molecule to be measured on the plate and drying. The derivatization may be labeling using a labeling compound. Hereinafter, derivatization will be described in detail.

A sugar chain (hereinafter referred to as a sugar chain) obtained by chemical or enzymatic release from a sugar, sugar chain, or complex carbohydrate is an aldehyde group of a sugar at the reducing end, an amino group or a hydrazide group (- A labeled compound which is a condensed polycyclic hydrocarbon derivative having CONHNH ₂ ) or the like is subjected to a condensation reaction to form a labeled intermediate, and then the labeled intermediate is reduced to give a reduction-labeled molecule. The condensation reaction is usually performed in an organic solvent such as methanol or DMSO by heating the sugar chain and the labeled compound in the presence or absence of acetic acid as a catalyst. Thereafter, a reducing agent such as NaCNBH ₃ or NaBH ₄ is added, and the reduction reaction is performed at warming or room temperature. Alternatively, a reducing agent may coexist from the beginning in the condensation reaction solution. In the method of labeling by condensing aldehyde group at the reducing end of a sugar and a PBH, [M ⁺ Na] + or [M-H] ^- stability of the parent ion, such is inferior sensitivity lower. For this reason, many parent ions can be produced | generated by performing a reductive reaction further.

  For sugars, sugar chains or complex carbohydrates containing sialic acid, a condensed polycyclic hydrocarbon derivative having an amino group, a hydrazide group, a diazomethyl group or the like is used as a labeling compound, and the carboxy group of sialic acid It can be labeled by reacting with an amino group, a hydrazide group, a diazomethyl group or the like of the labeling compound. This reaction is performed, for example, in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide.

Furthermore, sugars, sugar chains, or complex carbohydrates containing sialic acid can selectively oxidize only the C _7-9 position of sialic acid to generate an aldehyde group. Selective oxidation of the C _7-9 position of sialic acid can be carried out by reacting in 5 mM NaIO ₄ aqueous solution at 0 ° C. for 10-20 minutes. Using a labeled compound that is a condensed polycyclic hydrocarbon derivative having an amino group or hydrazide group, the aldehyde group produced by the selective oxidation and the amino group or hydrazide group of the labeled compound are subjected to a condensation reaction as described above. By forming a labeled intermediate and then reducing the labeled intermediate, a reduction-labeled molecule can be obtained.

Alternatively, many sugars, sugar chains, or glycoconjugates contain galactose at the non-reducing end. The non-reducing terminal galactose can be specifically oxidized 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 labeled compound which is a condensed polycyclic hydrocarbon derivative having an amino group or a hydrazide group, the aldehyde group generated by the above enzymatic oxidation and the amino group or hydrazide group of the labeled compound are subjected to a condensation reaction as described above. By forming a labeled intermediate and then reducing the labeled intermediate, a reduction-labeled molecule can be obtained.

  These methods of labeling galactose can be applied to sialic acid, galactose, etc. on glycoprotein (including glycopeptide) molecules. In that case, since the glycoprotein molecule can be labeled, the ionization efficiency of the molecule is increased.

  Proteins (including peptides) and glycoproteins (including glycopeptides) can be labeled with a carboxy group, amino group, or SH group contained therein. When labeling with a carboxy group, a condensed polycyclic hydrocarbon derivative having an amino group, a hydrazide group, a diazomethyl group, etc. is used as a labeling compound, and these groups react with a carboxy group in a protein or glycoprotein. To obtain a labeled molecule. On the other hand, when labeling with an amino group, a condensed polycyclic hydrocarbon derivative having a succinimidyl ester group, a sulfonyl chloride group or the like is used as a labeling compound, and these groups are combined with an amino group in a protein or glycoprotein. To give a labeled molecule. Furthermore, when labeling using the SH group of a cysteine residue contained in a protein or glycoprotein, a condensed polycyclic hydrocarbon derivative having an iodo group (-I) or the like is used as a labeling compound, Labeled molecules can be obtained by reacting with SH groups in proteins or glycoproteins.

  The labeling compound may be an aromatic compound or a condensed polycyclic hydrocarbon derivative. “Aromatic compound” means an aromatic ring part, a reactive functional group capable of binding to a molecule to be analyzed, and a spacer part that connects the aromatic ring part and the reactive functional group. The compound which has. Here, the aromatic ring moiety is a monocyclic aromatic ring such as a benzene ring, thiophene ring, pyrrole ring, pyridine ring, or naphthalene ring, anthracene ring, pyrene ring, quinoline ring, indole ring, acridine ring, benzothiazole. It may be a condensed aromatic ring such as a ring. “Condensed polycyclic hydrocarbon derivative” means a condensed polycyclic hydrocarbon moiety such as naphthalene, anthracene, pyrene, a reactive functional group capable of binding to a molecule to be analyzed, and the condensed polycyclic hydrocarbon And a spacer moiety that links the reactive functional group.

  The labeling compound used in the present invention is preferably a pyrene derivative compound. The “pyrene derivative compound” is a compound having a pyrene ring, a reactive functional group capable of binding to a molecule to be analyzed, and a spacer portion that connects the pyrene ring and the reactive functional group. Say. Specifically, 1-pyrenyldiazomethane, 1-pyrenebutanoic acid, hydrazide, 1-pyreneacetic acid, hydrazide, 1-pyrenepropionic acid hydrazide (1-pyrenepropionic acid, hydrazide), 1-pyreneacetic acid, succinimidyl ester, 1-pyrenepropionic acid, succinimidyl ester, 1-pyrenebutanoic acid Succinimidyl ester (1-pyrenebutanoic acid, succinimidyl ester), N- (1-pyrenebutanoyl) cysteic acid succinimidyl ester (N- (1-pyrenebutanoyl) cysteic acid, succinimidyl ester), N- (1-pyrene) iodo Acetamide (N- (1-pyrene) iodoacetamide), N- (1-pyrene) iodomaleimide (N- (1-pyrene) maleimide), N- (1-pyrene) Lenmethyl) 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 and the like. Among these, it is preferable to use 1-pyrenyldiazomethane in terms of high reactivity.

  Further, as an example of derivatization, alkylation that does not include an aromatic group may be performed.

(Biological sample placement method)
In the biological sample mounting step, the derivatized measurement molecule or the non-derivatized measurement molecule is dissolved in an appropriate solvent and dropped onto the plate with a dropping device such as a pipette or an automatic spotter. Drying may be left at room temperature or warmed if the molecule does not change. If necessary, a derivatization reaction is subsequently performed.

[Step of localizing target molecule on the plate]
(matrix)
The role of the matrix in the subsequent mass spectrometry is to absorb the light energy of the irradiated laser and cause the matrix itself to ionize and transfer protons or electrons to and from the molecule to be measured. Since the matrix has an inherent light energy absorption band depending on its type, the matrix used differs depending on the laser to be irradiated. In addition to the absorption of light energy, the matrix also has a role as a dispersing agent for uniformly dispersing the molecule to be measured at the molecular level. For this reason, it is important to select a matrix appropriately according to the difference in the molecule to be measured (protein, nucleic acid, saccharide, lipid, etc.).

  Examples of such a matrix include α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHBA), norharman, 6-aza-2-thiothymine ( 6-ATT), 2,4,6-trihydroxyacetophenone, etc. can be used. In order to achieve high localization, it is preferable to appropriately select the matrix concentration. Specifically, the matrix concentration needs to be selected depending on the measurement sample. The matrix concentration is particularly preferably from 0.1 to 20 mg / ml, for example, and particularly preferably from 2 to 10 mg / ml, since the crystal form can be controlled.

(solvent)
As the solvent for dissolving the matrix, acetone, methanol, ethanol, acetonitrile and the like, and a mixture of these with water can be used. In particular, the use of a mixed solution of acetonitrile and water is preferable in that it can be well mixed with the sample and the matrix to realize the subsequent localization. In order to achieve high localization, it is preferable to select a solvent concentration as appropriate. Specifically, the solvent concentration needs to be selected depending on the measurement sample. The solvent concentration, for example, acetonitrile concentration is preferably 0 to 100%, and particularly preferably 40 to 90%, from the viewpoint that the drying time and / or the drying process can be controlled.

(Localization method of target molecule)
Mixing of the matrix and the solvent is not particularly limited, and any known technique can be used. For example, 5 mg of DHBA (high purity matrix reagent, Shimadzu GL) is dissolved in 0.5 ml of water containing 40% acetonitrile, and a final concentration of 10 mg / ml solution is prepared at the time of use. At this time, the centrifuged supernatant is used if necessary.
In the step of localizing the molecule to be measured on the plate, first, the matrix solution is dropped onto the plate with a dropping device such as a pipette or an automatic spotter so as to cover the site where the measurement sample is placed. At this stage, the molecule to be measured is redissolved to form a mixed solution with the matrix.

  Next, by drying the mixed solution, the measurement target molecule is re-migrated to realize localization. Specifically, even if it is allowed to stand at room temperature, it may be heated if the molecule does not change. The drying is performed at a time when localization sufficiently occurs, depending on the measurement molecule. The drying time is preferably 2 minutes to 24 hours. Furthermore, from the viewpoint of shortening the analysis time and not changing the sample and crystal form, it is more preferable that the time is 5 minutes to 30 minutes.

  According to such a localization method (type 1) of a molecule to be measured on a plate, when the biological sample includes at least two kinds of molecules to be measured, in step (A), at least two of the plates In the step (C), different compounds to be measured can be localized in at least two regions by binding different compounds to the regions. Further, when the biological sample contains at least one kind of contaminating molecule, in step (A), different compounds are bound to at least two regions of the plate, and in step (C), molecules to be measured are detected in one region of the plate. In other areas, contaminating molecules can be localized.

<Localization method of molecule to be measured on plate (type 2)>
[Process for preparing plates]
In Type 2, the step of preparing a plate includes (A1) a step of binding a first compound that localizes a molecule to be measured on the first plate, and (A2) a molecule to be measured on the second plate. Binding a second compound that is localized and different from the first compound.

  The type of plate used in this step is the same as type 1 described above. However, different compounds are bound to the first plate and the second plate. Thereby, as will be described later, different kinds of molecules to be measured can be localized on different plates.

[Process for placing biological sample containing target molecule]
In Type 2, the step of placing the biological sample containing the molecule to be measured includes (B1) a step of sandwiching and drying the biological sample containing at least one type of molecule to be measured between the first plate and the second plate. And (B2) separating the first plate and the second plate.

  The kind of molecule to be measured used in this step is the same as that of Type 1 described above. When a biological sample containing a molecule to be measured is sandwiched between the first plate and the second plate, it is preferable to place a weight or the like so that the biological sample is placed in close contact with each other because the localization can be performed with high efficiency. In addition, when the biological sample is dried, it is preferable that the biological sample is dried over an appropriate time and further completely dried from the viewpoint that localization can be achieved with high efficiency. For example, it is preferable to dry at room temperature to 45 ° C. for 30 minutes to 24 hours.

[Step of localizing target molecule on the plate]
In Type 2, the step of localizing the molecule to be measured on the plate is as follows. (C) The solution containing the matrix is dropped on the first plate and the second plate and dried to localize the molecule to be measured on the plate. It is a process to convert.

  About the matrix used in this process, and the kind of solvent, it is the same as that of the above-mentioned type 1.

  According to such a localization method (type 2) of a molecule to be measured on the plate, when the biological sample includes at least two kinds of molecules to be measured, in step (C), the measurement target of the first plate A molecule to be measured different from the molecule can be localized on the second plate. Further, when the biological sample contains at least one kind of contaminating molecule, in the step (C), at least one kind of contaminating molecule can be localized on the second plate.

<Mass Spectrometry (Type 1)>
The method for mass spectrometry of the molecule to be measured of the present invention is as follows.
(1) a method of localizing a molecule to be measured on the plate by a method (type 1) for localizing the molecule to be measured on the plate, and (2) localized in the above step (1) The method includes the step of performing MS ⁿ measurement on a molecule to be measured, wherein n is an integer of 1 or more.

(Process (1))
Step 1 is a step of performing the above-described localization method of the molecule to be measured on the plate. This process is as described above.

(Process (2))
Step 2 is a step of performing MS ⁿ measurement on the molecule to be measured localized in Step 1, and n is a step in which n is an integer of 1 or more.

  “Mass spectrometry” means matrix-assisted laser desorption ionization (MALDI) method, laser desorption (LD) method, fast electron impact (FAB) method, electrospray ionization (ESI) method, atmospheric pressure chemistry (APCI) method Samples containing molecules are ionized by ionization methods such as time-of-flight method (TOF method), double-focusing method, quadrupole focusing method, etc. It is a method of separating and detecting according to the ratio (m / z).

  In the present invention, the ionization method is not limited, but is preferably the LD method, FAB method, or MALDI method, and more preferably the MALDI method.

  In the MALDI method, the ionization efficiency is increased because the methylated or condensed polycyclic hydrocarbon is bonded to the molecule.

Since molecules such as sugars, sugar chains, proteins, sugar-modified proteins, nucleic acids, glycolipids, etc. have structural isomers having the same molecular weight and composition, precursor ions are labeled after labeling with condensed polycyclic hydrocarbon derivatives. The structure information can be obtained by performing MS ⁿ (n> 1) analysis and generating ions specific to the isomer. When MS ⁿ (n> 1) analysis is performed on a molecule labeled on a sugar chain part of a glycopeptide or glycoprotein, a sugar chain binding position in the molecule can be determined by selecting a product ion containing the label.

  In the type 1 mass spectrometry method, any method usually used in the technical field can be used.

<Mass Spectrometry (Type 2)>
The method for mass spectrometry of the molecule to be measured of the present invention is as follows.
(1) Localizing the molecule to be measured in the biological sample on the plate by a method (type 2) for localizing the molecule to be measured on the plate; This is a method including the step of performing MS ⁿ measurement on the activated molecule to be measured, wherein n is an integer of 1 or more.

  (Step (1)), (Step (2)), the term “mass spectrometry” and the like are the same as the above-described method (type 1) for localizing a molecule to be measured on a plate.

  In the type 2 mass spectrometry method, any method usually used in the technical field can be used.

  Below, the effect of this invention is demonstrated based on an Example.

Example 1
1-1. Preparation of Phenyl Plate A phenyl group was introduced into the plate according to the Micro / Contact / Printing method. Specifically, a plate material made of polydimethylsiloxane was prepared, and dipped in an ethanol diluted solution (2 mM) of benzyl mercaptan [α-toluenethiol] for 10 minutes and air-dried. Next, stamping was performed for 3 seconds on a separately prepared Au substrate. Thereafter, the substrate was washed with ethanol and dried to obtain a phenyl plate. In this example, since the phenyl group was entirely coated on the plate, a plate larger than the Au substrate was used as the plate material.

1-2. Localization and Mass Spectrometry of Measurement Object Molecule Sugar chain 1 (500 fmol) was placed on the plate prepared in advance as described above and dried. On top of that, 0.25 μL of DMSO solution containing 500 pmol of 1-pyrenyldiazomethane was added dropwise and heated to 40 ° C. for 25 minutes to dry, thereby obtaining labeled sugar chain 1 partially labeled with pyrene. As a matrix, 0.5 μL of DHBA (10 mg / ml solution, 40% acetonitrile) was added dropwise so as to cover the sample and dried at room temperature to prepare a labeled sugar chain-containing measurement sample 1 shown in FIG.

  The obtained measurement sample 1 was divided into a total of 441 regions of X × Y = 21 × 21, and negative ions in each region were measured by a raster scan of a mass spectrometer (AXIMA-QIT, Shimadzu Corporation). The information of the measurement file of each region obtained by the raster scan was converted into text including measurement position coordinates (x, y), m / z, and signal intensity. Based on the text-converted data, the distribution of deprotonated ions derived from labeled sugar chain 1 (m / z = 2582) and the distribution of deprotonated ions derived from unlabeled sugar chain 1 (m / z = 2077) are disclosed. Displayed two-dimensionally using Soft Graph-R.

  FIG. 2 shows the signal intensity of the deprotonated ion derived from the labeled sugar chain 1, and FIG. 3 shows the signal intensity of the deprotonated ion derived from the unlabeled sugar chain 1. FIG. 4 shows a mass spectrum at point A in FIGS. FIG. 5 shows a mass spectrum at a point B in FIGS. 4 and 5 are shown such that the maximum values are the same.

  2 and 3, it was found that the regions where the labeled sugar chain ion and the unlabeled sugar chain ion are generated are different, in other words, the localization of the labeled sugar chain can be realized. Also, according to FIGS. 4 and 5, from the results of FIGS. 2 and 3, a predetermined region, that is, a region in which the point of FIG. 2 is darker among the points of FIGS. Only when () was selected, it was found that a high signal intensity compared with other ions of the desired ion (labeled sugar chain 1-derived deprotonated ion) was selectively obtained.

  The above effect was achieved by using a plate in which the plate was subjected to predetermined processing and bonded with an aryl group (phenyl group), thereby demonstrating the effect of the present application.

(Comparative Example 1)
Scanning was performed in the same manner as in Example 1 using a generally used stainless steel plate instead of the phenyl plate. The results are shown below.

  FIG. 6 shows the signal intensity of the deprotonated ion derived from the labeled sugar chain 1, and FIG. 7 shows the signal intensity of the deprotonated ion derived from the unlabeled sugar chain 1. FIG. 8 shows only the mass spectrum at point C in FIGS.

  According to FIGS. 6 and 7, it was found that the regions where the labeled sugar chain ion and the unlabeled sugar chain ion are generated are the same, in other words, the localization of the labeled sugar chain has not been realized. In FIG. 8, only the mass spectrum at the point C in FIGS. 6 and 7 is displayed. However, since there is no difference in the color intensity of each point in FIGS. It is expected that the result of FIG. 8 will be obtained.

  The above is a result obtained by using a conventional stainless steel plate without performing predetermined processing on the plate.

(Example 2)
A commercially available glycoprotein prostate specific antigen was electrophoresed and then digested in the gel with lysyl endoprotease C, digested with peptide N glycosidase F, and a C18 chip flow-through fraction was obtained. This fraction was placed as a sample on the phenyl plate prepared in Example 1, and scanning was performed in the same manner as in Example 1 below. The results are shown below.

  FIG. 9 shows the signal intensity of peptide ions (m / z = 1075), and FIG. 10 shows the signal intensity of labeled sugar chain 1-derived deprotonated ions (m / z = 2582). FIG. 11 shows a mass spectrum at a point D in FIGS. FIG. 12 shows a mass spectrum at point E in FIGS. 11 and 12 are shown such that the maximum values are the same.

  9 and 10, it was found that the regions where peptide ions and labeled sugar chain ions are generated are different, in other words, localization of peptide ions and labeled sugar chains can be realized. Further, according to FIGS. 11 and 12, a predetermined region based on the results of FIGS. 9 and 10, that is, a region with a darker color (for example, a point E in the point of FIG. Only when () was selected, it was found that a high signal intensity compared with other ions of the desired ion (labeled sugar chain 1-derived deprotonated ion) was selectively obtained.

  The above effect was achieved by using a plate in which the plate was subjected to predetermined processing and bonded with an aromatic compound, thereby demonstrating the effect of the present application.

  Furthermore, conventionally, when a peptide is mixed with a sugar chain in a measurement sample, since the peptide is easily ionized, sugar chain ions were hardly detected in mass spectrometry. However, in Example 2, separation of both substances by localization in a microregion is realized even if a peptide is mixed with a sugar chain, and even a trace sugar chain derived from a biological sample can be identified by mass spectrometry. It is expected that.

(Example 3)
Example 3 is an example in which a glycopeptide and a peptide are localized using a phenyl plate. First, purification of the PSA-ACT-derived glycopeptide was performed as follows. That is, 95 μL of 50 mM ammonium bicarbonate aqueous solution containing 0.5 μg of PSA-ACT and 5 μL of 10 U / μL thermolysin aqueous solution were added to a 0.6 mL tube, and the mixed solution was stirred and incubated at 56.5 ° C. overnight. This mixed solution is dried with a centrifugal evaporator to obtain a dried product, and 150 μL of 0.8% trifluoroacetic acid (TFA) is added to the dried product, and the mixture is reacted at 80 ° C. for 40 seconds using a heat block. Obtained samples were obtained.

Then, the sample was repeatedly dried by adding H ₂ O. Specifically, 20 μL of H ₂ O, 20 μL of ethanol, and 80 μL of butanol were added in this order and stirred well, and 10 μL of washed and equilibrated Sepharose CL-4B gel suspension (1: 1) was added and stirred well. Thereafter, it was shaken for 1 hour. The Sepharose CL-4B gel was washed and equilibrated three times with 50% ethanol and three times with a mixed solution of butanol / ethanol / water (4: 1: 1). After shaking, the supernatant was discarded by centrifugation, and this was repeated for washing. 100 μL of 50% ethanol was added and shaken for 30 seconds. After centrifugation, the supernatant was collected and dried with a centrifugal evaporator.

  Finally, localization and MS measurement using a phenyl plate were performed in the same manner as in Example 1. That is, 1 μL of 10 mg / mL DHBA dissolved in 60% acetonitrile was dropped, air-dried, and measured by AXIMA-QIT. As a result, negative ion MS spectra are shown in FIGS. 13 shows the result of MS measurement without pyrene labeling, and FIG. 14 shows the result of MS measurement with pyrene labeling.

  According to FIGS. 13 and 14, only peptide signal a was detected when pyrene labeling was not performed. In contrast, when pyrene labeling was performed, it was revealed that the signals b, c, and d of the glycopeptide were specifically detected. Similar results were obtained in the positive ion MS spectrum.

  Furthermore, FIG. 15 shows an example of the MS spectrum of a specific region of the measurement sample. Compared with the average spectrum of all measurement points shown in FIGS. 13 and 14, it was shown that there are clearly measurement points where the signal intensity of only the glycopeptide is relatively high, that is, localized. .

Example 4
Example 4 is an example in which localization was performed by sandwiching a glycopeptide and a peptide between a C18 plate and a phenyl plate. First, the C18 plate and the phenyl plate were produced as follows. That is, the phenyl plate was prepared in the same manner as in Example 1. In order to hold the measurement sample solution, a sheet made of PDMS (polydimethylsiloxane) with a hole having a diameter of 3 mm (thickness: 100 μm, outer shape is substantially the same as that of the phenyl plate) was prepared and attached. Adhesion is not used because of the self-adsorption of the PDMS sheet. A C18 plate was prepared in the same manner as the phenyl plate. A plate material made of polydimethylsiloxane was prepared and immersed in an ethanol dilution (2 mM) of [1-octadecanethiol] for 10 minutes and air-dried. Next, stamping was performed for 3 seconds on a separately prepared Au substrate. Thereafter, the substrate was washed with ethanol and dried. In this example, since C18 was coated on the entire surface of the plate, a material larger than the Au substrate was used as the plate material.
The glycopeptide was prepared in the same manner as in Example 3.

  Further, localization and MS measurement using C18 plate and phenyl plate were performed as follows. That is, 12.5 μL of 5% acetonitrile was added to the dried PSA-ACT-derived glycopeptide and dissolved. An acetonitrile solution containing 5% of 4 pmol / μL adrenocorticotropic hormone fragment (ACTH) fragment (Adrenocorticotropic Hormone Fragment 18-39 human, Sigma) was mixed with PSA-ACT-derived glycopeptide as a peptide to prepare a measurement sample. A sample solution of 0.5 μL was dropped onto a phenyl plate to which a PDMS sheet with a hole was attached, and a C18 plate was placed on top, and a weight was placed on the plate to bring it into close contact. The adhesion body was placed on a heat block and left in this state at 40 ° C. for 2.5 hours to evaporate the solvent. After peeling off the phenyl plate and the C18 plate, the PDMS sheet was peeled off, and a 0.25 μL dimethyl sulfoxide (DMSO) solution of 500 pmol of 1-pyrenyldiazomethane (PDAM) was dropped for pyrene labeling and left at 40 ° C. And reacted. After drying, it was further dried in a vacuum desiccator overnight.

  To this dried product, 1 μL of 10 mg / mL 2,5-dihydroxybenzoic acid (DHBA) dissolved in 60% acetonitrile was dropped, air-dried, and measured with AXIMA-QIT. As a result, a negative ion MS spectrum was shown in FIG. , 17 (for phenyl plates) and 18, 19 (for C18 plates). 16 and 18 show the results of MS measurement without pyrene labeling, and FIGS. 17 and 19 show the results of MS measurement with pyrene labeling.

  According to FIGS. 16-19, only the peptide signal a was detected in any plate, when pyrene labeling was not performed. In addition, according to FIG. 16, it turns out that the signal of a peptide has fallen to 1/2 of Example 3 (FIG. 13).

  On the other hand, when pyrene labeling is performed, it is understood that the signals b, c and d of the glycopeptide are not detected on the C18 plate, but are specifically detected on the phenyl plate. Similar results were obtained in the positive ion MS spectrum.

  From these results (Examples 3 and 4), in Example 4, the peptide, which is a contaminant molecule in the measurement sample on the phenyl plate, was captured by the C18 plate superimposed thereon, and the signal intensity decreased. It can be seen that the glycopeptide remains on the phenyl plate and is localized and does not reduce the signal. Further, in Example 4, it was shown that the signal / noise ratio was improved as compared with Example 3. That is, it can be said that Example 4 achieves more effective localization than Example 3. In addition, it is thought that this effect is acquired also in what arranged the C18 coupling | bond part and the phenyl group coupling | bond part on the same plate.

(Example 5)
The localization was performed by mounting the glycopeptide and peptide on the amino plate or sandwiching the glycopeptide and peptide between the C8 plate and amino plate. In these examples, the same operation as in Examples 3 and 4 was performed except for the type of plate. In Example 5, the results relating to the amino plate after localization, with the glycopeptide sandwiched between the C8 plate and the amino plate, and then separating these plates are shown in FIGS. In contrast, in Example 5, a glycopeptide or the like was placed on the amino plate for localization, and then the results relating to the amino plate are shown in FIGS. In these figures, FIGS. 20 and 22 show the results of MS measurement without pyrene labeling, and FIGS. 21 and 23 show the results of MS measurement with pyrene labeling.

  According to the results of measurement without pyrene labeling (FIGS. 20 and 22), only contaminating peptides were observed. On the other hand, according to the results of measurement using pyrene labeling (FIGS. 21 and 23), when the C8 plate was overlapped (FIG. 23), compared to the case of the amino plate localized without overlapping C8 (FIG. 23) FIG. 21) shows that the signal intensity of the glycopeptide is excellent as in Example 4. As described above, it was found that even when the C8 plate and the amino plate were used, the affinity between the glycopeptide and the amino plate was high, and the signal of the glycopeptide was observed even in the presence of the contaminating peptide.

  The localization method of the molecule to be measured on the plate of the present invention can secure a sufficient remaining amount on the plate without washing the molecule to be measured, and can be used for mass analysis of unknown molecules to be measured. In addition, it can also be used when a plurality of molecules to be measured are subjected to mass spectrometry at the same time. Therefore, the method is promising in that it can be suitably applied to mass spectrometry of various molecules, which are expected to be used more frequently in the fields of biochemistry and medicine.

As described above, one aspect of the present invention includes the following.
1. (A) preparing a plate to which at least one compound that localizes the molecule to be measured is bound;
(B) placing a biological sample containing at least one type of molecule to be measured on the plate;
(C) dropping and drying a solution containing a matrix on the biological sample to localize the molecule to be measured on the plate;
A method for localizing a molecule to be measured on a plate, comprising:
2. 2. The method for localizing a molecule to be measured on a plate according to 1, wherein the compound contains an aryl group, a dihydroxyboryl group, an alkyl group, an amino group, or a hydroxyl group.
3. 2. The aryl group is a phenyl group, a thienyl group, a pyrrolyl group, a pyridyl group, a naphthyl group, an anthryl group, a pyrenyl group, a quinolyl group, an indolyl group, an acrylidinyl group, or a benzothiazolyl group. A method for localizing a molecule to be measured on a plate.
4). 3. The method for localizing a molecule to be measured on a plate according to 2, wherein the alkyl group is a C4-C18 alkyl group.
5). The biological sample contains at least two kinds of molecules to be measured, and in the step (A), different compounds are bound to at least two regions of the plate, and in the step (C), different measurement subjects are used in at least two regions. 5. The method for localizing a molecule to be measured on a plate according to any one of 1 to 4, wherein the molecule is localized.
6). The biological sample further includes at least one contaminant molecule, and in the step (A), different compounds are bound to at least two regions of the plate, and in the step (C), measurement is performed in one region of the plate. 5. The method for localizing a measurement target molecule on a plate according to any one of 1 to 4, wherein the target molecule is localized in another region.
7). Between step (B) and step (C),
(B ′) The method for localizing a molecule to be measured on a plate according to any one of 1 to 6, further comprising a step of derivatizing the biological sample.
8). A method for mass spectrometry of a molecule to be measured,
(1) a step of localizing a molecule to be measured in a biological sample on a plate by the method according to any one of claims 1 to 7, and (2) a measurement localized in the step (1) A mass spectrometry method comprising a step of performing MS ⁿ measurement on a target molecule, wherein n is an integer of 1 or more.

Claims (15)

(A) preparing a plate to which at least one compound that localizes the molecule to be measured is bound;
(B) placing a biological sample containing at least one type of molecule to be measured on the plate;
(C) dropping and drying a solution containing a matrix on the biological sample to localize the molecule to be measured on the plate;
A method for localizing a molecule to be measured on a plate, comprising:   The method for localizing a molecule to be measured on a plate according to claim 1, wherein the compound contains an aryl group, a dihydroxyboryl group, an alkyl group, an amino group, or a hydroxyl group.   The aryl group is a phenyl group, a thienyl group, a pyrrolyl group, a pyridyl group, a naphthyl group, an anthryl group, a pyrenyl group, a quinolyl group, an indolyl group, an acrylidinyl group, or a benzothiazolyl group. A method for localizing a molecule to be measured on the plate.   The method for localizing a molecule to be measured on a plate according to claim 2, wherein the alkyl group is a C4-C18 alkyl group.   The biological sample contains at least two kinds of molecules to be measured, and in the step (A), different compounds are bound to at least two regions of the plate, and in the step (C), different measurement subjects are used in at least two regions. 5. The method for localizing a molecule to be measured on a plate according to claim 1, wherein the molecule is localized.   The biological sample further includes at least one contaminant molecule, and in the step (A), different compounds are bound to at least two regions of the plate, and in the step (C), measurement is performed in one region of the plate. The method for localizing a molecule to be measured on a plate according to any one of claims 1 to 4, wherein the molecule of interest is localized in another region. Between step (B) and step (C),
(B ′) The method for localizing a molecule to be measured on a plate according to any one of claims 1 to 6, further comprising a step of derivatizing the biological sample. A method for mass spectrometry of a molecule to be measured,
(1) A step of localizing a molecule to be measured in a biological sample on a plate by the method according to any one of claims 1 to 7, and (2) a measurement localized in the step (1) A mass spectrometry method comprising a step of performing MS ⁿ measurement on a target molecule, wherein n is an integer of 1 or more. (A1) binding a first compound that localizes the molecule to be measured on the first plate;
(A2) binding a second compound different from the first compound, which localizes the molecule to be measured on the second plate;
(B1) sandwiching and drying a biological sample containing at least one molecule to be measured between the first plate and the second plate;
(B2) separating the first plate and the second plate;
(C) dropping a solution containing a matrix on the first plate and the second plate and drying to localize the molecule to be measured on at least one of the first plate and the second plate; ,
A method for localizing a molecule to be measured on a plate, comprising:   Each of the first compound and the second compound includes an aryl group, a dihydroxyboryl group, an alkyl group, an amino group, or a hydroxyl group, according to claim 9. Localization method.   The aryl group is a phenyl group, a thienyl group, a pyrrolyl group, a pyridyl group, a naphthyl group, an anthryl group, a pyrenyl group, a quinolyl group, an indolyl group, an acrylidinyl group, or a benzothiazolyl group. A method for localizing a molecule to be measured on the plate.   The method according to claim 10, wherein the alkyl group is a C4 to C18 alkyl group.   The biological sample further includes at least one kind of contaminating molecule, and in the step (C), at least one kind of contaminating molecule is localized on the second plate. A method for localizing a molecule to be measured on a plate according to claim 1. Between step (B2) and step (C),
(B ′) The method further comprises derivatizing at least one of the biological sample on the first plate and the biological sample on the second plate. A method for localizing a molecule to be measured on the plate. A method for mass spectrometry of a molecule to be measured,
(1) By the method according to any one of claims 9 to 14, at least one of the measurement target molecule in the biological sample on the first plate and the measurement target molecule in the biological sample on the second plate is localized. And (2) a step of performing MS ⁿ measurement on the measurement target molecule localized in the step (1), wherein n is an integer of 1 or more. Characteristic mass spectrometry.

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