JPWO2014010700A1 - Amino compound and its sensitive mass spectrometry method and biomarker assay method - Google Patents

Abstract

Amino acid derivatives such as final glycation products and amino acid derivatives such as peptides, which are useful for diagnosing abnormal glucose tolerance, and amino compounds such as peptides, and mass capable of rapid, sensitive, and accurate quantification To provide an analysis method and a biomarker assay method using the same. The present invention provides a novel derivatized glycated protein degradation product derivative particularly suitable for mass spectrometry. In addition, the high-sensitivity mass spectrometry method for amino compounds such as amino acid derivatives and peptides according to the present invention comprises reacting the N-terminal amino group of an amino acid derivative or peptide with a trinitrophenylating agent or other derivatizing agent, A step of derivatizing the terminal amino group, and a step of mass spectrometry of the amino acid derivative or peptide in which the N-terminal amino group is derivatized. Furthermore, the present invention provides a biomarker assay method comprising a step of quantifying a biomarker concentration in a biological sample using the obtained mass spectrum intensity data.

Description

  The present invention relates to an amino compound, a high-sensitivity mass spectrometry method thereof, and a biomarker assay method. More specifically, an amino compound obtained by derivatization of an amino acid derivative such as a final saccharified product and a nitrobenzene compound and a salt thereof, and a mass spectrometric method capable of quantifying the amino compound quickly with high sensitivity and high accuracy , And a biomarker assay method using the same.

  Diabetes is a condition in which the blood sugar level is pathologically high. In addition to acute symptoms caused by abnormal blood sugar levels, various organs are damaged as hyperglycemia continues for a long period of time. When complications develop, it becomes very difficult to treat, so it is important to diagnose the onset at an early stage and start treatment such as lifestyle improvement, oral hypoglycemic drug administration, and insulin injection.

  However, the number of diabetic patients, mainly caused by lifestyle-related diseases, has been increasing year by year. According to estimates by the Ministry of Health, Labor and Welfare, over 9 million people are no longer a matter of time. In addition to the number of diabetic patients, it is estimated that there are more than 10 million so-called diabetic reserves, so it is not difficult to imagine that the number of diabetic patients will increase dramatically in the near future. However, only 3 million patients are actually undergoing treatment for diabetes, and the number of patients who suffer from nephropathy due to delay in treatment without sufficient diagnosis increases by 10,000 each year. This is the current situation. In addition, considering that about half of all diabetic patients die from ischemic diseases such as myocardial infarction and cerebral infarction, measures against diabetes and its complications, and so-called hidden diabetes (prediabetes), It is extremely important from the medical economy. In particular, there is an urgent need to establish a countermeasure for prediabetes, which is a potential diabetic army that is extremely difficult to determine only by measuring blood glucose levels in health examinations, that is, an early diagnosis method and a treatment system.

  Moreover, in recent years, among prediabetes patients, fasting blood glucose level (FPG) is normal, but postprandial blood glucose level becomes extremely high due to impaired glucose tolerance (IGT), thereby causing cardiovascular disorder It is pointed out that there is an increase. For this reason, the American Diabetes Association and the World Health Organization define pre-diabetes as a prevalent disease and advocate the importance of improving quality of life and improving IGT of pre-diabetes by drug treatment. As described above, such prediabetic patients are estimated to exceed an estimated 10 million people in Japan. Therefore, prevention and treatment measures for prediabetes are extremely important.

  The Japan Diabetes Society has guided the implementation of fasting blood glucose (FPG) in the primary health checkup and oral glucose tolerance test (OGTT) in the secondary health checkup as diagnostic criteria for prediabetes and diabetes. Glucose intolerance in pre-diabetic patients is difficult to accurately diagnose only by the FPG test and glycated hemoglobin (HbA1c) test in the primary health checkup. Is recommended to take a secondary health checkup for OGTT. However, this OGTT lacks the subject's long restraint time and convenience, and also has a problem such as imposing a physical burden of sugar (glucose) load and accompanying danger such as a rapid increase in blood glucose level, The reality is that it is not as widespread as the blood glucose testing method by blood tests in general health examinations. As a result, many potential prediabetics have failed to undergo a second OGTT health checkup, progressing to true diabetes without being aware of diabetes or its complications, and more severe Complications or necessitating renal dialysis.

  In the primary health examination, a test is usually performed using fasting blood glucose (FPG), occasional blood glucose or glycated hemoglobin (HbA1c) as a determination index, and the FPG value is 100 mg / dL or higher, or the HbA1c value is 5.2%. If it is above, it is diagnosed that there is a suspicion of insulin resistance or IGT caused by it. However, since IGT due to insulin resistance appears as a major initial change after postprandial hyperglycemia, it is necessary to say that any index used in the conventional primary health check is insufficient in terms of accuracy, Conventional primary health checkup methods cannot be used as an early diagnosis of prediabetes.

  Therefore, in reality, in a primary health checkup, a subject who is suspected of having insulin resistance or IGT using these indexes is subjected to a 75 g oral glucose tolerance test (secondary health checkup or subsequent health checkup ( 75 g OGTT), blood insulin, HOMA-R and the like are tested to determine whether insulin resistance or IGT is positive, and a definitive diagnosis of prediabetes has been made. Of course, these inspections are expensive and troublesome.

  Therefore, if there is a method that can confirm the diagnosis of prediabetes with the same accuracy as OGTT with the same blood sample for blood glucose measurement used in the primary health checkup, the detection rate of prediabetic patients will improve dramatically, The OGTT test in the secondary health checkup is unnecessary, and it is possible to strongly promote the strategy for preventing diabetes by early diagnosis and treatment of prediabetes proposed by the World Health Organization.

  On the other hand, in recent years, various final glycation products have attracted attention as risk factors for chronic complications of diabetes. Final glycated products (AGEs) are also called glycated proteins, Maillard reaction products, and the like, and are protein derivatives having various structures generated by non-enzymatic reaction between a reducing sugar such as glucose and the amino group of the protein. AGEs are the result of disruption of the functions of these proteins resulting from glycation modification of extracellular matrix proteins, membrane proteins, and intracellular proteins, and cell functions dependent on them, or the cellular responses caused by receptors with AGEs as ligands. Involved in the development and exacerbation of various lesions. For example, when AGEs are recognized by RAGEs, which are one of the AGE receptors, the production of intracellular oxidative stress substances by intracellular NADPH oxidase is enhanced, which changes gene expression in epithelial cells, It is thought that the diabetic vascular disorder of the other will develop (refer nonpatent literature 1).

  Among AGEs, those derived from glyceraldehyde and methylglyoxal (MG), which are produced as glycolytic intermediates or by-products, are expected to be diagnostic markers for the onset and prognosis of diabetes and impaired glucose tolerance. (Non-Patent Documents 2, 3, 4, 5).

  Non-patent document 2 shows that the serum level of methylglyoxal-derived hydroimidazole was measured in an ELISA system using a polyclonal antibody of methylglyoxal-derived hydroimidazole for the serum of type 2 diabetic patients. It has been reported that it has increased. The non-patent document 2 shows that there is a significant correlation between the serum level of methylglyoxal-derived hydroimidazole and the serum level of Nε- (carbonylmethyl) lysine, which is one of AGEs, Serum levels of methylglyoxal-derived hydroimidazoles have been reported to be unrelated to fasting blood glucose plasma levels or hemoglobin levels.

  In Non-patent Document 3, as a result of measuring plasma of a type 1 diabetic patient using LC / MS / MS, the concentration of an adduct due to glycation or oxidation of protein and the concentration of its free adduct increased. It shows that. For example, methyl glyoxal-derived hydroimidazolone, glyoxal-derived hydroimidazolone or other AGEs such as Nε- (carbonylmethyl) lysine, Nε- (carbonylethyl) lysine, and free adducts thereof are increased. It has been reported that

  Non-patent document 4 suggests that methylglyoxal-derived hydroimidazolone free adduct is an excellent marker for diabetic patients as a result of tandem mass spectrometry of plasma of type 1 diabetic patients.

  Non-patent document 5 shows that as a result of measuring plasma and urine samples of type 1 diabetic patients by LC-MS / MS, in addition to an increase in blood glucose level and insulin level after glucose loading, methylglyoxal-derived hydroimidazole free adduct was added. First, Nε- (carbonylmethyl) lysine, Nε- (carbonylethyl) lysine, glyoxal-derived hydroimidazolone, Nδ- [5- (2,3,4-trihydroxybutyl) -5-hydro-4-imidazolone 2- Ill] -ornithine (3DG-H1) and other free adducts have also been reported to increase. That is, this paper shows that not only hyperglycemia occurs after meals, but also a high carbonyl stress state is induced. Furthermore, this document describes tandem mass spectrometry results for many AGEs including argypirimidine and amino acids (arginine, lysine, methionine, tyrosine and tryptophan) in addition to methylglyoxal-derived hydroimidazolone free adduct. ing.

  As described above, a technique for analyzing these AGEs by tandem mass (MS / MS) has been reported. However, there is currently no effective method for measuring these AGEs other than the total amount evaluation using an immunochemical method such as an ELISA method (Patent Document 1). There is almost no established measurement method for products, and it must be said that there is no measurement method that can be applied clinically. Since the quantification of these individual AGEs is useful not only in the diagnosis of diabetes and impaired glucose tolerance but also in metabolome analysis, etc., individual compounds can be measured more quickly and with high sensitivity and high accuracy for AGEs. An assay is highly desired.

  In addition to AGE analysis, amino acid analysis is also very important for clinical fields such as innate metabolic disorder assays and has the potential to be used for diagnostic biomarker searches. Amino acid analysis is used, for example, as a diagnosis of congenital amino acidosis, the severity of liver dysfunction, and a guideline for treatment. In recent reports, amino acid profiles have been applied to the diagnosis of progressive fibrosis and the like in patients with chronic hepatitis C. Furthermore, the possibility of analyzing the degree of health of living organisms using an amino acid profile as an index has been reported (Non-patent Document 6).

  An amino acid automatic analyzer has been developed for amino acid analysis as described above. This apparatus measures the colorimetric color of amino acids using a ninhydrin reagent and requires a very long time for analysis.

  In addition to the colorimetric measurement method as described above, a precolumn derivatization method has been developed, and various derivatizing agents used for the precolumn derivatization method have been developed (Non-patent Document 6). However, this derivatization method is a complicated and time-consuming technique. Furthermore, an automatic amino acid analysis method combining this derivatization method and a mass spectrometry method has also been developed, but further improvement has been demanded for this method.

JP 2008-224453 A

Marie-Paule Wautier et al., "Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE", American Journal of Physiology-Endocrinology and Metabolism, (USA), American Physiological Society, 2001, 5 Moon, Volume 280, E685-E694 Kilhovd, B.K., et al. Metabolism, Vol 52, No 2 (February), 2003: pp. 163-167 Rabbani, N., and Thornalley, P.J.Amino Acids DOI 10.1007 / s00726-010-0783-0 Ahmed, N., et al. Diabetologia (2005) 48: 1590-1603 Ahmed, N., et al. Diabetes Care, Vol. 28, No. 10, October 2005, pp. 2465-2471 Shimbo, K., et al. Biomed. Chromatogr. 2010; 24: 683-691.

  Therefore, as a result of intensive studies in view of such circumstances, the present inventors have determined that the terminal amino groups of AGEs, such as methylglyoxal-derived hydroimidazolone derivatives (MG-H1) and argypirimidine (AP), are nitrobenzene compounds. By derivatizing, the sensitivity in mass spectrometry was greatly improved, and in addition to AGEs, it was found that it could be applied to other amino acid derivatives or amino compounds such as peptides, and the present invention was completed. .

  Therefore, the present invention enables amino acid compounds and salts thereof obtained by derivatization of amino acid derivatives, particularly final glycation products, and nitrobenzene compounds, and quantification of amino compounds quickly and with high sensitivity. It is an object of the present invention to provide a mass spectrometric method and a biomarker assay method using the same.

  In order to achieve the above object, the present invention provides an amino compound represented by the general formula [I] or a salt thereof.

  In the formula [I], R means an amino substituent and Z means a nitrophenyl group.

  In a preferred embodiment of the present invention, an amino compound or a salt thereof in which the amino substituent is an amino acid residue or a substituted amino acid residue or a peptide residue in which the amino acid residue and / or the substituted amino acid residue is a peptide bond. provide.

  In a preferred embodiment of the present invention, the amino acid residue or substituted amino acid residue or the constituent amino acid of the peptide residue is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine. , Lysine, leucine, methionine, proline, arginine, serine, threonine, valine, tryptophan or tyrosine or a substitute thereof, preferably arginine, lysine, valine, tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan or a substitute thereof An amino compound or a salt thereof is provided.

  As another preferred embodiment of the present invention, an amino compound or a salt thereof in which the substituted amino acid residue is a residue of a final glycation product (AGE), preferably an arginine derivative or a lysine derivative.

  As another preferred embodiment, the present invention provides an amino compound or a salt thereof, wherein the peptide of the above peptide residue is a peptide bond of 2 to 10 amino acids.

  As another preferred embodiment of the present invention, an amino compound or a salt thereof, in which the nitrophenyl group is represented by the general formula [IIIb], is provided.

In the formula [IIIb], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.

  As a more preferred embodiment of the present invention, the amino compound is an amino compound represented by the general formula [Ia] or a salt thereof.

In the formula [Ia], R, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ each have the same meaning as described above.

  As another preferred embodiment of the present invention, the amino compound is an amino compound represented by the general formula [Ii] or a salt thereof.

    In the formula [Ii], R has the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib] or a nitrophenylated argpyrimidine derivative represented by the general formula [Ic]. An amino compound or a salt thereof is provided.

In the formulas [Ib] and [Ic], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a trinitrophenylated argon represented by the structural formula [Iiii]. An amino compound which is a pyrimidine derivative or a salt thereof is provided.

  This invention provides the mass spectrometry method of the amino compound or its salt which measures the amino compound or its salt represented with general formula [I] by mass spectrometry as another form.

  In the formula [I], R means an amino substituent, for example, an amino acid residue or a substituted amino acid residue or a peptide residue, and Z means a nitrophenyl group.

  As a preferred embodiment, the present invention provides a mass spectrometric method for the amino compound represented by the general formula [Ia] or a salt thereof.

In the formula [Ia], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group, and R has the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib] or a nitrophenylated argpyrimidine derivative represented by the general formula [Ic]. A method for mass spectrometry of an amino compound or a salt thereof is provided.

In the formulas [Ib] and [Ic], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or an amino compound represented by the structural formula [Iiii] or a salt thereof. A mass spectrometry method is provided.

As another form of the present invention, the amino acid derivative represented by the general formula [II] or a salt thereof and the nitrobenzene compound represented by the general formula [III] are reacted to represent the general formula [I]. Obtaining an amino compound or a salt thereof;
Provided is a method for mass spectrometry of an amino compound or a salt thereof, which comprises a step of mass-analyzing the amino compound or a salt thereof obtained in the above step.

  In the formula [II], R means an amino substituent, for example, an amino acid residue or a substituted amino acid residue or a peptide residue.

  In the formula [III], X means an amino group-reactive functional group, and Z means a nitrophenyl group.

  In the formula [I], R and Z have the same meaning as described above.

  As a more preferred embodiment of the present invention, the reaction of the amino acid derivative represented by the general formula [II] or a salt thereof and the nitrobenzene compound represented by the general formula [III] is carried out by using an alkaline aqueous solution or water and an organic solvent. Provided is a mass spectrometric method for an amino compound or a salt thereof, which is a mixed solution and the pH of the solution is 8 to 10.

  As a preferred embodiment, the present invention provides a mass spectrometric method for amino compounds or salts thereof in which the above reaction is carried out in a sample.

  As a more preferred embodiment, the present invention provides a mass spectrometric method for amino compounds in which the amino acid derivative or salt thereof is a biomarker.

  As another embodiment, the present invention provides a method for mass spectrometry of an amino compound or a salt thereof, wherein the amino compound represented by the general formula [I ′] or a salt thereof is subjected to mass spectrometry, wherein the derivatization residue is derivatized. Agents are sulfonyl compounds such as 1-pyrenesulfonyl chloride (PSC) and dansyl chloride (DNS-Cl); 9-fluorenylmethyl chloroformate (FMOC), 4-dimethylaminosulfonyl-7-fluorobenzoxadiazole (DBD-F), 4-fluoro-7-nitrobenzofurazan (NBD-F), 2,3-naphthalenedialdehyde (NDA); 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), etc. Carbamate compound; 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2-quinoxalinone ( MEQ-COCl), 3-nitrophenyl isothiocyanate, 3-pyridyl isothiocyanate, 4- (dimethylamino) phenyl isothiocyanate, fluorescein-5-isothiocyanate (FITC), and the like; diethyl ethoxymethylene malonate, etc. Mass spectrometric method of amino compound or salt thereof which is a malonate compound, a phosphonium compound such as (5-succinimidoxy-5-oxopentyl) triphenylphosphonium bromide (SPTPP); or a pyridinium compound such as 2-chloro-1-methylpyridinium salt I will provide a.

  In the formula [I ′], R ′ means a substituted amino acid residue or peptide residue having the same meaning as R, and Z ′ means a derivatized residue.

  As yet another embodiment, the present invention provides a biomarker assay method for quantifying the biomarker concentration in a biological sample using the intensity data of the mass spectrum obtained by the mass spectrometry method for amino compounds described above.

  By derivatizing the N-terminal amino group of an amino acid derivative according to the present invention, ionization efficiency in mass spectrometry is improved, and mass spectrometry is also performed for molecular biomarkers such as final glycation products and peptide hormones that exist only in a small amount in a sample. The method enables rapid, highly sensitive and accurate quantitative analysis. Accordingly, the present invention provides a novel amino compound that is a derivatization product of an amino acid derivative and a derivatizing agent such as a nitrobenzene compound, preferably a glycated protein degradation product that is a reaction intermediate product such as protein glycolysis is a nitrobenzene compound or the like. The present invention provides a novel glycated protein hydrolyzate derivative that is derivatized with a derivatizing agent useful for diagnosing abnormal glucose tolerance. The present invention also provides a mass spectrometric method capable of rapid, highly sensitive and highly accurate quantification of the amino compound, and a biomarker assay method using the same.

It is a mass spectrum (MS, MS / MS) of a methylglyoxal-derived hydroimidazolone derivative (MG-H1). It is a mass spectrum (MS, MS / MS) of a 2,4,6-trinitrophenyl derivative of MG-H1 (MG-H1-TNP). It is a mass spectrum (MS, MS / MS) of argypirimidine (AP). 2 is a mass spectrum (MS, MS / MS) of a 2,4,6-trinitrophenyl derivative of AP (AP-TNP). It is a figure which shows the fragmentation pattern of the 2,4,6-trinitrophenyl derivative (MG-H1-TNP) of MG-H1 estimated from a mass spectrum. It is a figure which shows the fragmentation pattern of the 2,4,6-trinitrophenyl derivative (AP-TNP) of AP estimated from a mass spectrum. (A) is a mass chromatogram showing the results of LC-MS analysis of MG-H1, and (B) is the 2,4,6-trinitrophenyl derivative of MG-H1 (MG-H1-TNP). (A) is a mass chromatogram showing the results of LC-MS analysis of AP, and (B) is the results of LC-MS analysis of 2,4,6-trinitrophenyl derivative of AP (AP-TNP). It is the calibration curve produced using the internal standard method about the density | concentration of MG-H1-TNP and the peak intensity of a mass spectrum. It is the calibration curve produced using the internal standard method about the density | concentration of AP-H1-TNP, and the peak intensity of a mass spectrum. It is a graph which shows the influence of pH which acts on 2,4,6- trinitrophenylation of MG-H1. It is a graph which shows the influence of pH which acts on 2,4,6- trinitrophenylation of AP. It is a chart which shows the protocol of pretreatment of rat plasma.

  Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments, and the following embodiments are exemplarily described in order to specifically describe the present invention. Therefore, it should be understood that forms conceived from the following embodiments are also included in the scope of the present invention.

  In the present specification, in order to simplify the explanation, a nitrobenzene compound will be described as an example of the derivatizing agent. However, in the present invention, the derivatizing agent is not limited to the nitrobenzene compound at all. It should be understood that other derivatizing agents are naturally included within the scope of the present invention. Therefore, in this specification, although the nitrophenyl group which is a residue of a nitrobenzene compound is explained also about the residue which derivatized the terminal amino group of the amino acid derivative with the derivatizing agent, this is also limited at all. Rather, it should be understood that residues of other derivatizing agents are included as well.

  The amino compound according to the present invention can be represented by the general formula [I].

  In the formula [I], R means an amino substituent and Z means a nitrophenyl group.

In the above general formula, the term “amino substituent” means an amino acid residue or a substituted amino acid residue or a peptide residue.
Here, the term “amino acid residue” means a residue obtained by removing an amino group from an unsubstituted amino acid. For example, in the case of alanine, since it is also called 2-aminopropanoic acid, the amino acid residue excluding the amino group from 2-aminopropanoic acid is a carboxyethyl group. Similarly, the amino acid residue in the case of lysine is a 1-carboxyl-5-aminopentyl group.

  In the above general formula, the term “substituted amino acid residue” refers to an amino acid residue obtained by removing an amino group from the amino acid, and a residue other than a carboxyl group constituting the amino acid, that is, an atomic group is a substituent. It means an atomic group formed by substitution with an amino acid residue having a substitution moiety (moiety). Examples of the substituted amino acid residue include a 1-carboxyl-5- (carboxylmethyl) aminopentyl group in which a 5-methyl group of the lysine residue is substituted with a carboxymethyl group. Another example includes a substituted amino acid residue obtained by converting an amino-iminomethyl group of an arginine residue into a 2-pyrimidinyl group.

  Furthermore, in the above general formula, the term “peptide residue” means a residue in which a terminal amino group is excluded from a terminal amino acid of a peptide formed by peptide bonding of the amino acid and / or a substituted amino acid. . For example, in the case of the peptide Val-Tyr, the peptide residue means a residue in which the amino group is excluded from the terminal amino acid valine.

  In the present invention, the term “amino acid derivative” means an unsubstituted amino acid derivative, a substituted amino acid derivative in which an amino acid moiety other than the N-terminal amino group is modified with a substituent or the like, unless otherwise specified. doing.

  Therefore, unless otherwise specified, the term “amino compound” used in the present invention is a nitrophenylation in which the N-terminal amino group of the amino acid derivative [II] or a salt thereof is nitrophenylated by the nitrobenzene compound [III]. An amino compound is meant.

  In the present invention, amino acids of amino acid residues or substituted amino acid residues, or amino acids of peptide constituent amino acids of peptide residues are not particularly limited as long as they are compounds having both amino group and carboxy functional groups. Although not limited, for example, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine, valine, Examples include amino acids that are constituents of proteins such as tryptophan and tyrosine, preferably arginine and lysine, and substituted amino acid residues having a substitution moiety formed by substituting residues other than the carboxyl group constituting them with a substituent. .

  Preferred amino acids include, for example, preferably arginine, lysine, valine, tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan.

  The term “substituted amino acid residue” used in the present invention is a residue of a substituted amino acid derivative having another atomic group formed by bonding an arbitrary substituent to a residue other than the terminal amino group of the amino acid. Means. Such amino acid derivatives are not particularly limited as long as they do not depart from the object of the present invention, and include, for example, degradation products, intermediate products, or metabolites of biological components such as amino acids. Glycation end products (AGEs) produced by the saccharification reaction, ornithine, the degradation product of arginine, creatinine, a metabolite of creatinine phosphate, which is an energy source for muscles, and biotechnology that shows glutathione deficiency in the liver In addition to α-aminobutyric acid (AABA), an intermediate of biosynthesis of ophthalmic acid as a marker, γ-aminobutyric acid (GABA), which mainly acts as a neurotransmitter, and other molecules used for diagnosis and prognosis prediction of various diseases Examples include biomarkers and hippuric acid. Of these amino acids, the above AGEs and the like are preferable. More preferably, for example, it is produced by a reaction between a guanosine group on the side chain of arginine and methylglyoxal, or an amino group of lysine and a reaction intermediate product such as lipid peroxidation such as glyoxal or methylglyoxal, Maillard reaction, glycolysis. AGEs and the like. Of these AGEs, preferred are substituted arginine derivatives and substituted lysine derivatives.

  Examples of substituted arginine derivatives include alkyl-substituted arginine derivatives in which the guanidino group of arginine is modified with a substituent such as carboxylmethyl group and carboxylethyl group, guanidino groups such as imidazolones, pyrimidines, and imidazopyridines in a cyclic structure. And cyclic arginine derivatives.

  More specifically, examples of the alkyl-substituted arginine derivative include carboxymethylarginine (CMA). Examples of imidazolones of cyclic arginine derivatives include imidazolone, glyoxal hydroimidazolone (GH), methylglyoxal hydroimidazolone (MG-H), Nδ- [5- (2,3,4-trihydroxy). Butyl) -5-hydro-4-imidazolone-2-yl] -ornithine (3-DG-H) and the like. Examples of pyrimidines include argpyrimidine and tetrahydropyrimidine. Examples of imidazopyridines include pentosidine.

  Among these substituted arginine derivatives, for example, a methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [IIa] (MG-H1: Nδ- (5-hydro-5-methyl-4-imidazolone 2) is particularly preferable. -Yl) -ornithine), or argpyrimidine (AP) represented by the structural formula [IIb].

  More specifically, the amino acid derivative has a three-dimensional structure and can be represented by the structural formula [IIc] or the structural formula [IId].

  Among the above AGEs, examples of substituted lysine derivatives include alkyl-substituted lysine derivatives in which the amino group of lysine is modified with a substituent such as carboxylmethyl group or carboxylethyl group, and the amino group of lysine has a cyclic structure. And cyclic lysine derivatives.

  Examples of the alkyl-substituted lysine derivative include carboxymethyl lysine (CML) and carboxyethyl lysine (CML). Examples of the cyclic lysine derivative include viralin, pyrrole derivatives such as FTP (Formyl Therosyl Pyrrol), GOLD (glyoxal-derived lysine dimer), MOLD (methylglyoxal-derived lysine dimer), DOLD (3-deoxyglucose). Imidazole derivatives such as lysine-derived lysine dimer), pyridine derivatives such as GLAP (Glyceraldehyde-derived pyridinium) and GA-pyridine (glycolaldehyde-pyridine), and imidazopyridine derivatives such as pentosidine.

  Further, in the general formula [I], the peptide residue peptide represented by the substituent R is not particularly limited in the number of the amino acids constituting the peptide, but preferably 2 to 10 amino acids, preferably May be a peptide composed of 2 to 6, more preferably 2 to 4, peptide bonds. Examples of such peptides include dipeptides such as valyltyrosine, lysyltyrosine, methionyltyrosine, glycyltyrosine, histidyltyrosine, glutamyltyrosine, tryptophanyltyrosine, histidinyltryptophan, carnosine and anserine, and tripeptides such as glutathione. Etc. In addition, enkephalin, caxitocin, vasopressin and the like can be mentioned. Of these peptides, dipeptides such as valyltyrosine, lysyltyrosine, methionyltyrosine, glycyltyrosine, histidyltyrosine, glutamyltyrosine, tryptophanyltyrosine and histidinyltryptophan are preferred. The peptide may be any of a digested protein, a metabolite such as a final glycation product, and a physiologically active peptide such as a peptide hormone, and is a molecular biomarker used for diagnosis and prognosis prediction of various diseases. Also good.

  On the other hand, in the above general formula [I], the substituent Z is a residue of a nitrobenzene compound obtained by nitrophenylating the terminal amino group of the amino acid derivative [II], and can be represented by the general formula [IIIb].

In the formula [IIIb], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.

  Here, examples of the nitrophenyl group include a mono-, di- or tri-nitrophenyl group, preferably a tri-nitrophenyl group, particularly preferably a 2,4,6-nitrophenyl group. The position of the nitro substituent is not particularly limited, but in the case of one, the 2-position, in the case of 2, the 2-position and the 4-position, in the case of 3, the 2-position, the 4-position and It should be in the 6th position.

  Therefore, the amino compound according to the present invention is generally represented by the general formula [I], and more specifically can be represented by the general formula [Ia].

In the formula [Ia], R, Z, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  In the present invention, preferred examples of the amino compound represented by the structural formula [Ia] include, for example, a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Ib] or the structural formula [Ic]. And nitrophenylated argpyrimidine derivatives represented.

In the formulas [Ib] and [Ic], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  A more preferred amino compound can be represented, for example, by the general formula [Ii].

  In the formula [Ii], R has the same meaning as described above.

  Therefore, as the amino compound, for example, a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a nitrophenylated argpyrimidine derivative represented by the general formula [Iiii] is more preferable.

  The three-dimensional structural formulas ([Iiv] and [Iv]) of the above-mentioned trinitrophenylated methylglyoxal-derived hydroimidazolone derivative and trinitrophenylated argpyrimidine derivative can be represented as follows.

  The amino compound [I] according to the present invention can be obtained by derivatizing an amino acid derivative represented by the general formula [II] or a salt thereof with a nitrobenzene compound represented by the general formula [III].

  In the formula [II], R has the same meaning as described above.

  In the formula [III], X means an amino group-reactive functional group, and Z has the same meaning as described above.

  More specifically, the amino compound [Ia] of the present invention can be obtained by a derivatization reaction between an amino acid derivative [II] or a salt thereof and a nitrobenzene compound represented by the general formula [IIIa].

In the formula [IIIa], X, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  More specifically, the amino compound [Ii] of the present invention can be obtained by a derivatization reaction between an amino acid derivative [II] or a salt thereof and a trinitrobenzene compound represented by the general formula [IIIc].

  In the formula [IIIc], X has the same meaning as described above.

  Here, the amino group-reactive functional group represented by X is not particularly limited as long as it is a reactive functional group that derivatizes an amino acid or a substituted amino acid ([II]) with a nitrophenyl. Aromatic nucleophilicity of nitrobenzene derivatives such as atoms, halogen atoms selected from bromine atoms or iodine atoms, or sulfonic acids or sulfonates, alkyl sulfonate groups, aryl sulfonate groups, perfluoroalkyl sulfonate groups, etc. Any functional group that can be a leaving group in the substitution reaction is exemplified.

  Accordingly, preferred nitrobenzene compounds include, for example, 2,4,6-trinitrofluorobenzene, 2,4,6-trinitrochlorobenzene, 2,4,6-trinitrobromobenzene, 2,4,6-trinitro. Examples thereof include iodobenzene and 2,4,6-trinitrobenzenesulfonic acid or a salt thereof. Among these, 2,4,6-trinitrobenzenesulfonic acid or a salt thereof is particularly preferable. Examples of the salt include alkali metal salts such as sodium salt, alkaline earth metal salts such as potassium salt, and the like.

  In the present invention, in order to derivatize the N-terminal amino group of the amino acid derivative with a nitrobenzene compound, the amino acid derivative and the nitrobenzene compound are tens of several hours at room temperature in an alkaline aqueous solution or a mixed solution of water and an organic solvent. This can be done by mixing for a minute. Preferably it is carried out in an alkaline aqueous solution. Moreover, when using the mixed solution of water and an organic solvent, the organic solvent with high affinity with water, such as ethanol, is preferably used as an organic solvent. The pH of these solutions is preferably 8 to 10, more preferably 8.5 to 9.5. If the pH of the solution is too high, the possibility of decomposition of the amino acid derivative to be analyzed or this compound increases, and if it is too low, the nitrophenylation reaction does not proceed rapidly. Here, the pH is a value directly measured by a pH meter. The room temperature is a temperature of about 15 ° C. to 40 ° C., and the reaction is preferably performed at around 30 ° C. The mixing may be carried out by any method, and mixing is performed for 1 minute or more and 1 hour or less, preferably 5 minutes to 40 minutes. Usually, mixing for 30 minutes is sufficient.

  One of the features of the present invention is that the nitrophenylation reaction can be performed under such mild conditions. A substance to be analyzed in a sample may be isolated and subjected to nitrophenylation, or may be subjected to nitrophenylation as it is in a sample containing impurities. Since the present invention can directly perform nitrophenylation in such a sample, loss due to isolation can be reduced, and even in the case where only a small amount of a substance to be analyzed is contained. It is an excellent method that can be measured. Further, it is not necessary to perform a complicated operation, and measurement can be performed easily. In particular, when the target sample is a biological sample or the like, it is effective because there are few substances to be analyzed and there are many impurities.

In addition, for example, in measuring an amino acid derivative in a sample, it is possible to derivatize an amino acid derivative as a biomarker in a sample with a nitrobenzene compound by adding a nitrobenzene compound to the biological sample, Naturally, such derivatized amino acid derivatives can be measured as biomarkers.
What is used as a sample in the present invention is not limited as long as it contains a final glycation product to be measured and the like that become an amino compound obtained by derivatization of an amino acid derivative and a nitrobenzene compound according to the present invention. A sample derived from a living body is called a biological sample.

  Furthermore, as a salt of an amino acid derivative, for example, an alkali metal salt such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a lithium salt, an ammonium salt, or the like can be used.

  In the present invention, as a derivatizing agent, in addition to the above nitrobenzene compound, other derivatizing agents that derivatize amino groups can naturally be used. Examples of such derivatizing agents include nitrobenzene compounds such as nitrobenzene sulfonic acid compounds and fluoro-nitrobenzene, sulfonyl compounds such as 1-pyrenesulfonyl chloride (PSC) and dansyl chloride (DNS-Cl), 9-fluorene, and the like. Nylmethyl chloroformate (FMOC), 4-dimethylaminosulfonyl-7-fluorobenzoxadiazole (DBD-F), 4-fluoro-7-nitrobenzofurazan (NBD-F), 2,3-naphthalene Carbamate compounds such as aldehyde (NDA), 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2-quinoxalinone (DMEQ-COCl), 3 -Nitrophenylisothiosi , 3-pyridyl isothiocyanate, 4- (dimethylamino) phenyl isothiocyanate, isothiocyanate compounds such as fluorescein-5-isothiocyanate (FITC), malonate compounds such as diethyl ethoxymethylene malonate, (5-succinimidoxy-5- Examples thereof include phosphonium compounds such as oxopentyl) triphenylphosphonium bromide (SPTPP), and pyridinium compounds such as 2-chloro-1-methylpyridinium salt. These derivatizing agents can also be used in substantially the same manner as the above nitrobenzene compound.

  That is, in the present invention, when the derivatizing agent is used, the amino compound according to the present invention is represented by the general formula [I ′].

  In the formula [I ′], R ′ means an amino acid residue or substituted amino acid residue or peptide residue having the same meaning as R above, and Z ′ means a derivatized residue.

Here, the derivatized residue means a residue obtained by derivatizing the amino group of the substituted amino acid derivative (R′-NH ₂ ) with the derivatizing agent. For example, when dansyl chloride is used as a derivatizing agent, the amino group of the substituted amino acid derivative (R′-NH ₂ ) is dansylated, that is, dimethylaminonaphthalenesulfonylated, and this dansyl group is called a derivatized residue. be able to.

  Therefore, also in this case, in this specification, the description about the case where nitrobenzenesulfonic acid is used as a derivatizing agent can be applied substantially in the same manner.

In the present invention, the ionization method of the sample used for mass spectrometry is not particularly limited, but it is preferable to use MALDI method, ESI method, APCI method and the like widely used for detection and quantification of biomolecules. In LC / MS, GC / MS, etc. combined with a separation technique, it is preferable to use an ESI method or an APCI method as a sample ionization method. For the analysis, a tandem mass spectrometry method such as MS / MS or MS ³ can be used. In this case, for the detection of product ions, for example, it is preferable to use multiple reaction monitoring (MRM) which can eliminate the influence of impurities and enable highly sensitive detection.

Next, a biomarker assay method using the mass spectrometry method according to the present invention will be described.
The present invention can provide an assay method capable of detecting and quantifying a biomarker rapidly, with high sensitivity and high accuracy by selecting the biomarker as an analysis target. That is, the biomarker assay method of the present invention comprises reacting an N-terminal amino group of a biomarker that is an amino acid derivative with a derivatizing agent such as a nitrobenzene compound to derivatize the N-terminal amino group such as nitrophenylation. And mass spectrometry of the amino acid derivative or its salt in which the N-terminal amino group is derivatized such as nitrophenylation, and the intensity data of the mass spectrum obtained by using the mass spectrometry method, A step of quantifying the biomarker concentration.

  In the present invention, the biomarker is not particularly limited as long as it is an amine compound having an amino group. Examples of the method to which the assay method of the present invention can be particularly preferably applied include amino acid derivatives and sugar metabolites. Final glycation products (AGEs) produced by the reaction, biogenic amines catecholamine (dopamine, noradrenaline, adrenaline), indoleamine (serotonin, melatonin), imidazoleamine (histamine), acetylcholine, polyamine (putrescine, spermidine, Spermine) and the like.

  Examples of AGEs that are particularly preferable as biomarkers to be analyzed by the assay method of the present invention include, for example, trinitrophenylated methylglyoxal-derived hydroimidazolone derivatives (MG-H1) represented by the structural formula [Iii], and structural formulas And trinitrophenylated argpyrimidine derivative (AP) represented by [Iiii].

  These are MG-H1 and N-2,4,6-trinitrophenyl derivatives of AP, which are AGEs that can be detected and quantified with high sensitivity by mass spectrometry, and are useful as diagnostic indicators of impaired glucose tolerance It is.

  In the present invention, for example, a standard curve method or an isotope dilution method is used to quantify the concentration of the amino acid derivative or peptide to be assayed from the area intensity of the mass spectrum. In mass spectrometry, one of the techniques for quantitative analysis of a substance to be analyzed is a calibration curve method. The calibration curve method shows that the relationship (calibration curve) between several standard samples containing known amounts of standard substances and different concentrations and the signal intensities obtained from these standard samples is theoretically a linear relationship. This is a method of using and quantifying the concentration of the sample from the signal intensity obtained from the unknown sample. The calibration curve method is roughly classified into an external standard method, an internal standard method, and a standard addition method depending on the preparation method of the standard sample. Any method may be used in the present assay method. The isotope dilution method focuses on the isotope ratio of a specific element that constitutes the measurement target compound, and after adding a certain amount of spikes with a known isotope ratio to reach the equilibrium of the isotope composition of the measurement target compound In this method, the isotope ratio of the sample before and after spike addition is measured, and the element concentration of the sample is obtained from the change in the isotope composition. The advantage is that the detection limit is as low as about 1 pg.

  The quantitative data of the biomarker obtained as described above can be used for prediction and diagnosis of disease onset, progression, prognosis and the like. That is, as one embodiment of the present invention, an N-terminal amino group of a biomarker that is an amino acid derivative or peptide is reacted with a derivatizing agent such as a trinitrophenylating agent, and the N-terminal amino group is trinitrophenylated or the like. Using the mass spectrum intensity data obtained by using the mass spectrometric method, mass spectrometric analysis of the amino acid derivative or peptide in which the N-terminal amino group is derivatized such as trinitrophenylation, etc. A method for diagnosing a disease comprising a step of quantifying a biomarker concentration in a biological sample and a step of diagnosing the onset, progression, prognosis, etc. of the disease based on the biomarker concentration data thus obtained It is about.

  The diagnostic method of the present invention is useful because it can simultaneously measure and quantify a plurality of types of AGEs. That is, the present invention is a method capable of performing nitrophenylation in a sample, and is analyzed by mass spectrometry. Therefore, when measuring target substances having different mass numbers, it can be combined with mass spectrometry. The present invention provides an excellent method capable of simultaneously measuring (quantifying). According to the present invention, costs and labor can be greatly reduced as compared with the conventional early diagnosis method.

  Hereinafter, based on the Example of this invention, the characteristic and effect of this invention are demonstrated more concretely. These examples are illustrative only and are not intended to limit the scope of the present invention.

Trinitrophenylated methylglyoxal-derived hydroimidazolone derivative (MG-H1-TNP) [Iii] was prepared as follows.
Methylglyoxal-derived hydroimidazolone derivative (MG-H1) [IIa] was dissolved in 0.1 M boric acid solution (pH 9.3), and 30 mM 2,4,6-trinitrobenzenesulfonic acid sodium salt (TNBS) -0. After adding 10 μL of 1M boric acid solution (pH 9.3), the mixture was thoroughly mixed. The resulting mixture was incubated at 30 ° C. for 20 minutes for derivatization. Sep-Pak Plus C ₁₈ derivatized MG-H1 (MG-H1-TNP) with 100% CH ₃ CN / 0.1% formic acid (3 mL) and 0.1% formic acid (3 mL), respectively. After loading on a cartridge (Waters) and washing with 0.1% FA (3 mL), the fraction eluted with 100% CH ₃ CN / 0.1% formic acid (3 mL) was dried to dryness with a centrifugal evaporator. The fraction dried product was redissolved in 1.5 mL of deionized water, freeze-dried, and stored at -40 ° C.

The trinitrophenylated argpyrimidine derivative (AP-TNP) [Iiii] was prepared as follows.
Argpyrimidine [IIb] was treated in substantially the same manner as in Example 1 to obtain a trinitrophenylated argpyrimidine derivative (AP-TNP) [Iiii].

Measurement of Mass Spectra MG-H1 and AP, and N-2,4,6-trinitrophenyl derivatives of MG-H1 and AP prepared as described above (“MG-H1-TNP” and “AP-TNP respectively”) )) Was dissolved in 0.1% formic acid, and the mass spectrum was measured. The measurement conditions are as follows.
・ Ionization mode ESI-positive
・ Scanning range 100-500m / z
-Dry gas 330 ° C, 8.0 L / min-Nebulizer gas 276 kPa
・ Capillary voltage -2750V
-Skimmer voltage 40V
・ Capillary outlet voltage 124V

The mass spectra (MS and MS / MS) of MG-H1, AP, MG-H1-TNP and AP-TNP are shown in FIGS. 2 and 4, MG-H1 and N-2,4,6-trinitrophenyl derivatives of AP are denoted as “MG-H1-TNP” and “AP-TNP”, respectively. Precursor ions used for MS / MS measurement are [M + H] ⁺ ions for each compound. Among the obtained mass spectra, the one having the highest peak intensity was selected as an ion peak to be monitored in the LC-MRM-MS / MS analysis described later.

  Moreover, the fragmentation patterns of MG-H1-TNP and AP-TNP predicted from the MS spectrum and the MS / MS spectrum shown in FIGS. 3 and 4 are shown in FIGS. 5 and 6, respectively.

LC-MRM-MS / MS analysis LC-MRM-MS / MS analysis of MG-H1, AP, MG-H1-TNP and AP-TNP was performed under the following conditions. Each sample concentration was 10 nmol / L, and all were dissolved in 0.1% formic acid aqueous solution.
<HPLC conditions>
-Mobile phase MeOH-0.1% formic acid aqueous solution-Flow rate 0.20 mL / min-Gradient 0-100% (25 min)
・ Stationary phase Waters Atlantis T3 (φ2.1 × 100mm)
・ Column oven temperature 40 ℃

<MS conditions>
・ Ionization mode ESI-positive
・ Scanning range 100-500m / z
-Dry gas 330 ° C, 8.0 L / min-Nebulizer gas 276 kPa
・ Capillary voltage -2750V
-Skimmer voltage 40V
・ Capillary outlet voltage 124V
・ Injection volume 25μL

  The mass chromatograms of MG-H1, AP, MG-H1-TNP, and AP-TNP are shown in FIGS. 7 and 8, respectively. From the results shown in FIG. 7, it was confirmed that the peak intensity increased to about 1500 times when the N-terminal amino group of MG-H1 was 2,4,6-trinitrophenylated.

Preparation of calibration curve and determination of detection limit and quantification limit MG-H1-TNP and AP-TNP calibration curves using deuterated form of MG-H1-TNP (MG-H1-d3-TNP) as an internal standard Was made. In order to avoid the influence of decomposition by acid, dilution was performed by a procedure (see Table 1) in which a 0.1% formic acid aqueous solution was added immediately before injection, and measurement was performed from the high concentration side. First, a 1 μmol / mL aqueous solution of MG-H1-TNP and AP-TNP was prepared as a stock solution, and diluted with water so that the final concentrations were 400, 200, 40, 20, 4, 2 pmol / mL. For each concentration, add 25 μL of a 400 pmol / mL aqueous solution of MG-H1-d3-TNP to 25 μL of these equimolar mixtures, add 50 μL of a 0.2% aqueous formic acid solution, and mix well, 25 μL of which for LC-MS analysis. Using.

  By plotting the peak intensity ratio of MG-H1-TNP or AP-TNP and MG-H1-d3-TNP in the obtained mass spectrum against the concentration of MG-H1-TNP or AP-TNP, FIG. A calibration curve shown in FIGS. From these, for MG-H1-TNP, the detection limit was 2.05 pmol / mL, the limit of quantification was 6.20 pmol / mL, and for AP-TNP, the detection limit was 0.11 pmol / mL and the limit of quantification was 0.33 pmol / mL. It was. Similarly, when a calibration curve was prepared for CML-TNP and the concentration was converted, the detection limit was 5.3 nmol / L and the quantification limit was 16 nmol / L. For CEL-TNP, the detection limit was 2.7 nmol / L and the quantification limit was 8.0 nmol / L. Regarding the calibration curve, it was confirmed that good linearity with a correlation coefficient of 0.996 or more was obtained, and that quantitative analysis at the pmol level (concentration nM / L level) was possible for each. These numerical values are 1/100 to 1/200 of the detection limit in dansyl chloride and NDA (2,3-naphthalenedialdehyde) which are conventionally used derivatizing agents, and 2,4,6-tri It shows that nitrophenylation can greatly improve sensitivity.

Effect of pH on 2,4,6-trinitrophenylation In the preparation of MG-H1-TNP and AP-TNP in Examples 1 and 2, the pH of the reaction solution was adjusted to 7.5, 8.0, 8. The derivatization rate in the case of 5 was calculated using LC-MS analysis. Dissolve the dried product obtained by the centrifugal evaporator treatment in 100 μL of 0.2% formic acid aqueous solution, add 50 μL of 200 pmol / mL aqueous solution of MG-H1-d3-TNP as an internal standard, mix thoroughly, and then add 25 μL to LC. The sample was subjected to -MS analysis, and the concentration of 2,4,6-trinitrophenyl compound was determined by a calibration curve method. The results are as shown in FIGS. 11 and 12, and it is confirmed that derivatization can be quantitatively performed at pH 8.5 to 9.3 in either case of MG-H1-TNP or AP-TNP. It was done.

Rat Plasma Spike Test MG-H1, AP and MG-H1-d3, which is an internal standard, were spiked into control plasma of SD rats, and after recovery, 2,4,6-trinitrophenylation was performed. Plasma pretreatment was performed according to the protocol shown in FIG. 13 (see EMN Nakashima et al., Anal. Biochem., 414, 109-116 (2011)), and spiked MG-H1, AP and MG-H1. Attempted simultaneous detection of -d3. LC-MS analysis using a calibration curve prepared by the standard addition method confirmed that the plasma concentration of MG-H1 and AP could be quantified.

Examination using Val-Tyr Using the dipeptide Val-Tyr instead of AGEs MG-H1 and AP, the change in detection sensitivity due to 2,4,6-trinitrophenylation was performed in the same procedure as above. As a result of the examination, it was confirmed that the peak intensity increased about 50 times by 2,4,6-trinitrophenylation as in the case of MG-H1 (Table 2).

Examination using dipeptide The following dipeptide was prepared in substantially the same manner as in Example 8, and the change in detection sensitivity was examined. The results are shown in Table 2.

  The present invention is, for example, an amino compound obtained by derivatizing an amino acid derivative including a metabolite such as a final glycation product or an intermediate product obtained by saccharifying a protein that is an in vivo component in a metabolic process with a derivatizing agent such as a nitrobenzene compound. Since the compound is particularly useful for diagnosis of impaired glucose tolerance, it is a mass spectrometric method capable of quantifying these compounds quickly, with high sensitivity and high accuracy. Therefore, it can be expected that assaying these amino compounds as biomarkers will be useful for the prevention and diagnosis of various diseases, and thereby useful for the development of preventive and therapeutic agents for the diseases.

式中、Ｒはアミノ置換基を意味し、
Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味する。
前記一般式［Ｉａ］で表されるアミノ化合物またはその塩のアミノ置換基が、アミノ酸残基もしくは置換アミノ酸残基またはペプチド残基であり、
前記アミノ酸残基もしくは前記置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アラニン、アスパラギン、アスパラギン酸、システイン、グルタミン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リジン、ロイシン、メチオニン、プロリン、アルギニン、セリン、トレオニン、バリン、トリプトファンもしくはチロシンまたはそれらの置換体であることを特徴とするアミノ化合物またはその塩の質量分析方法。
［２］ 前記［１］に記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ酸残基もしくは置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アルギニン、リジン、バリン、チロシン、メチオニン、グリシン、ヒスチジン、グルタミン酸もしくはトリプトファンまたはそれらの置換体であることを特徴とするアミノ化合物またはその塩の質量分析方法。
［３］ 前記［１］または［２］に記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ化合物が、一般式［Ｉｉ］で表されることを特徴とするアミノ化合物またはその塩の質量分析方法。
［化１８−２］

式中、Ｒは前記と同じ意味を有する。
［４］ 前記［１］〜［３］のいずれかに記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ化合物が、一般式［Ｉｂ］で表されるニトロフェニル化メチルグリオキサール由来ヒドロイミダゾロン誘導体または一般式［Ｉｃ］で表されるニトロフェニル化アルグピリミジン誘導体であることを特徴とするアミノ化合物またはその塩の質量分析方法。
［化１８−３］

［化１８−４］

式中、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はいずれも前記と同じ意味を有する。
［５］ 前記［１］〜［４］のいずれかに記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ化合物が、構造式［Ｉｉｉ］で表されるトリニトロフェニル化メチルグリオキサール由来ヒドロイミダゾロン誘導体または構造式［Ｉｉｉｉ］で表されるトリニトロフェニル化アルグピリミジン誘導体であることを特徴とするアミノ化合物またはその塩の質量分析方法。
［化１８−５］

［化１８−６］

［００７２−２］
［６］ 一般式［ＩＩ］で表されるアミノ酸誘導体またはその塩において、アミノ置換基がアミノ酸残基もしくは置換アミノ酸残基またはペプチド残基であり、前記アミノ酸残基もしくは前記置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アラニン、アスパラギン、アスパラギン酸、システイン、グルタミン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リジン、ロイシン、メチオニン、プロリン、アルギニン、セリン、トレオニン、バリン、トリプトファンもしくはチロシンまたはそれらの置換体であるアミノ酸誘導体またはその塩と、一般式［ＩＩＩａ］で表されるニトロベンゼン化合物を反応させて一般式［Ｉａ］で表されるアミノ化合物またはその塩を得る工程と、
上記工程で得られたアミノ化合物またはその塩を質量分析する工程を有することを特徴とするアミノ化合物またはその塩の質量分析方法。
［化１８−７］

式中、Ｒはアミノ置換基を意味する。
［化１８−８］

式中、Ｘはアミノ基反応性官能基を意味し、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味する。
［化１８−９］

式中、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味し、Ｒは前記と同じ意味を有する。
［７］ 前記一般式［ＩＩ］で表されるアミノ酸誘導体またはその塩と、一般式［ＩＩＩａ］で表されるニトロベンゼン化合物の反応を、アルカリ性水溶液または水と有機溶媒の混合溶液であり、かつ前記溶液のｐＨが８〜１０でおこなうことを特徴とする前記［６］に記載のアミノ化合物またはその塩の質量分析方法。
［８］ 前記［６］または［７］に記載のアミノ化合物またはその塩の質量分析方法であって、前記反応が試料中で行われることを特徴とするアミノ化合物またはその塩の質量分析方法。
［９］ 前記［６］〜［８］のいずれかに記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ酸誘導体またはその塩がバイオマーカーであることを特徴とするアミノ化合物の質量分析方法。
［００７２−３］
［１０］ 前記［１］〜［９］のいずれかに記載のアミノ化合物またはその塩の質量分析方法により得られる質量スペクトルの強度データを用いて、生体試料中のバイオマーカー濃度の定量を行うことを特徴とするバイオマーカーのアッセイ方法。
［００７２−４］
［１１］ 一般式［Ｉａ］で表されるアミノ化合物またはその塩であって、式［Ｉａ］におけるアミノ置換基が、アミノ酸残基もしくは置換アミノ酸残基またはペプチド残基であり、アミノ酸残基もしくは置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アラニン、アスパラギン、アスパラギン酸、システイン、グルタミン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リジン、ロイシン、メチオニン、プロリン、アルギニン、セリン、トレオニン、バリン、トリプトファンもしくはテロシンまたはそれらの置換体である質量分析用のアミノ化合物またはその塩。
［化１８−１０］

式中、Ｒはアミノ置換基を意味し、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味する。
［１２］ 前記［１１］に記載のアミノ化合物またはその塩であって、前記アミノ酸残基もしくは置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アルギニン、リジン、バリン、チロシン、メチオニン、グリシン、ヒスチジン、グルタミン酸もしくはトリプトファンまたはそれらの置換体であることを特徴とする質量分析用のアミノ化合物またはその塩。
［１３］ 前記［１１］または［１２］に記載のアミノ化合物またはその塩であって、前記置換アミノ酸残基が、最終糖化産物（ＡＧＥ）の残基であることを特徴とする質量分析用のアミノ化合物またはその塩。
［１４］ 前記［１３］に記載のアミノ化合物またはその塩であって、前記最終糖化産物（ＡＧＥ）が、アルギニン誘導体またはリジン誘導体であることを特徴とする質量分析用のアミノ化合物またはその塩。
［１５］ 前記［１１］または［１２］に記載のアミノ化合物またはその塩であって、前記ペプチド残基のペプチドが、２個ないし１０個のアミノ酸がペプチド結合していることを特徴とする質量分析用のアミノ化合物またはその塩。
［１６］ 前記［１１］〜［１５］のいずれかに記載のアミノ化合物またはその塩であって、前記アミノ化合物が、一般式［Ｉｉ］で表されることを特徴とする質量分析用のアミノ化合物またはその塩。
［化１８−１１］

式中、Ｒは前記と同じ意味を有する。
［００７２−５］
［１７］ 一般式［Ｉｂ］で表されるニトロフェニル化メチルグリオキサール由来ヒドロイミダゾロン誘導体または一般式［Ｉｃ］で表されるニトロフェニル化アルグピリミジン誘導体であることを特徴とするアミノ化合物またはその塩。
［化１８−１２］

［化１８−１３］

式中、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はいずれも前記と同じ意味を有する。
［１８］ 前記［１７］に記載のアミノ化合物またはその塩であって、前記アミノ化合物が、構造式［Ｉｉｉ］で表されるトリニトロフェニル化メチルグリオキサール由来ヒドロイミダゾロン誘導体または構造式［Ｉｉｉｉ］で表されるトリニトロフェニル化アルグピリミジン誘導体であることを特徴とするアミノ化合物またはその塩。
［化１８−１４］

［化１８−１５］

発明の効果
［００７３］
本発明によって、アミノ酸誘導体のＮ末端アミノ基を誘導体化することにより、質量分析におけるイオン化効率が向上し、試料中に少量しか存在しない最終糖化産物等の分子バイオマーカーやペプチドホルモンについても、質量分析方法を用いて迅速、高感度かつ高精度な定量分析が可能になる。したがって、本発明は、アミノ酸誘導体とニトロベンゼン化合物等の誘導体化剤との誘導体化生成物である新規なアミノ化合物、好ましくはタンパク質の解糖等の反応中間産物である糖化タンパク質分解物がニトロベンゼン化合物等の誘導体化剤で誘導体化された、耐糖能異常の診断に有用な新規な糖化タンパク質分解物誘導体を提供する。また、本発明は、そのアミノ化合物についての迅速、高感度かつ高精度な定量が可能な質量分析方法およびそれを用いたバイオマーカーのアッセイ方法を提供する。
図面の簡単な説明
［００７４］
［図１］メチルグリオキサール由来ヒドロイミダゾロン誘導体（ＭＧ−Ｈ１）の質量スペクトル（ＭＳ、ＭＳ／ＭＳ）である。
［図２］ＭＧ−Ｈ１の２，４，６−トリニトロフェニル誘導体（ＭＧ−Ｈ１−ＴＮＰ）の質量スペクトル（ＭＳ、ＭＳ／ＭＳ）である。
［図３］アルグピリミジン（ＡＰ）の質量スペクトル（ＭＳ、ＭＳ／ＭＳ）である。
［図４］ＡＰの２，４，６−トリニトロフェニル誘導体（ＡＰ−ＴＮＰ）の質量スペクトル（ＭＳ、ＭＳ／ＭＳ）である。
［図５］質量スペクトルより予測されるＭＧ−Ｈ１の２，４，６−トリニトロフェニル誘導体（ＭＧ−Ｈ１−ＴＮＰ）のフラグメンテーションパターンを示す[0014]
[0071]
In the formula [I ′], R ′ means a substituted amino acid residue or peptide residue having the same meaning as R, and Z ′ means a derivatized residue.
[0072]
As yet another embodiment, the present invention provides a biomarker assay method for quantifying the biomarker concentration in a biological sample using the intensity data of the mass spectrum obtained by the mass spectrometry method for amino compounds described above. In the formula [I ′], R ′ means a substituted amino acid residue or peptide residue having the same meaning as R, and Z ′ means a derivatized residue.
[0072-1]
The present invention provides the following mass spectrometry method and biomarker assay method of an amino compound or a salt thereof, and an amino compound or a salt thereof.
[1] In a mass spectrometry method for an amino compound or a salt thereof, wherein the amino compound represented by the general formula [Ia] or a salt thereof is measured by mass spectrometry.
[Formula 18-1]

Where R represents an amino substituent,
R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.
The amino substituent of the amino compound represented by the general formula [Ia] or a salt thereof is an amino acid residue, a substituted amino acid residue or a peptide residue;
The amino acid residue or amino acid of the substituted amino acid residue or the constituent amino acid of the peptide residue is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline , Arginine, serine, threonine, valine, tryptophan or tyrosine, or a substitute thereof, A mass spectrometric method for an amino compound or a salt thereof
[2] The mass spectrometric method of the amino compound or the salt thereof according to [1], wherein the amino acid residue or the substituted amino acid residue or the constituent amino acid of the peptide residue is arginine, lysine, valine, A method for mass spectrometry of an amino compound or a salt thereof, which is tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan, or a substituted product thereof.
[3] A mass spectrometric method of the amino compound or salt thereof according to [1] or [2], wherein the amino compound is represented by the general formula [Ii], or an amino compound thereof Salt mass spectrometry.
[Chemical 18-2]

In the formula, R has the same meaning as described above.
[4] A mass spectrometric method of the amino compound or salt thereof according to any one of [1] to [3], wherein the amino compound is derived from nitrophenylated methylglyoxal represented by the general formula [Ib] A method for mass spectrometry of an amino compound or a salt thereof, which is a hydroimidazolone derivative or a nitrophenylated argypyrimidine derivative represented by the general formula [Ic].
[Chemical Formula 18-3]

[Formula 18-4]

In the formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.
[5] A mass spectrometric method of the amino compound or salt thereof according to any one of [1] to [4], wherein the amino compound is trinitrophenylated methylglyoxal represented by the structural formula [Iiii] A method for mass spectrometry of an amino compound or a salt thereof, which is a hydronitroazolone derivative or a trinitrophenylated argpyrimidine derivative represented by the structural formula [Iiii].
[Formula 18-5]

[Formula 18-6]

[0072-2]
[6] In the amino acid derivative represented by the general formula [II] or a salt thereof, the amino substituent is an amino acid residue, a substituted amino acid residue or a peptide residue, and the amino acid residue or the amino acid of the substituted amino acid residue Or the constituent amino acids of the peptide residues are alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine, valine, tryptophan or tyrosine Or reacting an amino acid derivative or a salt thereof as a substitute thereof with a nitrobenzene compound represented by the general formula [IIIa] to obtain an amino compound represented by the general formula [Ia] or a salt thereof;
A method for mass spectrometry of an amino compound or a salt thereof, comprising a step of mass-analyzing the amino compound or a salt thereof obtained in the above step.
[Chemical Formula 18-7]

In the formula, R means an amino substituent.
[Formula 18-8]

In the formula, X represents an amino group-reactive functional group, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group To do. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.
[Formula 18-9]

In the formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group, and R has the same meaning as described above.
[7] The reaction of the amino acid derivative represented by the general formula [II] or a salt thereof and the nitrobenzene compound represented by the general formula [IIIa] is an alkaline aqueous solution or a mixed solution of water and an organic solvent, and The method for mass spectrometry of an amino compound or a salt thereof according to the above [6], wherein the pH of the solution is 8 to 10.
[8] A mass spectrometric method for the amino compound or salt thereof according to [6] or [7], wherein the reaction is carried out in a sample.
[9] A mass spectrometry method of the amino compound or salt thereof according to any one of [6] to [8], wherein the amino acid derivative or salt thereof is a biomarker. Analysis method.
[0072-3]
[10] Quantifying the biomarker concentration in the biological sample using the intensity data of the mass spectrum obtained by the mass spectrometry method of the amino compound or salt thereof according to any one of [1] to [9] A biomarker assay method characterized by the above.
[0072-4]
[11] An amino compound represented by the general formula [Ia] or a salt thereof, wherein the amino substituent in the formula [Ia] is an amino acid residue, a substituted amino acid residue or a peptide residue, The amino acid of the substituted amino acid residue or the constituent amino acid of the peptide residue is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine , An amino compound for mass spectrometry which is valine, tryptophan or telosin or a substitute thereof, or a salt thereof.
[Chemical Formula 18-10]

In the formula, R represents an amino substituent, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.
[12] The amino compound or a salt thereof according to [11] above, wherein the amino acid residue or substituted amino acid residue or the constituent amino acid of the peptide residue is arginine, lysine, valine, tyrosine, methionine, An amino compound or a salt thereof for mass spectrometry, which is glycine, histidine, glutamic acid, tryptophan, or a substituted product thereof.
[13] The amino compound or salt thereof according to [11] or [12] above, wherein the substituted amino acid residue is a residue of a final glycation product (AGE). An amino compound or a salt thereof.
[14] The amino compound or salt thereof for mass spectrometry, wherein the amino compound or salt thereof according to the above [13], wherein the final glycation product (AGE) is an arginine derivative or a lysine derivative.
[15] The amino compound or the salt thereof according to [11] or [12], wherein the peptide of the peptide residue is a peptide bond of 2 to 10 amino acids. An amino compound for analysis or a salt thereof.
[16] The amino compound or the salt thereof according to any one of [11] to [15], wherein the amino compound is represented by the general formula [Ii] Compound or salt thereof.
[Chemical Formula 18-11]

In the formula, R has the same meaning as described above.
[0072-5]
[17] An amino compound or a salt thereof, which is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib] or a nitrophenylated argpyrimidine derivative represented by the general formula [Ic] .
[Chemical Formula 18-12]

[Chemical Formula 18-13]

In the formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.
[18] The amino compound or the salt thereof according to [17], wherein the amino compound is a trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iiii] or the structural formula [Iiii] An amino compound or a salt thereof, which is a trinitrophenylated argypyrimidine derivative represented by the formula:
[Formula 18-14]

[Chemical Formula 18-15]

Effects of the Invention [0073]
By derivatizing the N-terminal amino group of an amino acid derivative according to the present invention, ionization efficiency in mass spectrometry is improved, and mass spectrometry is also performed for molecular biomarkers such as final glycation products and peptide hormones that exist only in a small amount in a sample. The method enables rapid, highly sensitive and accurate quantitative analysis. Accordingly, the present invention provides a novel amino compound that is a derivatization product of an amino acid derivative and a derivatizing agent such as a nitrobenzene compound, preferably a glycated protein degradation product that is a reaction intermediate product such as protein glycolysis is a nitrobenzene compound or the like. The present invention provides a novel glycated protein hydrolyzate derivative that is derivatized with a derivatizing agent useful for diagnosing abnormal glucose tolerance. The present invention also provides a mass spectrometric method capable of rapid, highly sensitive and highly accurate quantification of the amino compound, and a biomarker assay method using the same.
BRIEF DESCRIPTION OF THE DRAWINGS [0074]
FIG. 1 is a mass spectrum (MS, MS / MS) of methylglyoxal-derived hydroimidazolone derivative (MG-H1).
FIG. 2 is a mass spectrum (MS, MS / MS) of a 2,4,6-trinitrophenyl derivative (MG-H1-TNP) of MG-H1.
FIG. 3 is a mass spectrum (MS, MS / MS) of argypirimidine (AP).
FIG. 4 is a mass spectrum (MS, MS / MS) of a 2,4,6-trinitrophenyl derivative of AP (AP-TNP).
FIG. 5 shows a fragmentation pattern of a 2,4,6-trinitrophenyl derivative of MG-H1 (MG-H1-TNP) predicted from a mass spectrum.

  The present invention relates to an amino compound, a high-sensitivity mass spectrometry method thereof, and a biomarker assay method. More specifically, an amino compound obtained by derivatization of an amino acid derivative such as a final saccharified product and a nitrobenzene compound and a salt thereof, and a mass spectrometric method capable of quantifying the amino compound quickly with high sensitivity and high accuracy , And a biomarker assay method using the same.

  Diabetes is a condition in which the blood sugar level is pathologically high. In addition to acute symptoms caused by abnormal blood sugar levels, various organs are damaged as hyperglycemia continues for a long period of time. When complications develop, it becomes very difficult to treat, so it is important to diagnose the onset at an early stage and start treatment such as lifestyle improvement, oral hypoglycemic drug administration, and insulin injection.

  However, the number of diabetic patients, mainly caused by lifestyle-related diseases, has been increasing year by year. According to estimates by the Ministry of Health, Labor and Welfare, over 9 million people are no longer a matter of time. In addition to the number of diabetic patients, it is estimated that there are more than 10 million so-called diabetic reserves, so it is not difficult to imagine that the number of diabetic patients will increase dramatically in the near future. However, only 3 million patients are actually undergoing treatment for diabetes, and the number of patients who suffer from nephropathy due to delay in treatment without sufficient diagnosis increases by 10,000 each year. This is the current situation. In addition, considering that about half of all diabetic patients die from ischemic diseases such as myocardial infarction and cerebral infarction, measures against diabetes and its complications, and so-called hidden diabetes (prediabetes), It is extremely important from the medical economy. In particular, there is an urgent need to establish a countermeasure for prediabetes, which is a potential diabetic army that is extremely difficult to determine only by measuring blood glucose levels in health examinations, that is, an early diagnosis method and a treatment system.

  Moreover, in recent years, among prediabetes patients, fasting blood glucose level (FPG) is normal, but postprandial blood glucose level becomes extremely high due to impaired glucose tolerance (IGT), thereby causing cardiovascular disorder It is pointed out that there is an increase. For this reason, the American Diabetes Association and the World Health Organization define pre-diabetes as a prevalent disease and advocate the importance of improving quality of life and improving IGT of pre-diabetes by drug treatment. As described above, such prediabetic patients are estimated to exceed an estimated 10 million people in Japan. Therefore, prevention and treatment measures for prediabetes are extremely important.

  The Japan Diabetes Society has guided the implementation of fasting blood glucose (FPG) in the primary health checkup and oral glucose tolerance test (OGTT) in the secondary health checkup as diagnostic criteria for prediabetes and diabetes. Glucose intolerance in pre-diabetic patients is difficult to accurately diagnose only by the FPG test and glycated hemoglobin (HbA1c) test in the primary health checkup. Is recommended to take a secondary health checkup for OGTT. However, this OGTT lacks the subject's long restraint time and convenience, and also has a problem such as imposing a physical burden of sugar (glucose) load and accompanying danger such as a rapid increase in blood glucose level, The reality is that it is not as widespread as the blood glucose testing method by blood tests in general health examinations. As a result, many potential prediabetics have failed to undergo a second OGTT health checkup, progressing to true diabetes without being aware of diabetes or its complications, and more severe Complications or necessitating renal dialysis.

  In the primary health examination, a test is usually performed using fasting blood glucose (FPG), occasional blood glucose or glycated hemoglobin (HbA1c) as a determination index, and the FPG value is 100 mg / dL or higher, or the HbA1c value is 5.2%. If it is above, it is diagnosed that there is a suspicion of insulin resistance or IGT caused by it. However, since IGT due to insulin resistance appears as a major initial change after postprandial hyperglycemia, it is necessary to say that any index used in the conventional primary health check is insufficient in terms of accuracy, Conventional primary health checkup methods cannot be used as an early diagnosis of prediabetes.

  Therefore, in reality, in a primary health checkup, a subject who is suspected of having insulin resistance or IGT using these indexes is subjected to a 75 g oral glucose tolerance test (secondary health checkup or subsequent health checkup ( 75 g OGTT), blood insulin, HOMA-R and the like are tested to determine whether insulin resistance or IGT is positive, and a definitive diagnosis of prediabetes has been made. Of course, these inspections are expensive and troublesome.

  Therefore, if there is a method that can confirm the diagnosis of prediabetes with the same accuracy as OGTT with the same blood sample for blood glucose measurement used in the primary health checkup, the detection rate of prediabetic patients will improve dramatically, The OGTT test in the secondary health checkup is unnecessary, and it is possible to strongly promote the strategy for preventing diabetes by early diagnosis and treatment of prediabetes proposed by the World Health Organization.

  On the other hand, in recent years, various final glycation products have attracted attention as risk factors for chronic complications of diabetes. Final glycated products (AGEs) are also called glycated proteins, Maillard reaction products, and the like, and are protein derivatives having various structures generated by non-enzymatic reaction between a reducing sugar such as glucose and the amino group of the protein. AGEs are the result of disruption of the functions of these proteins resulting from glycation modification of extracellular matrix proteins, membrane proteins, and intracellular proteins, and cell functions dependent on them, or the cellular responses caused by receptors with AGEs as ligands. Involved in the development and exacerbation of various lesions. For example, when AGEs are recognized by RAGEs, which are one of the AGE receptors, the production of intracellular oxidative stress substances by intracellular NADPH oxidase is enhanced, which changes gene expression in epithelial cells, It is thought that the diabetic vascular disorder of the other will develop (refer nonpatent literature 1).

  Among AGEs, those derived from glyceraldehyde and methylglyoxal (MG), which are produced as glycolytic intermediates or by-products, are expected to be diagnostic markers for the onset and prognosis of diabetes and impaired glucose tolerance. (Non-Patent Documents 2, 3, 4, 5).

  Non-patent document 2 shows that the serum level of methylglyoxal-derived hydroimidazole was measured in an ELISA system using a polyclonal antibody of methylglyoxal-derived hydroimidazole for the serum of type 2 diabetic patients. It has been reported that it has increased. The non-patent document 2 shows that there is a significant correlation between the serum level of methylglyoxal-derived hydroimidazole and the serum level of Nε- (carbonylmethyl) lysine, which is one of AGEs, Serum levels of methylglyoxal-derived hydroimidazoles have been reported to be unrelated to fasting blood glucose plasma levels or hemoglobin levels.

  In Non-patent Document 3, as a result of measuring plasma of a type 1 diabetic patient using LC / MS / MS, the concentration of an adduct due to glycation or oxidation of protein and the concentration of its free adduct increased. It shows that. For example, methyl glyoxal-derived hydroimidazolone, glyoxal-derived hydroimidazolone or other AGEs such as Nε- (carbonylmethyl) lysine, Nε- (carbonylethyl) lysine, and free adducts thereof are increased. It has been reported that

  Non-patent document 4 suggests that methylglyoxal-derived hydroimidazolone free adduct is an excellent marker for diabetic patients as a result of tandem mass spectrometry of plasma of type 1 diabetic patients.

  Non-patent document 5 shows that as a result of measuring plasma and urine samples of type 1 diabetic patients by LC-MS / MS, in addition to an increase in blood glucose level and insulin level after glucose loading, methylglyoxal-derived hydroimidazole free adduct was added. First, Nε- (carbonylmethyl) lysine, Nε- (carbonylethyl) lysine, glyoxal-derived hydroimidazolone, Nδ- [5- (2,3,4-trihydroxybutyl) -5-hydro-4-imidazolone 2- Ill] -ornithine (3DG-H1) and other free adducts have also been reported to increase. That is, this paper shows that not only hyperglycemia occurs after meals, but also a high carbonyl stress state is induced. Furthermore, this document describes tandem mass spectrometry results for many AGEs including argypirimidine and amino acids (arginine, lysine, methionine, tyrosine and tryptophan) in addition to methylglyoxal-derived hydroimidazolone free adduct. ing.

  As described above, a technique for analyzing these AGEs by tandem mass (MS / MS) has been reported. However, there is currently no effective method for measuring these AGEs other than the total amount evaluation using an immunochemical method such as an ELISA method (Patent Document 1). There is almost no established measurement method for products, and it must be said that there is no measurement method that can be applied clinically. Since the quantification of these individual AGEs is useful not only in the diagnosis of diabetes and impaired glucose tolerance but also in metabolome analysis, etc., individual compounds can be measured more quickly and with high sensitivity and high accuracy for AGEs. An assay is highly desired.

  In addition to AGE analysis, amino acid analysis is also very important for clinical fields such as innate metabolic disorder assays and has the potential to be used for diagnostic biomarker searches. Amino acid analysis is used, for example, as a diagnosis of congenital amino acidosis, the severity of liver dysfunction, and a guideline for treatment. In recent reports, amino acid profiles have been applied to the diagnosis of progressive fibrosis and the like in patients with chronic hepatitis C. Furthermore, the possibility of analyzing the degree of health of living organisms using an amino acid profile as an index has been reported (Non-patent Document 6).

  An amino acid automatic analyzer has been developed for amino acid analysis as described above. This apparatus measures the colorimetric color of amino acids using a ninhydrin reagent and requires a very long time for analysis.

  In addition to the colorimetric measurement method as described above, a precolumn derivatization method has been developed, and various derivatizing agents used for the precolumn derivatization method have been developed (Non-patent Document 6). However, this derivatization method is a complicated and time-consuming technique. Furthermore, an automatic amino acid analysis method combining this derivatization method and a mass spectrometry method has also been developed, but further improvement has been demanded for this method.

JP 2008-224453 A

Marie-Paule Wautier et al., "Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE", American Journal of Physiology-Endocrinology and Metabolism, (USA), American Physiological Society, 2001, 5 Moon, Volume 280, E685-E694 Kilhovd, B.K., et al. Metabolism, Vol 52, No 2 (February), 2003: pp. 163-167 Rabbani, N., and Thornalley, P.J.Amino Acids DOI 10.1007 / s00726-010-0783-0 Ahmed, N., et al. Diabetologia (2005) 48: 1590-1603 Ahmed, N., et al. Diabetes Care, Vol. 28, No. 10, October 2005, pp. 2465-2471 Shimbo, K., et al. Biomed. Chromatogr. 2010; 24: 683-691.

  Therefore, as a result of intensive studies in view of such circumstances, the present inventors have determined that the terminal amino groups of AGEs, such as methylglyoxal-derived hydroimidazolone derivatives (MG-H1) and argypirimidine (AP), are nitrobenzene compounds. By derivatizing, the sensitivity in mass spectrometry was greatly improved, and in addition to AGEs, it was found that it could be applied to other amino acid derivatives or amino compounds such as peptides, and the present invention was completed. .

  Therefore, the present invention enables amino acid compounds and salts thereof obtained by derivatization of amino acid derivatives, particularly final glycation products, and nitrobenzene compounds, and quantification of amino compounds quickly and with high sensitivity. It is an object of the present invention to provide a mass spectrometric method and a biomarker assay method using the same.

  In order to achieve the above object, the present invention provides an amino compound represented by the general formula [I] or a salt thereof.

  In the formula [I], R means an amino substituent and Z means a nitrophenyl group.

  In a preferred embodiment of the present invention, an amino compound or a salt thereof in which the amino substituent is an amino acid residue or a substituted amino acid residue or a peptide residue in which the amino acid residue and / or the substituted amino acid residue is a peptide bond. provide.

  In a preferred embodiment of the present invention, the amino acid residue or substituted amino acid residue or the constituent amino acid of the peptide residue is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine. , Lysine, leucine, methionine, proline, arginine, serine, threonine, valine, tryptophan or tyrosine or a substitute thereof, preferably arginine, lysine, valine, tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan or a substitute thereof An amino compound or a salt thereof is provided.

  As another preferred embodiment of the present invention, an amino compound or a salt thereof in which the substituted amino acid residue is a residue of a final glycation product (AGE), preferably an arginine derivative or a lysine derivative.

  As another preferred embodiment, the present invention provides an amino compound or a salt thereof, wherein the peptide of the above peptide residue is a peptide bond of 2 to 10 amino acids.

  As another preferred embodiment of the present invention, an amino compound or a salt thereof, in which the nitrophenyl group is represented by the general formula [IIIb], is provided.

In the formula [IIIb], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.

  As a more preferred embodiment of the present invention, the amino compound is an amino compound represented by the general formula [Ia] or a salt thereof.

In the formula [Ia], R, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ each have the same meaning as described above.

  As another preferred embodiment of the present invention, the amino compound is an amino compound represented by the general formula [Ii] or a salt thereof.

    In the formula [Ii], R has the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib] or a nitrophenylated argpyrimidine derivative represented by the general formula [Ic]. An amino compound or a salt thereof is provided.

In the formulas [Ib] and [Ic], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a trinitrophenylated argon represented by the structural formula [Iiii]. An amino compound which is a pyrimidine derivative or a salt thereof is provided.

  This invention provides the mass spectrometry method of the amino compound or its salt which measures the amino compound or its salt represented with general formula [I] by mass spectrometry as another form.

  In the formula [I], R means an amino substituent, for example, an amino acid residue or a substituted amino acid residue or a peptide residue, and Z means a nitrophenyl group.

  As a preferred embodiment, the present invention provides a mass spectrometric method for the amino compound represented by the general formula [Ia] or a salt thereof.

In the formula [Ia], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group, and R has the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib] or a nitrophenylated argpyrimidine derivative represented by the general formula [Ic]. A method for mass spectrometry of an amino compound or a salt thereof is provided.

In the formulas [Ib] and [Ic], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  In a more preferred embodiment of the present invention, the amino compound is a trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or an amino compound represented by the structural formula [Iiii] or a salt thereof. A mass spectrometry method is provided.

As another form of the present invention, the amino acid derivative represented by the general formula [II] or a salt thereof and the nitrobenzene compound represented by the general formula [III] are reacted to represent the general formula [I]. Obtaining an amino compound or a salt thereof;
Provided is a method for mass spectrometry of an amino compound or a salt thereof, which comprises a step of mass-analyzing the amino compound or a salt thereof obtained in the above step.

  In the formula [II], R means an amino substituent, for example, an amino acid residue or a substituted amino acid residue or a peptide residue.

  In the formula [III], X means an amino group-reactive functional group, and Z means a nitrophenyl group.

  In the formula [I], R and Z have the same meaning as described above.

  As a more preferred embodiment of the present invention, the reaction of the amino acid derivative represented by the general formula [II] or a salt thereof and the nitrobenzene compound represented by the general formula [III] is carried out by using an alkaline aqueous solution or water and an organic solvent. Provided is a mass spectrometric method for an amino compound or a salt thereof, which is a mixed solution and the pH of the solution is 8 to 10.

  As a preferred embodiment, the present invention provides a mass spectrometric method for amino compounds or salts thereof in which the above reaction is carried out in a sample.

  As a more preferred embodiment, the present invention provides a mass spectrometric method for amino compounds in which the amino acid derivative or salt thereof is a biomarker.

  As another embodiment, the present invention provides a method for mass spectrometry of an amino compound or a salt thereof, wherein the amino compound represented by the general formula [I ′] or a salt thereof is subjected to mass spectrometry, wherein the derivatization residue is derivatized. Agents are sulfonyl compounds such as 1-pyrenesulfonyl chloride (PSC) and dansyl chloride (DNS-Cl); 9-fluorenylmethyl chloroformate (FMOC), 4-dimethylaminosulfonyl-7-fluorobenzoxadiazole (DBD-F), 4-fluoro-7-nitrobenzofurazan (NBD-F), 2,3-naphthalenedialdehyde (NDA); 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), etc. Carbamate compound; 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2-quinoxalinone ( MEQ-COCl), 3-nitrophenyl isothiocyanate, 3-pyridyl isothiocyanate, 4- (dimethylamino) phenyl isothiocyanate, fluorescein-5-isothiocyanate (FITC), and the like; diethyl ethoxymethylene malonate, etc. Mass spectrometric method of amino compound or salt thereof which is a malonate compound, a phosphonium compound such as (5-succinimidoxy-5-oxopentyl) triphenylphosphonium bromide (SPTPP); or a pyridinium compound such as 2-chloro-1-methylpyridinium salt I will provide a.

  In the formula [I ′], R ′ means a substituted amino acid residue or peptide residue having the same meaning as R, and Z ′ means a derivatized residue.

  As yet another embodiment, the present invention provides a biomarker assay method for quantifying the biomarker concentration in a biological sample using the intensity data of the mass spectrum obtained by the mass spectrometry method for amino compounds described above.

（式中、Ｒはアミノ置換基を意味し、
Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味する。）
式［Ia］で表されるアミノ化合物またはその塩におけるアミノ置換基が、置換アミノ酸残基またはペプチド残基であり、
前記置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アラニン、アスパラギン、アスパラギン酸、システイン、グルタミン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リジン、ロイシン、メチオニン、プロリン、アルギニン、セリン、トレオニン、バリン、トリプトファンもしくはチロシンまたはそれらの置換体であり、
かつ、前記置換アミノ酸残基が、最終糖化産物（ＡＧＥ）の残基である、または前記ペプチド残基のペプチドが、２個ないし１０個のアミノ酸がペプチド結合しているペプチドであることを特徴とするアミノ化合物またはその塩の質量分析方法。

［２］ 前記［１］に記載のアミノ化合物またはその塩の質量分析方法であって、
前記最終糖化産物（ＡＧＥ）が、置換アルギニン誘導体または置換リジン誘導体であることを特徴とするアミノ化合物またはその塩の質量分析方法。

［３］ 前記［１］に記載のアミノ化合物またはその塩の質量分析方法であって、
前記最終糖化産物（ＡＧＥ）が、グリオキサールヒドロイミダゾロン、メチルグリオキサールヒドロイミダゾロン、アルグピリミジン、カルボキシメチルリジン、カルボキシエチルリジンからなる群から選択されるいずれかの最終糖化産物（ＡＧＥ）であることを特徴とするアミノ化合物またはその塩の質量分析方法。

［４］ 前記［１］に記載のアミノ化合物またはその塩の質量分析方法であって、
前記置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アルギニン、リジン、バリン、チロシン、メチオニン、グリシン、ヒスチジン、グルタミン酸もしくはトリプトファンまたはそれらの置換体であることを特徴とする質量分析用のアミノ化合物またはその塩。

［５］ 前記［１］〜［４］のいずれかに記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ化合物が、一般式［Ii］で表されることを特徴とするアミノ化合物またはその塩の質量分析方法。

（式中、Ｒは前記と同じ意味を有する。）

［６］ 前記［１］〜［５］のいずれかに記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ化合物が、一般式［Ib］で表されるニトロフェニル化メチルグリオキサール由来ヒドロイミダゾロン誘導体または一般式［Ic］で表されるニトロフェニル化アルグピリミジン誘導体であることを特徴とするアミノ化合物またはその塩の質量分析方法。

（式中、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はいずれも前記と同じ意味を有する。）

［７］ 前記［１］〜［６］のいずれかに記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ化合物が、
構造式［Iii］で表されるトリニトロフェニル化メチルグリオキサール由来ヒドロイミダゾロン誘導体または構造式［Iiii］で表されるトリニトロフェニル化アルグピリミジン誘導体であることを特徴とするアミノ化合物またはその塩の質量分析方法。

That is, the present invention relates to the following inventions.
[1] In a mass spectrometry method for an amino compound or a salt thereof, wherein the amino compound represented by the general formula [Ia] or a salt thereof is measured by mass spectrometry,

(Wherein R represents an amino substituent,
R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group. )
Amino substituent in the amino compound represented by the formula [Ia] is a substitution amino acid residue or peptide residue,
Constituent amino acids of an amino acid or said peptide residue before Ki置 changeover amino residue, alanine, asparagine, aspartate, cysteine, glutamine, glutamate, phenylalanine, glycine, histidine, isoleucine lysine, a leucine, methionine, proline, arginine, serine, threonine, valine, tryptophan or tyrosine, or substitutions thereof der is,
And, wherein the substituted amino acid residue is a residue of advanced glycation products (AGE), or a peptide of the peptide residues, a Oh Rukoto with peptides 2 to 10 amino acids are a peptide bond A mass spectrometric method of the amino compound or salt thereof.

[2] A mass spectrometric method of the amino compound or salt thereof according to [1],
A method for mass spectrometry of an amino compound or a salt thereof, wherein the final glycation product (AGE) is a substituted arginine derivative or a substituted lysine derivative.

[3] A mass spectrometric method of the amino compound or salt thereof according to [1],
The final glycation product (AGE) is any final glycation product (AGE) selected from the group consisting of glyoxal hydroimidazolone, methylglyoxal hydroimidazolone, argypirimidine, carboxymethyl lysine, and carboxyethyl lysine. A mass spectrometric method for a characteristic amino compound or a salt thereof.

[ 4 ] A mass spectrometric method for the amino compound or salt thereof according to [ 1 ] above,
The amino acid of the substituted amino acid residue or the constituent amino acid of the peptide residue is arginine, lysine, valine, tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan or a substituted product thereof for mass spectrometry An amino compound or a salt thereof.

[ 5 ] A mass spectrometry method of the amino compound or salt thereof according to any one of [1] to [4] , wherein the amino compound is represented by the general formula [Ii] A method for mass spectrometry of a compound or a salt thereof.

(In the formula, R has the same meaning as described above.)

[ 6 ] A mass spectrometry method of the amino compound or a salt thereof according to any one of [1] to [ 5 ], wherein the amino compound is derived from nitrophenylated methylglyoxal represented by the general formula [Ib] A method for mass spectrometry of an amino compound or a salt thereof, which is a hydroimidazolone derivative or a nitrophenylated argypyrimidine derivative represented by the general formula [Ic].

(In the formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.)

[ 7 ] The mass spectrometric method of the amino compound or the salt thereof according to any one of [1] to [ 6 ], wherein the amino compound is
A trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a trinitrophenylated argpyrimidine derivative represented by the structural formula [Iiii] Mass spectrometry method.

（式中、Ｒはアミノ置換基を意味する。）

（式中、Ｘはアミノ基反応性官能基を意味し、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味する。）

（式中、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味し、Ｒは前記と同じ意味を有する。）

［９］ 前記一般式［II］で表されるアミノ酸誘導体またはその塩と、一般式［IIIa］で表されるニトロベンゼン化合物の反応を、アルカリ性水溶液または水と有機溶媒の混合溶液であり、かつ前記溶液のｐＨが８〜１０でおこなうことを特徴とする前記［８］に記載のアミノ化合物またはその塩の質量分析方法。
［１０］ 前記［８］または［９］に記載のアミノ化合物またはその塩の質量分析方法であって、前記反応が試料中で行われることを特徴とするアミノ化合物またはその塩の質量分析方法。
［１１］ 前記［８］〜［１０］のいずれかに記載のアミノ化合物またはその塩の質量分析方法であって、前記アミノ酸誘導体またはその塩がバイオマーカーであることを特徴とするアミノ化合物の質量分析方法。
[8] In the formula amino acid derivative or a salt thereof [II], amino substituent is substitution amino acid residue or peptide residue,
Constituent amino acids of an amino acid or said peptide residue before Ki置 conversion amino acid residue is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine, valine, tryptophan or tyrosine, or substitutions thereof der is,
And, wherein the substituted amino acid residue is a residue of advanced glycation products (AGE), or a peptide of the peptide residues, two to 10 amino acids amino acid derivative Ru Oh a peptide which is a peptide bond or With its salt,
Reacting with a nitrobenzene compound represented by the general formula [IIIa] to obtain an amino compound represented by the general formula [Ia] or a salt thereof;
A method for mass spectrometry of an amino compound or a salt thereof, comprising a step of mass-analyzing the amino compound or a salt thereof obtained in the above step.

(In the formula, R means an amino substituent.)

(In the formula, X means an amino group-reactive functional group, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different, and each represents a hydrogen atom or a nitro group. (However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ represents a nitro group.)

(Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group, provided that R ₁ , R ₂ , R ₃ , at least one of R ₄ and R ₅ represents a nitro group, and R has the same meaning as described above.)

[ 9 ] The reaction of the amino acid derivative represented by the general formula [II] or a salt thereof and the nitrobenzene compound represented by the general formula [IIIa] is an alkaline aqueous solution or a mixed solution of water and an organic solvent, and The method for mass spectrometry of an amino compound or a salt thereof according to the above [ 8 ], wherein the pH of the solution is 8 to 10.
[ 10 ] A mass spectrometric method for the amino compound or salt thereof according to [ 8 ] or [ 9 ], wherein the reaction is performed in a sample.
[ 11 ] The mass spectrometric method of the amino compound or salt thereof according to any one of [ 8 ] to [ 10 ], wherein the amino acid derivative or salt thereof is a biomarker. Analysis method.

[ 12 ] Using the intensity data of the mass spectrum obtained by the mass spectrometry method of the amino compound or salt thereof according to any one of [1] to [ 11 ], the biomarker concentration in the biological sample is quantified. A biomarker assay method characterized by the above.

（式中、Ｒはアミノ置換基を意味し、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味する。）

［１４］ 前記［１３］に記載のアミノ化合物またはその塩であって、
前記最終糖化産物（ＡＧＥ）が、置換アルギニン誘導体または置換リジン誘導体であることを特徴とする質量分析用のアミノ化合物またはその塩。

［１５］ 前記［１３］に記載のアミノ化合物またはその塩であって、
前記置換アミノ酸残基のアミノ酸または前記ペプチド残基の構成アミノ酸が、アルギニン、リジン、バリン、チロシン、メチオニン、グリシン、ヒスチジン、グルタミン酸もしくはトリプトファンまたはそれらの置換体であることを特徴とする質量分析用のアミノ化合物またはその塩。

［１６］ 前記［１３］〜［１５］のいずれかに記載のアミノ化合物またはその塩であって、
前記アミノ化合物が、一般式［Ii］で表されることを特徴とする質量分析用のアミノ化合物またはその塩。

（式中、Ｒは前記と同じ意味を有する。）
[ 13 ] An amino compound represented by the general formula [Ia] or a salt thereof,
Amino substituent in formula [Ia] is a substitution amino acid residue or peptide residue,
Constituent amino acids of an amino acid or said peptide residue before Ki置 conversion amino acid residue is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine, valine, tryptophan or tyrosine, or substitutions thereof der is,
And, wherein the substituted amino acid residue is a residue of advanced glycation products (AGE), or a peptide of the peptide residues, for Oh Ru mass spectrometry peptide 2 to 10 amino acids are a peptide bond An amino compound or a salt thereof.

(Wherein R represents an amino substituent, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different from each other, and represent a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.)

[ 14 ] The amino compound or salt thereof according to [ 13 ] above,
An amino compound for mass spectrometry or a salt thereof, wherein the final glycation product (AGE) is a substituted arginine derivative or a substituted lysine derivative.

[ 15 ] The amino compound or salt thereof according to [ 13 ] above,
The amino acid of the substituted amino acid residue or the constituent amino acid of the peptide residue is arginine, lysine, valine, tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan or a substituted product thereof for mass spectrometry An amino compound or a salt thereof.

[ 16 ] The amino compound or salt thereof according to any one of [ 13 ] to [ 15 ],
An amino compound for mass spectrometry or a salt thereof, wherein the amino compound is represented by the general formula [Ii].

(In the formula, R has the same meaning as described above.)

（式中、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５はそれぞれ同じであってもまたは異なっていてもよく、水素原子またはニトロ基を意味する。ただし、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４およびＲ_５の少なくとも１個はニトロ基を意味する。）

［１８］ 前記［１７］に記載のアミノ化合物またはその塩であって、
前記アミノ化合物が、構造式［Iii］で表されるトリニトロフェニル化メチルグリオキサール由来ヒドロイミダゾロン誘導体または構造式［Iiii］で表されるトリニトロフェニル化アルグピリミジン誘導体であることを特徴とするアミノ化合物またはその塩。

[ 17 ] An amino compound or a salt thereof, which is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib] or a nitrophenylated argpyrimidine derivative represented by the general formula [Ic] .

(Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group, provided that R ₁ , R ₂ , R ₃ , at least one of R ₄ and R ₅ means a nitro group.)

[ 18 ] The amino compound or salt thereof according to [17 ] above,
The amino compound is a trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a trinitrophenylated argpyrimidine derivative represented by the structural formula [Iiii] Compound or salt thereof.

  By derivatizing the N-terminal amino group of an amino acid derivative according to the present invention, ionization efficiency in mass spectrometry is improved, and mass spectrometry is also performed for molecular biomarkers such as final glycation products and peptide hormones that exist only in a small amount in a sample. The method enables rapid, highly sensitive and accurate quantitative analysis. Accordingly, the present invention provides a novel amino compound that is a derivatization product of an amino acid derivative and a derivatizing agent such as a nitrobenzene compound, preferably a glycated protein degradation product that is a reaction intermediate product such as protein glycolysis is a nitrobenzene compound or the like. The present invention provides a novel glycated protein hydrolyzate derivative that is derivatized with a derivatizing agent useful for diagnosing abnormal glucose tolerance. The present invention also provides a mass spectrometric method capable of rapid, highly sensitive and highly accurate quantification of the amino compound, and a biomarker assay method using the same.

It is a mass spectrum (MS, MS / MS) of a methylglyoxal-derived hydroimidazolone derivative (MG-H1). It is a mass spectrum (MS, MS / MS) of a 2,4,6-trinitrophenyl derivative of MG-H1 (MG-H1-TNP). It is a mass spectrum (MS, MS / MS) of argypirimidine (AP). 2 is a mass spectrum (MS, MS / MS) of a 2,4,6-trinitrophenyl derivative of AP (AP-TNP). It is a figure which shows the fragmentation pattern of the 2,4,6-trinitrophenyl derivative (MG-H1-TNP) of MG-H1 estimated from a mass spectrum. It is a figure which shows the fragmentation pattern of the 2,4,6-trinitrophenyl derivative (AP-TNP) of AP estimated from a mass spectrum. (A) is a mass chromatogram showing the results of LC-MS analysis of MG-H1, and (B) is the 2,4,6-trinitrophenyl derivative of MG-H1 (MG-H1-TNP). (A) is a mass chromatogram showing the results of LC-MS analysis of AP, and (B) is the results of LC-MS analysis of 2,4,6-trinitrophenyl derivative of AP (AP-TNP). It is the calibration curve produced using the internal standard method about the density | concentration of MG-H1-TNP and the peak intensity of a mass spectrum. It is the calibration curve produced using the internal standard method about the density | concentration of AP-H1-TNP, and the peak intensity of a mass spectrum. It is a graph which shows the influence of pH which acts on 2,4,6- trinitrophenylation of MG-H1. It is a graph which shows the influence of pH which acts on 2,4,6- trinitrophenylation of AP. It is a chart which shows the protocol of pretreatment of rat plasma.

  Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments, and the following embodiments are exemplarily described in order to specifically describe the present invention. Therefore, it should be understood that forms conceived from the following embodiments are also included in the scope of the present invention.

  In the present specification, in order to simplify the explanation, a nitrobenzene compound will be described as an example of the derivatizing agent. However, in the present invention, the derivatizing agent is not limited to the nitrobenzene compound at all. It should be understood that other derivatizing agents are naturally included within the scope of the present invention. Therefore, in this specification, although the nitrophenyl group which is a residue of a nitrobenzene compound is explained also about the residue which derivatized the terminal amino group of the amino acid derivative with the derivatizing agent, this is also limited at all. Rather, it should be understood that residues of other derivatizing agents are included as well.

  The amino compound according to the present invention can be represented by the general formula [I].

  In the formula [I], R means an amino substituent and Z means a nitrophenyl group.

In the above general formula, the term “amino substituent” means an amino acid residue or a substituted amino acid residue or a peptide residue.
Here, the term “amino acid residue” means a residue obtained by removing an amino group from an unsubstituted amino acid. For example, in the case of alanine, since it is also called 2-aminopropanoic acid, the amino acid residue excluding the amino group from 2-aminopropanoic acid is a carboxyethyl group. Similarly, the amino acid residue in the case of lysine is a 1-carboxyl-5-aminopentyl group.

  In the above general formula, the term “substituted amino acid residue” refers to an amino acid residue obtained by removing an amino group from the amino acid, and a residue other than a carboxyl group constituting the amino acid, that is, an atomic group is a substituent. It means an atomic group formed by substitution with an amino acid residue having a substitution moiety (moiety). Examples of the substituted amino acid residue include a 1-carboxyl-5- (carboxylmethyl) aminopentyl group in which a 5-methyl group of the lysine residue is substituted with a carboxymethyl group. Another example includes a substituted amino acid residue obtained by converting an amino-iminomethyl group of an arginine residue into a 2-pyrimidinyl group.

  Furthermore, in the above general formula, the term “peptide residue” means a residue in which a terminal amino group is excluded from a terminal amino acid of a peptide formed by peptide bonding of the amino acid and / or a substituted amino acid. . For example, in the case of the peptide Val-Tyr, the peptide residue means a residue in which the amino group is excluded from the terminal amino acid valine.

  In the present invention, the term “amino acid derivative” means an unsubstituted amino acid derivative, a substituted amino acid derivative in which an amino acid moiety other than the N-terminal amino group is modified with a substituent or the like, unless otherwise specified. doing.

  Therefore, unless otherwise specified, the term “amino compound” used in the present invention is a nitrophenylation in which the N-terminal amino group of the amino acid derivative [II] or a salt thereof is nitrophenylated by the nitrobenzene compound [III]. An amino compound is meant.

  In the present invention, amino acids of amino acid residues or substituted amino acid residues, or amino acids of peptide constituent amino acids of peptide residues are not particularly limited as long as they are compounds having both amino group and carboxy functional groups. Although not limited, for example, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine, valine, Examples include amino acids that are constituents of proteins such as tryptophan and tyrosine, preferably arginine and lysine, and substituted amino acid residues having a substitution moiety formed by substituting residues other than the carboxyl group constituting them with a substituent. .

  Preferred amino acids include, for example, preferably arginine, lysine, valine, tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan.

  The term “substituted amino acid residue” used in the present invention is a residue of a substituted amino acid derivative having another atomic group formed by bonding an arbitrary substituent to a residue other than the terminal amino group of the amino acid. Means. Such amino acid derivatives are not particularly limited as long as they do not depart from the object of the present invention, and include, for example, degradation products, intermediate products, or metabolites of biological components such as amino acids. Glycation end products (AGEs) produced by the saccharification reaction, ornithine, the degradation product of arginine, creatinine, a metabolite of creatinine phosphate, which is an energy source for muscles, and biotechnology that shows glutathione deficiency in the liver In addition to α-aminobutyric acid (AABA), an intermediate of biosynthesis of ophthalmic acid as a marker, γ-aminobutyric acid (GABA), which mainly acts as a neurotransmitter, and other molecules used for diagnosis and prognosis prediction of various diseases Examples include biomarkers and hippuric acid. Of these amino acids, the above AGEs and the like are preferable. More preferably, for example, it is produced by a reaction between a guanosine group on the side chain of arginine and methylglyoxal, or an amino group of lysine and a reaction intermediate product such as lipid peroxidation such as glyoxal or methylglyoxal, Maillard reaction, glycolysis. AGEs and the like. Of these AGEs, preferred are substituted arginine derivatives and substituted lysine derivatives.

  Examples of substituted arginine derivatives include alkyl-substituted arginine derivatives in which the guanidino group of arginine is modified with a substituent such as carboxylmethyl group and carboxylethyl group, guanidino groups such as imidazolones, pyrimidines, and imidazopyridines in a cyclic structure. And cyclic arginine derivatives.

  More specifically, examples of the alkyl-substituted arginine derivative include carboxymethylarginine (CMA). Examples of imidazolones of cyclic arginine derivatives include imidazolone, glyoxal hydroimidazolone (GH), methylglyoxal hydroimidazolone (MG-H), Nδ- [5- (2,3,4-trihydroxy). Butyl) -5-hydro-4-imidazolone-2-yl] -ornithine (3-DG-H) and the like. Examples of pyrimidines include argpyrimidine and tetrahydropyrimidine. Examples of imidazopyridines include pentosidine.

  Among these substituted arginine derivatives, for example, a methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [IIa] (MG-H1: Nδ- (5-hydro-5-methyl-4-imidazolone 2) is particularly preferable. -Yl) -ornithine), or argpyrimidine (AP) represented by the structural formula [IIb].

  More specifically, the amino acid derivative has a three-dimensional structure and can be represented by the structural formula [IIc] or the structural formula [IId].

  Among the above AGEs, examples of substituted lysine derivatives include alkyl-substituted lysine derivatives in which the amino group of lysine is modified with a substituent such as carboxylmethyl group or carboxylethyl group, and the amino group of lysine has a cyclic structure. And cyclic lysine derivatives.

  Examples of the alkyl-substituted lysine derivative include carboxymethyl lysine (CML) and carboxyethyl lysine (CML). Examples of the cyclic lysine derivative include viralin, pyrrole derivatives such as FTP (Formyl Therosyl Pyrrol), GOLD (glyoxal-derived lysine dimer), MOLD (methylglyoxal-derived lysine dimer), DOLD (3-deoxyglucose). Imidazole derivatives such as lysine-derived lysine dimer), pyridine derivatives such as GLAP (Glyceraldehyde-derived pyridinium) and GA-pyridine (glycolaldehyde-pyridine), and imidazopyridine derivatives such as pentosidine.

  Further, in the general formula [I], the peptide residue peptide represented by the substituent R is not particularly limited in the number of the amino acids constituting the peptide, but preferably 2 to 10 amino acids, preferably May be a peptide composed of 2 to 6, more preferably 2 to 4, peptide bonds. Examples of such peptides include dipeptides such as valyltyrosine, lysyltyrosine, methionyltyrosine, glycyltyrosine, histidyltyrosine, glutamyltyrosine, tryptophanyltyrosine, histidinyltryptophan, carnosine and anserine, and tripeptides such as glutathione. Etc. In addition, enkephalin, caxitocin, vasopressin and the like can be mentioned. Of these peptides, dipeptides such as valyltyrosine, lysyltyrosine, methionyltyrosine, glycyltyrosine, histidyltyrosine, glutamyltyrosine, tryptophanyltyrosine and histidinyltryptophan are preferred. The peptide may be any of a digested protein, a metabolite such as a final glycation product, and a physiologically active peptide such as a peptide hormone, and is a molecular biomarker used for diagnosis and prognosis prediction of various diseases. Also good.

  On the other hand, in the above general formula [I], the substituent Z is a residue of a nitrobenzene compound obtained by nitrophenylating the terminal amino group of the amino acid derivative [II], and can be represented by the general formula [IIIb].

In the formula [IIIb], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group. However, at least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ means a nitro group.

  Here, examples of the nitrophenyl group include a mono-, di- or tri-nitrophenyl group, preferably a tri-nitrophenyl group, particularly preferably a 2,4,6-nitrophenyl group. The position of the nitro substituent is not particularly limited, but in the case of one, the 2-position, in the case of 2, the 2-position and the 4-position, in the case of 3, the 2-position, the 4-position and It should be in the 6th position.

  Therefore, the amino compound according to the present invention is generally represented by the general formula [I], and more specifically can be represented by the general formula [Ia].

In the formula [Ia], R, Z, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  In the present invention, preferred examples of the amino compound represented by the structural formula [Ia] include, for example, a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Ib] or the structural formula [Ic]. And nitrophenylated argpyrimidine derivatives represented.

In the formulas [Ib] and [Ic], R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  A more preferred amino compound can be represented, for example, by the general formula [Ii].

  In the formula [Ii], R has the same meaning as described above.

  Therefore, as the amino compound, for example, a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a nitrophenylated argpyrimidine derivative represented by the general formula [Iiii] is more preferable.

  The three-dimensional structural formulas ([Iiv] and [Iv]) of the above-mentioned trinitrophenylated methylglyoxal-derived hydroimidazolone derivative and trinitrophenylated argpyrimidine derivative can be represented as follows.

  The amino compound [I] according to the present invention can be obtained by derivatizing an amino acid derivative represented by the general formula [II] or a salt thereof with a nitrobenzene compound represented by the general formula [III].

  In the formula [II], R has the same meaning as described above.

  In the formula [III], X means an amino group-reactive functional group, and Z has the same meaning as described above.

  More specifically, the amino compound [Ia] of the present invention can be obtained by a derivatization reaction between an amino acid derivative [II] or a salt thereof and a nitrobenzene compound represented by the general formula [IIIa].

In the formula [IIIa], X, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.

  More specifically, the amino compound [Ii] of the present invention can be obtained by a derivatization reaction between an amino acid derivative [II] or a salt thereof and a trinitrobenzene compound represented by the general formula [IIIc].

  In the formula [IIIc], X has the same meaning as described above.

  Here, the amino group-reactive functional group represented by X is not particularly limited as long as it is a reactive functional group that derivatizes an amino acid or a substituted amino acid ([II]) with a nitrophenyl. Aromatic nucleophilicity of nitrobenzene derivatives such as atoms, halogen atoms selected from bromine atoms or iodine atoms, or sulfonic acids or sulfonates, alkyl sulfonate groups, aryl sulfonate groups, perfluoroalkyl sulfonate groups, etc. Any functional group that can be a leaving group in the substitution reaction is exemplified.

  Accordingly, preferred nitrobenzene compounds include, for example, 2,4,6-trinitrofluorobenzene, 2,4,6-trinitrochlorobenzene, 2,4,6-trinitrobromobenzene, 2,4,6-trinitro. Examples thereof include iodobenzene and 2,4,6-trinitrobenzenesulfonic acid or a salt thereof. Among these, 2,4,6-trinitrobenzenesulfonic acid or a salt thereof is particularly preferable. Examples of the salt include alkali metal salts such as sodium salt, alkaline earth metal salts such as potassium salt, and the like.

  In the present invention, in order to derivatize the N-terminal amino group of the amino acid derivative with a nitrobenzene compound, the amino acid derivative and the nitrobenzene compound are tens of several hours at room temperature in an alkaline aqueous solution or a mixed solution of water and an organic solvent. This can be done by mixing for a minute. Preferably it is carried out in an alkaline aqueous solution. Moreover, when using the mixed solution of water and an organic solvent, the organic solvent with high affinity with water, such as ethanol, is preferably used as an organic solvent. The pH of these solutions is preferably 8 to 10, more preferably 8.5 to 9.5. If the pH of the solution is too high, the possibility of decomposition of the amino acid derivative to be analyzed or this compound increases, and if it is too low, the nitrophenylation reaction does not proceed rapidly. Here, the pH is a value directly measured by a pH meter. The room temperature is a temperature of about 15 ° C. to 40 ° C., and the reaction is preferably performed at around 30 ° C. The mixing may be carried out by any method, and mixing is performed for 1 minute or more and 1 hour or less, preferably 5 minutes to 40 minutes. Usually, mixing for 30 minutes is sufficient.

  One of the features of the present invention is that the nitrophenylation reaction can be performed under such mild conditions. A substance to be analyzed in a sample may be isolated and subjected to nitrophenylation, or may be subjected to nitrophenylation as it is in a sample containing impurities. Since the present invention can directly perform nitrophenylation in such a sample, loss due to isolation can be reduced, and even in the case where only a small amount of a substance to be analyzed is contained. It is an excellent method that can be measured. Further, it is not necessary to perform a complicated operation, and measurement can be performed easily. In particular, when the target sample is a biological sample or the like, it is effective because there are few substances to be analyzed and there are many impurities.

In addition, for example, in measuring an amino acid derivative in a sample, it is possible to derivatize an amino acid derivative as a biomarker in a sample with a nitrobenzene compound by adding a nitrobenzene compound to the biological sample, Naturally, such derivatized amino acid derivatives can be measured as biomarkers.
What is used as a sample in the present invention is not limited as long as it contains a final glycation product to be measured and the like that become an amino compound obtained by derivatization of an amino acid derivative and a nitrobenzene compound according to the present invention. A sample derived from a living body is called a biological sample.

  Furthermore, as a salt of an amino acid derivative, for example, an alkali metal salt such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a lithium salt, an ammonium salt, or the like can be used.

  In the present invention, as a derivatizing agent, in addition to the above nitrobenzene compound, other derivatizing agents that derivatize amino groups can naturally be used. Examples of such derivatizing agents include nitrobenzene compounds such as nitrobenzene sulfonic acid compounds and fluoro-nitrobenzene, sulfonyl compounds such as 1-pyrenesulfonyl chloride (PSC) and dansyl chloride (DNS-Cl), 9-fluorene, and the like. Nylmethyl chloroformate (FMOC), 4-dimethylaminosulfonyl-7-fluorobenzoxadiazole (DBD-F), 4-fluoro-7-nitrobenzofurazan (NBD-F), 2,3-naphthalene Carbamate compounds such as aldehyde (NDA), 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2-quinoxalinone (DMEQ-COCl), 3 -Nitrophenylisothiosi , 3-pyridyl isothiocyanate, 4- (dimethylamino) phenyl isothiocyanate, isothiocyanate compounds such as fluorescein-5-isothiocyanate (FITC), malonate compounds such as diethyl ethoxymethylene malonate, (5-succinimidoxy-5- Examples thereof include phosphonium compounds such as oxopentyl) triphenylphosphonium bromide (SPTPP), and pyridinium compounds such as 2-chloro-1-methylpyridinium salt. These derivatizing agents can also be used in substantially the same manner as the above nitrobenzene compound.

  That is, in the present invention, when the derivatizing agent is used, the amino compound according to the present invention is represented by the general formula [I ′].

  In the formula [I ′], R ′ means an amino acid residue or substituted amino acid residue or peptide residue having the same meaning as R above, and Z ′ means a derivatized residue.

Here, the derivatized residue means a residue obtained by derivatizing the amino group of the substituted amino acid derivative (R′-NH ₂ ) with the derivatizing agent. For example, when dansyl chloride is used as a derivatizing agent, the amino group of the substituted amino acid derivative (R′-NH ₂ ) is dansylated, that is, dimethylaminonaphthalenesulfonylated, and this dansyl group is called a derivatized residue. be able to.

  Therefore, also in this case, in this specification, the description about the case where nitrobenzenesulfonic acid is used as a derivatizing agent can be applied substantially in the same manner.

In the present invention, the ionization method of the sample used for mass spectrometry is not particularly limited, but it is preferable to use MALDI method, ESI method, APCI method and the like widely used for detection and quantification of biomolecules. In LC / MS, GC / MS, etc. combined with a separation technique, it is preferable to use an ESI method or an APCI method as a sample ionization method. For the analysis, a tandem mass spectrometry method such as MS / MS or MS ³ can be used. In this case, for the detection of product ions, for example, it is preferable to use multiple reaction monitoring (MRM) which can eliminate the influence of impurities and enable highly sensitive detection.

Next, a biomarker assay method using the mass spectrometry method according to the present invention will be described.
The present invention can provide an assay method capable of detecting and quantifying a biomarker rapidly, with high sensitivity and high accuracy by selecting the biomarker as an analysis target. That is, the biomarker assay method of the present invention comprises reacting an N-terminal amino group of a biomarker that is an amino acid derivative with a derivatizing agent such as a nitrobenzene compound to derivatize the N-terminal amino group such as nitrophenylation. And mass spectrometry of the amino acid derivative or its salt in which the N-terminal amino group is derivatized such as nitrophenylation, and the intensity data of the mass spectrum obtained by using the mass spectrometry method, A step of quantifying the biomarker concentration.

  In the present invention, the biomarker is not particularly limited as long as it is an amine compound having an amino group. Examples of the method to which the assay method of the present invention can be particularly preferably applied include amino acid derivatives and sugar metabolites. Final glycation products (AGEs) produced by the reaction, biogenic amines catecholamine (dopamine, noradrenaline, adrenaline), indoleamine (serotonin, melatonin), imidazoleamine (histamine), acetylcholine, polyamine (putrescine, spermidine, Spermine) and the like.

  Examples of AGEs that are particularly preferable as biomarkers to be analyzed by the assay method of the present invention include, for example, trinitrophenylated methylglyoxal-derived hydroimidazolone derivatives (MG-H1) represented by the structural formula [Iii], and structural formulas And trinitrophenylated argpyrimidine derivative (AP) represented by [Iiii].

  These are MG-H1 and N-2,4,6-trinitrophenyl derivatives of AP, which are AGEs that can be detected and quantified with high sensitivity by mass spectrometry, and are useful as diagnostic indicators of impaired glucose tolerance It is.

  In the present invention, for example, a standard curve method or an isotope dilution method is used to quantify the concentration of the amino acid derivative or peptide to be assayed from the area intensity of the mass spectrum. In mass spectrometry, one of the techniques for quantitative analysis of a substance to be analyzed is a calibration curve method. The calibration curve method shows that the relationship (calibration curve) between several standard samples containing known amounts of standard substances and different concentrations and the signal intensities obtained from these standard samples is theoretically a linear relationship. This is a method of using and quantifying the concentration of the sample from the signal intensity obtained from the unknown sample. The calibration curve method is roughly classified into an external standard method, an internal standard method, and a standard addition method depending on the preparation method of the standard sample. Any method may be used in the present assay method. The isotope dilution method focuses on the isotope ratio of a specific element that constitutes the measurement target compound, and after adding a certain amount of spikes with a known isotope ratio to reach the equilibrium of the isotope composition of the measurement target compound In this method, the isotope ratio of the sample before and after spike addition is measured, and the element concentration of the sample is obtained from the change in the isotope composition. The advantage is that the detection limit is as low as about 1 pg.

  The quantitative data of the biomarker obtained as described above can be used for prediction and diagnosis of disease onset, progression, prognosis and the like. That is, as one embodiment of the present invention, an N-terminal amino group of a biomarker that is an amino acid derivative or peptide is reacted with a derivatizing agent such as a trinitrophenylating agent, and the N-terminal amino group is trinitrophenylated or the like. Using the mass spectrum intensity data obtained by using the mass spectrometric method, mass spectrometric analysis of the amino acid derivative or peptide in which the N-terminal amino group is derivatized such as trinitrophenylation, etc. A method for diagnosing a disease comprising a step of quantifying a biomarker concentration in a biological sample and a step of diagnosing the onset, progression, prognosis, etc. of the disease based on the biomarker concentration data thus obtained It is about.

  The diagnostic method of the present invention is useful because it can simultaneously measure and quantify a plurality of types of AGEs. That is, the present invention is a method capable of performing nitrophenylation in a sample, and is analyzed by mass spectrometry. Therefore, when measuring target substances having different mass numbers, it can be combined with mass spectrometry. The present invention provides an excellent method capable of simultaneously measuring (quantifying). According to the present invention, costs and labor can be greatly reduced as compared with the conventional early diagnosis method.

  Hereinafter, based on the Example of this invention, the characteristic and effect of this invention are demonstrated more concretely. These examples are illustrative only and are not intended to limit the scope of the present invention.

Trinitrophenylated methylglyoxal-derived hydroimidazolone derivative (MG-H1-TNP) [Iii] was prepared as follows.
Methylglyoxal-derived hydroimidazolone derivative (MG-H1) [IIa] was dissolved in 0.1 M boric acid solution (pH 9.3), and 30 mM 2,4,6-trinitrobenzenesulfonic acid sodium salt (TNBS) -0. After adding 10 μL of 1M boric acid solution (pH 9.3), the mixture was thoroughly mixed. The resulting mixture was incubated at 30 ° C. for 20 minutes for derivatization. Sep-Pak Plus C ₁₈ derivatized MG-H1 (MG-H1-TNP) with 100% CH ₃ CN / 0.1% formic acid (3 mL) and 0.1% formic acid (3 mL), respectively. After loading on a cartridge (Waters) and washing with 0.1% FA (3 mL), the fraction eluted with 100% CH ₃ CN / 0.1% formic acid (3 mL) was dried to dryness with a centrifugal evaporator. The fraction dried product was redissolved in 1.5 mL of deionized water, freeze-dried, and stored at -40 ° C.

The trinitrophenylated argpyrimidine derivative (AP-TNP) [Iiii] was prepared as follows.
Argpyrimidine [IIb] was treated in substantially the same manner as in Example 1 to obtain a trinitrophenylated argpyrimidine derivative (AP-TNP) [Iiii].

Measurement of Mass Spectra MG-H1 and AP, and N-2,4,6-trinitrophenyl derivatives of MG-H1 and AP prepared as described above (“MG-H1-TNP” and “AP-TNP respectively”) )) Was dissolved in 0.1% formic acid, and the mass spectrum was measured. The measurement conditions are as follows.
・ Ionization mode ESI-positive
・ Scanning range 100-500m / z
-Dry gas 330 ° C, 8.0 L / min-Nebulizer gas 276 kPa
・ Capillary voltage -2750V
-Skimmer voltage 40V
・ Capillary outlet voltage 124V

The mass spectra (MS and MS / MS) of MG-H1, AP, MG-H1-TNP and AP-TNP are shown in FIGS. 2 and 4, MG-H1 and N-2,4,6-trinitrophenyl derivatives of AP are denoted as “MG-H1-TNP” and “AP-TNP”, respectively. Precursor ions used for MS / MS measurement are [M + H] ⁺ ions for each compound. Among the obtained mass spectra, the one having the highest peak intensity was selected as an ion peak to be monitored in the LC-MRM-MS / MS analysis described later.

  Moreover, the fragmentation patterns of MG-H1-TNP and AP-TNP predicted from the MS spectrum and the MS / MS spectrum shown in FIGS. 3 and 4 are shown in FIGS. 5 and 6, respectively.

LC-MRM-MS / MS analysis LC-MRM-MS / MS analysis of MG-H1, AP, MG-H1-TNP and AP-TNP was performed under the following conditions. Each sample concentration was 10 nmol / L, and all were dissolved in 0.1% formic acid aqueous solution.
<HPLC conditions>
-Mobile phase MeOH-0.1% formic acid aqueous solution-Flow rate 0.20 mL / min-Gradient 0-100% (25 min)
・ Stationary phase Waters Atlantis T3 (φ2.1 × 100mm)
・ Column oven temperature 40 ℃

<MS conditions>
・ Ionization mode ESI-positive
・ Scanning range 100-500m / z
-Dry gas 330 ° C, 8.0 L / min-Nebulizer gas 276 kPa
・ Capillary voltage -2750V
-Skimmer voltage 40V
・ Capillary outlet voltage 124V
・ Injection volume 25μL

  The mass chromatograms of MG-H1, AP, MG-H1-TNP, and AP-TNP are shown in FIGS. 7 and 8, respectively. From the results shown in FIG. 7, it was confirmed that the peak intensity increased to about 1500 times when the N-terminal amino group of MG-H1 was 2,4,6-trinitrophenylated.

Preparation of calibration curve and determination of detection limit and quantification limit MG-H1-TNP and AP-TNP calibration curves using deuterated form of MG-H1-TNP (MG-H1-d3-TNP) as an internal standard Was made. In order to avoid the influence of decomposition by acid, dilution was performed by a procedure (see Table 1) in which a 0.1% formic acid aqueous solution was added immediately before injection, and measurement was performed from the high concentration side. First, a 1 μmol / mL aqueous solution of MG-H1-TNP and AP-TNP was prepared as a stock solution, and diluted with water so that the final concentrations were 400, 200, 40, 20, 4, 2 pmol / mL. For each concentration, add 25 μL of a 400 pmol / mL aqueous solution of MG-H1-d3-TNP to 25 μL of these equimolar mixtures, add 50 μL of a 0.2% aqueous formic acid solution, and mix well, 25 μL of which for LC-MS analysis. Using.

  By plotting the peak intensity ratio of MG-H1-TNP or AP-TNP and MG-H1-d3-TNP in the obtained mass spectrum against the concentration of MG-H1-TNP or AP-TNP, FIG. A calibration curve shown in FIGS. From these, for MG-H1-TNP, the detection limit was 2.05 pmol / mL, the limit of quantification was 6.20 pmol / mL, and for AP-TNP, the detection limit was 0.11 pmol / mL and the limit of quantification was 0.33 pmol / mL. It was. Similarly, when a calibration curve was prepared for CML-TNP and the concentration was converted, the detection limit was 5.3 nmol / L and the quantification limit was 16 nmol / L. For CEL-TNP, the detection limit was 2.7 nmol / L and the quantification limit was 8.0 nmol / L. Regarding the calibration curve, it was confirmed that good linearity with a correlation coefficient of 0.996 or more was obtained, and that quantitative analysis at the pmol level (concentration nM / L level) was possible for each. These numerical values are 1/100 to 1/200 of the detection limit in dansyl chloride and NDA (2,3-naphthalenedialdehyde) which are conventionally used derivatizing agents, and 2,4,6-tri It shows that nitrophenylation can greatly improve sensitivity.

Effect of pH on 2,4,6-trinitrophenylation In the preparation of MG-H1-TNP and AP-TNP in Examples 1 and 2, the pH of the reaction solution was adjusted to 7.5, 8.0, 8. The derivatization rate in the case of 5 was calculated using LC-MS analysis. Dissolve the dried product obtained by the centrifugal evaporator treatment in 100 μL of 0.2% formic acid aqueous solution, add 50 μL of 200 pmol / mL aqueous solution of MG-H1-d3-TNP as an internal standard, mix thoroughly, and then add 25 μL to LC. The sample was subjected to -MS analysis, and the concentration of 2,4,6-trinitrophenyl compound was determined by a calibration curve method. The results are as shown in FIGS. 11 and 12, and it is confirmed that derivatization can be quantitatively performed at pH 8.5 to 9.3 in either case of MG-H1-TNP or AP-TNP. It was done.

Rat Plasma Spike Test MG-H1, AP and MG-H1-d3, which is an internal standard, were spiked into control plasma of SD rats, and after recovery, 2,4,6-trinitrophenylation was performed. Plasma pretreatment was performed according to the protocol shown in FIG. 13 (see EMN Nakashima et al., Anal. Biochem., 414, 109-116 (2011)), and spiked MG-H1, AP and MG-H1. Attempted simultaneous detection of -d3. LC-MS analysis using a calibration curve prepared by the standard addition method confirmed that the plasma concentration of MG-H1 and AP could be quantified.

Examination using Val-Tyr Using the dipeptide Val-Tyr instead of AGEs MG-H1 and AP, the change in detection sensitivity due to 2,4,6-trinitrophenylation was performed in the same procedure as above. As a result of the examination, it was confirmed that the peak intensity increased about 50 times by 2,4,6-trinitrophenylation as in the case of MG-H1 (Table 2).

Examination using dipeptide The following dipeptide was prepared in substantially the same manner as in Example 8, and the change in detection sensitivity was examined. The results are shown in Table 2.

  The present invention is, for example, an amino compound obtained by derivatizing an amino acid derivative including a metabolite such as a final glycation product or an intermediate product obtained by saccharifying a protein that is an in vivo component in a metabolic process with a derivatizing agent such as a nitrobenzene compound. Since the compound is particularly useful for diagnosis of impaired glucose tolerance, it is a mass spectrometric method capable of quantifying these compounds quickly, with high sensitivity and high accuracy. Therefore, it can be expected that assaying these amino compounds as biomarkers will be useful for the prevention and diagnosis of various diseases, and thereby useful for the development of preventive and therapeutic agents for the diseases.

Claims (25)

An amino compound represented by the general formula [I] or a salt thereof.

(In the formula, R means an amino substituent, and Z means a nitrophenyl group.)   2. The amino compound or salt thereof according to claim 1, wherein the amino substituent is an amino acid residue, a substituted amino acid residue, or a plurality of the amino acid residues and / or a peptide residue in which the substituted amino acid residues are peptide-bonded. An amino compound or a salt thereof, wherein An amino compound or a salt thereof according to claim 2,
The amino acid residue or the amino acid of the substituted amino acid residue or the constituent amino acid of the peptide residue is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, An amino compound or a salt thereof which is arginine, serine, threonine, valine, tryptophan or tyrosine, or a substituted product thereof. The amino compound or a salt thereof according to claim 2 or 3,
The amino acid of the amino acid residue or substituted amino acid residue or the constituent amino acid of the peptide residue is arginine, lysine, valine, tyrosine, methionine, glycine, histidine, glutamic acid or tryptophan, or a substituted product thereof An amino compound or a salt thereof. The amino compound or a salt thereof according to any one of claims 2 to 4,
The amino compound or a salt thereof, wherein the substituted amino acid residue is a residue of a final glycation product (AGE).   6. The amino compound or salt thereof according to claim 5, wherein the final glycation product (AGE) is an arginine derivative or a lysine derivative. An amino compound or a salt thereof according to claim 2,
2. An amino compound or a salt thereof, wherein the peptide of the peptide residue is a peptide bond of 2 to 10 amino acids.
The amino compound or a salt thereof according to any one of claims 1 to 7, wherein the nitrophenyl group is represented by the general formula [IIIb].

(Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group, provided that R ₁ , R ₂ , R ₃ , at least one of R ₄ and R ₅ means a nitro group.)
The amino compound or salt thereof according to any one of claims 1 to 8, wherein the amino compound is represented by the general formula [Ia].

(In the formula, R, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.)
The amino compound or a salt thereof according to claim 9, wherein the amino compound is represented by the general formula [Ii].

(In the formula, R has the same meaning as described above.)
The amino compound or a salt thereof according to any one of claims 1 to 10, wherein the amino compound is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib] or a general formula [ An amino compound or a salt thereof, which is a nitrophenylated argypyrimidine derivative represented by Ic].

(In the formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.)
The amino compound or a salt thereof according to claim 11, wherein the amino compound is represented by a trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a structural formula [Iiii]. An amino compound or a salt thereof, which is a trinitrophenylated argpyrimidine derivative.

A method for mass spectrometry of an amino compound or a salt thereof, comprising measuring the amino compound represented by the general formula [I] or a salt thereof by mass spectrometry.

(In the formula, R means an amino substituent, and Z means a nitrophenyl group.)
A method for mass spectrometry of an amino compound or a salt thereof according to claim 13,
A method for mass spectrometry of an amino compound or a salt thereof, wherein the amino substituent is an amino acid residue, a substituted amino acid residue or a peptide residue. A method for mass spectrometry of an amino compound or a salt thereof according to claim 13 or 14,
The amino compound is represented by the general formula [Ia], A method for mass spectrometry of an amino compound or a salt thereof.

(Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different and each represents a hydrogen atom or a nitro group, provided that R ₁ , R ₂ , R ₃ , at least one of R ₄ and R ₅ represents a nitro group, and R has the same meaning as described above.)
16. The method for mass spectrometry of an amino compound or a salt thereof according to any one of claims 13 to 15, wherein the amino compound is a nitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the general formula [Ib]. Alternatively, a mass spectrometric method for an amino compound or a salt thereof, which is a nitrophenylated argypyrimidine derivative represented by the general formula [Ic].

(In the formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ all have the same meaning as described above.)
The method for mass spectrometry of an amino compound or a salt thereof according to any one of claims 13 to 16, wherein the amino compound is
A trinitrophenylated methylglyoxal-derived hydroimidazolone derivative represented by the structural formula [Iii] or a trinitrophenylated argpyrimidine derivative represented by the structural formula [Iiii] Mass spectrometry method.

A step of reacting an amino acid derivative represented by the general formula [II] or a salt thereof with a nitrobenzene compound represented by the general formula [III] to obtain an amino compound represented by the general formula [I] or a salt thereof;
A method for mass spectrometry of an amino compound or a salt thereof, comprising a step of mass-analyzing the amino compound or a salt thereof obtained in the above step.

(In the formula, R means an amino substituent.)

(In the formula, X means an amino group-reactive functional group, and Z means a nitrophenyl group.)

(In the formula, R and Z have the same meaning as described above.)
  The reaction of the amino acid derivative represented by the general formula [II] or a salt thereof and the nitrobenzene compound represented by the general formula [III] is an alkaline aqueous solution or a mixed solution of water and an organic solvent, and the pH of the solution The method for mass spectrometry of an amino compound or a salt thereof according to claim 18, wherein is carried out at 8 to 10. A method for mass spectrometry of an amino compound or a salt thereof according to claim 18 or 19,
A method for mass spectrometry of an amino compound or a salt thereof, wherein the amino substituent is an amino acid residue, a substituted amino acid residue or a peptide residue.   21. A mass spectrometry method for an amino compound or a salt thereof according to any one of claims 18 to 20, wherein the reaction is carried out in a sample. The mass spectrometry method according to any one of claims 18 to 21, comprising:
A method for mass spectrometry of an amino compound, wherein the amino acid derivative or a salt thereof is a biomarker. A method for mass spectrometry of an amino compound or a salt thereof, comprising mass-analyzing an amino compound represented by the general formula [I ′] or a salt thereof.

(In the formula, R ′ means a substituted amino acid residue or peptide residue, and Z ′ means a derivatized residue.)
A method for mass spectrometry of an amino compound or a salt thereof according to claim 23,
A derivatizing agent for the derivatized residue,
Sulfonyl compounds such as 1-pyrenesulfonyl chloride (PSC) and dansyl chloride (DNS-Cl);
9-fluorenylmethyl chloroformate (FMOC), 4-dimethylaminosulfonyl-7-fluorobenzoxadiazole (DBD-F), 4-fluoro-7-nitrobenzofurazan (NBD-F), 2, 3-naphthalenedialdehyde (NDA);
Carbamate compounds such as 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS);
3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2-quinoxalinone (DMEQ-COCl);
Isothiocyanate compounds such as 3-nitrophenyl isothiocyanate, 3-pyridyl isothiocyanate, 4- (dimethylamino) phenyl isothiocyanate, fluorescein-5-isothiocyanate (FITC);
Malonate compounds such as diethyl ethoxymethylene malonate;
Phosphonium compounds such as (5-succinimidoxy-5-oxopentyl) triphenylphosphonium bromide (SPTPP);
Or a mass spectrometric method for an amino compound or a salt thereof, which is at least one derivatizing agent selected from pyridinium compounds such as 2-chloro-1-methylpyridinium salt.
  25. A biomarker concentration characterized in that the biomarker concentration in a biological sample is quantified using the intensity data of the mass spectrum obtained by the mass spectrometry method of the amino compound according to any one of claims 13 to 24. Assay method.

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