WO2013147096A1 - 同位体標識ピリリウム化合物 - Google Patents
同位体標識ピリリウム化合物 Download PDFInfo
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- WO2013147096A1 WO2013147096A1 PCT/JP2013/059405 JP2013059405W WO2013147096A1 WO 2013147096 A1 WO2013147096 A1 WO 2013147096A1 JP 2013059405 W JP2013059405 W JP 2013059405W WO 2013147096 A1 WO2013147096 A1 WO 2013147096A1
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- 0 O*(C(F)(F)F)=O Chemical compound O*(C(F)(F)F)=O 0.000 description 2
- ITMCEJHCFYSIIV-UHFFFAOYSA-N OS(C(F)(F)F)(=O)=O Chemical compound OS(C(F)(F)F)(=O)=O ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 2
- SZDZOWCXDSNCPC-UHFFFAOYSA-M CCc(c(C)c1CC)c(C)c(CC)[oH+]1[O]=S(C(F)(F)F)([O-])=O Chemical compound CCc(c(C)c1CC)c(C)c(CC)[oH+]1[O]=S(C(F)(F)F)([O-])=O SZDZOWCXDSNCPC-UHFFFAOYSA-M 0.000 description 1
- KATOGPYEINSODE-UHFFFAOYSA-N CCc(c(C)c1CC)c(C)c(CC)[oH+]1[O]=S(C)(C(F)(F)F)=O Chemical compound CCc(c(C)c1CC)c(C)c(CC)[oH+]1[O]=S(C)(C(F)(F)F)=O KATOGPYEINSODE-UHFFFAOYSA-N 0.000 description 1
- ARUIMKUOHIINGI-UHFFFAOYSA-N CS(C(F)(F)F)(=O)=O Chemical compound CS(C(F)(F)F)(=O)=O ARUIMKUOHIINGI-UHFFFAOYSA-N 0.000 description 1
- SFEBPWPPVGRFOA-UHFFFAOYSA-N OS(C(F)(F)F)=O Chemical compound OS(C(F)(F)F)=O SFEBPWPPVGRFOA-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
- C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
- G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
Definitions
- the present invention generally relates to biological sample analysis. More specifically, the present invention relates to a novel isotope-labeled pyrylium compound, a method for quantitatively analyzing amino group-containing compounds in two or more samples using the compound, and the like.
- R 1 , R 2 and R 3 are the same or different and each represents hydrogen, halogen or alkyl
- Patent Document 1 a method using a salt thereof as a labeling compound has been reported.
- the three compounds having a mass difference from each other by isotope labeling (these are collectively referred to as Py compounds) have been synthesized.
- a protein in two or more analytical samples is labeled with one of Py compounds to give a mass difference to the same protein between the samples, and then the sample is mixed to hydrolyze the protein .
- the obtained labeled peptide is subjected to mass spectrometry to identify a protein derived from the structural analysis of the peptide, and the abundance ratio of the identified protein between samples is calculated based on the mass spectrum.
- Patent Document 2 describes a method for producing various pyrylium compounds.
- two types of isomers are formed.
- the detection depends on the NMR analysis of the pyridine compound formed by reaction with ammonia, and the acid used in the reaction It is stated that Ho of -10 to -5 can be used.
- the molecular weight of peptides subjected to mass spectrometry is often in the range of 500 to 3000 Da. Therefore, the mass spectra of these peptides are observed not as a single peak but as multiple peaks having a mass difference from each other due to naturally occurring isotopes.
- the size of each peak depends on the molecular weight of the peptide, but the monoisotopic peak is the main peak, the peak where the mass is 1 to 3 larger than that is large, and the subsequent peaks gradually become smaller.
- the present inventors have intensively studied to solve the above problems. Therefore, the present inventors first searched for a better compound for protein labeling using a Py compound as a lead compound. As a result, it has been found that a pyrylium compound having an ethyl group at the 2nd, 4th and 6th positions and a methyl group at the 3rd and 5th positions has excellent properties such as reactivity with proteins and water solubility. Therefore, further attempts were made to synthesize the isotope-labeled compounds, and three types of compounds having a mass difference of 6 (PyII-0, PyII-6 and PyII-12 described later) were successfully synthesized with high purity. .
- the Py derivative of an amino group-containing non-peptide compound has a low binding property to a trap column (ODS column) that is once introduced before introduction into an analytical column, which is essential in a nano liquid chromatography system. It has been confirmed by the present inventors that it is not easy to connect to a high-sensitivity automatic analysis using the. However, since the PyII compound has a higher hydrophobicity of the ring side chain than the Py compound and contributes to the improvement of the hydrophobic adsorption capacity, an ODS column is used as a trap column to increase the amino group-containing non-peptide compound. It has been found that it opens up the possibility of automating sensitivity analysis. As a result of further investigations based on the above findings, the present inventors have completed the present invention.
- ODS column trap column
- a kit for quantification of a target substance containing an amino group in a biological sample using a mass spectrometer which comprises a compound represented by the above or a salt thereof as a labeling compound.
- a compound represented by the formula (I) containing no carbon atom having a mass number of 13 The compound represented by the formula (I) having 6 carbon atoms having a mass number of 13 and the compound represented by the formula (I) having 12 carbon atoms having a mass number of 13 or a salt thereof [5] ] Kit of description.
- the kit according to [6] above comprising PyII-0, PyII-6 and PyII-12 represented by the formula (wherein the atoms indicated by black circles represent carbon atoms having a mass number of 13).
- the kit according to [4] above comprising two or more compounds represented by the formula (I) having a mass difference of 2 or more, or a salt thereof, and the target substance is an amino group-containing non-peptide compound. .
- a method comprising a step of determining an amount ratio of the target substance between samples subjected to preparation of the mixture from the obtained abundance ratio and the mixing ratio of the sample in the step (3).
- the labeled compound comprises two or more compounds represented by the formula (I) having a mass difference of 2 or more or a salt thereof.
- the labeled compound is A compound represented by the formula (I) containing no carbon atom having a mass number of 13, A compound represented by formula (I) having two carbon atoms having a mass number of 13, A compound represented by formula (I) having four carbon atoms having a mass number of 13, A compound represented by formula (I) having 6 carbon atoms having a mass number of 13, A compound represented by formula (I) having 8 carbon atoms having a mass number of 13; Two or more selected from the group consisting of a compound represented by formula (I) having 10 carbon atoms having a mass number of 13 and a compound represented by formula (I) having 12 carbon atoms having a mass number of 13 The method of the above-mentioned [13], comprising a compound or a salt thereof.
- the labeled compound has the formula (II
- One of the samples prepared in step (1) is an internal standard sample containing a known concentration of a target substance, and Determining the abundance ratio in step (4) includes determining a ratio of peak intensities of target substances derived from samples other than the internal standard sample and target substances derived from the internal standard sample; The method according to [11] above.
- a method for quantitatively analyzing proteins in two or more biological samples using a mass spectrometer (1) a step of preparing two or more biological samples for analysis; (2) Two or more formulas (I) having different masses by isotope labeling so as to give a mass difference to the same protein between samples:
- the labeled compound is A compound represented by the formula (I) containing no carbon atom having a mass number of 13, 3 organisms comprising a compound represented by formula (I) having 6 carbon atoms having a mass number of 13 and a compound represented by formula (I) having 12 carbon atoms having a mass number of 13 or salts thereof
- the labeled compound is represented by the following formula (III):
- step (1) One of the samples prepared in step (1) is an internal standard sample obtained by mixing all the other prepared samples, and Determining the abundance ratio in step (5) comprises determining a ratio of peak intensities of peptides derived from each sample other than the internal standard sample and peptides derived from the internal standard sample; The method according to [17] above.
- step (2) In the presence of an acid anhydride, 3-ethyl-3-pentanol or 3-ethyl-2-pentene is condensed with propionic anhydride to obtain a compound of formula (I):
- R represents an optionally substituted hydrocarbon group
- a salt thereof excluding compounds having a peptide bond
- the isotope-labeled compound of the present invention can provide a plurality of compounds having a large mass difference (eg, 6 or more) with respect to each other, even if the peptide has a relatively high molecular weight in the comprehensive expression difference analysis of proteins.
- the problem of mutual interference between body peaks can be solved.
- none of the conventional stable isotope labeling reagents can provide three types of compounds with a mass difference of 6, but according to the present invention, three types of stable isotope labeling reagents are provided with a mass difference of 6, Mass spectrometry enables highly sensitive multiplex simultaneous protein quantitative analysis between three samples, which contributes to much higher efficiency in protein quantitative analysis.
- the isotope-labeled compound of the present invention also makes it possible to simultaneously analyze amino group-containing non-peptide compounds contained in a plurality of samples (eg, 7 types) with high sensitivity. Furthermore, a system capable of automatically performing high-sensitivity analysis at a femtomole level for 24 hours can be established by automation using a trap column.
- the EI-MS spectrum of each synthesized PyII compound is shown.
- 1 H-NMR (270 MHz, D 2 O) spectrum of each synthesized PyII compound is shown.
- the elution curves of bovine serum albumin (BSA) derived peptide and human serum transferrin (TRFE) derived peptide are shown.
- the nano LC / MS / MS analysis results of fraction 5 (including BSA-derived peptide) (left column) and fraction 9 (including TRFE-derived peptide) (right column) in FIG. 3 are shown.
- the intensity ratio between the PyII-0-labeled peptide-derived peak, the PyII-6-labeled peptide-derived peak, and the PyII-12-labeled peptide-derived peak in the mass spectrum of FIG. 4 is shown (upper: BSA, lower: TRFE).
- An exemplary protocol in a verification experiment using an actual human plasma specimen of a comparative quantification method using a PyII reagent is shown.
- the result of the comparative experiment of the quantitative accuracy between the cICAT reagent and the PyII reagent is shown.
- the outline of the experimental proof method of protein labeling of the PyII reagent by the fluorescent DIGE method (left figure) and the result of two-dimensional electrophoresis of all the extracted proteins from HeLa cells labeled with two colors (right figure) are shown.
- the mass spectrum of dopamine labeled with each PyII compound is shown.
- the calibration curve prepared for dopamine measurement using PyII reagent is shown.
- the result of the measurement experiment of dopamine content in a striatum level is shown.
- Each square represents a site in the brain tissue, and the number in the square represents the dopamine content in the site in fmol units.
- the ion chromatograph about PyII derivative of eight types of amines / amino acids is shown.
- the ion chromatograph about PyII derivative of eight types of amines / amino acids is shown.
- the ion chromatograph about PyII derivative of eight types of amines / amino acids is shown.
- the ion chromatograph about PyII derivative of eight types of amines / amino acids is shown.
- the mass spectrum of the peak part of FIG. 12 is shown.
- the mass spectrum of the peak part of FIG. 12 is shown.
- the mass spectrum of the peak part of FIG. 12 is shown.
- the mass spectrum of the peak part of FIG. 12 is shown.
- the compound or a salt thereof may be one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 by carbon having a mass number of 13, for example. 11, 12, 13) areotope-labeled.
- the compound represented by the formula (I) or a salt thereof, including those not isotopically labeled is also referred to as an isotope-labeled compound of the present invention.
- the isotope-labeled compound of the present invention is usually used in the form of a salt.
- the salt may be any anionic atom or anionic molecule as long as it does not interfere with the labeling reaction to the amino group, such as trifluoromethanesulfonate, hexafluorophosphate, tetrafluoroborate, perchlorine.
- hexafluorophosphate can be easily obtained by adding sodium hexafluorophosphate to tetrafluoroborate.
- PyII compounds (Wherein the atoms indicated by black circles represent carbon atoms having a mass number of 13) PyII-0, PyII-2, PyII-4, PyII-6, PyII-8, PyII-10 and PyII-12 (hereinafter collectively referred to as PyII compounds).
- the isotope-labeled compound of the present invention can be obtained by condensing 3-ethyl-3-pentanol or 3-ethyl-2-pentene and propionic anhydride in the presence of anhydride. According to the production method of the present invention, since a single compound is formed without producing an isomer, the subsequent separation operation is simple.
- the anhydride include trifluoromethanesulfonic acid, hexafluorophosphoric acid, tetrafluoroboric acid, perchloric acid, trimethylsilyl trifluoromethanesulfonate, methanesulfonic acid, etc. Among them, the crystallinity of the salt is good and the safety is relatively high.
- the reaction temperature is usually 40 to 100 ° C., preferably 50 to 65 ° C., and the reaction time is usually 10 minutes to 24 hours, preferably 1 to 1.5 hours.
- the amount of the raw material compound is usually 3 to 20 molar equivalents, preferably about 4 molar equivalents of propionic anhydride with respect to 3-ethyl-2-pentanol.
- propionic anhydride is usually 3 to 3-ethyl-2-pentene.
- It can be 10 molar equivalents, preferably about 3 molar equivalents.
- An acid anhydride such as trifluoromethanesulfonic acid is usually added in an amount of 0.8 to 1.2 molar equivalents relative to 3-ethyl-2-pentanol or 3-ethyl-2-pentene.
- the reaction solution after the condensation reaction is concentrated, neutralized, extracted and the like by a method known per se, and then recrystallized to obtain a desired compound.
- Any isotope-labeled compound of the present invention having one or more carbons having a mass number of 13 can also be produced in the same manner as described above except that a 13 C-labeled compound is used as a raw material compound.
- the above-mentioned isotope-labeled compound of the present invention which is a target substance containing an amino group in a biological sample using a mass spectrometer by combining two or more of the same compounds except that the mass number is different due to isotope labeling Quantification kit (hereinafter also referred to as the kit of the present invention).
- the target substance may be any compound as long as it contains an amino group, and is typically an amino group-containing non-peptide compound (eg, amine, amino acid, etc.), an amino group-containing peptide compound, and a protein.
- the combination of the isotope labeled compounds of the present invention included in the kit of the present invention is not particularly limited.
- the target substance is a protein
- a combination in which the mass difference between the compounds is 6 or more is preferable in that interference between isotopic peaks can be avoided in the mass spectrum.
- Examples of such combinations include a compound represented by the formula (I) not containing a carbon atom having a mass number of 13, a compound represented by the formula (I) having 6 carbon atoms having a mass number of 13, and a mass number.
- Examples include compounds represented by the formula (I) having 12 13 carbon atoms, or salts thereof.
- Specific examples of preferred combinations include those containing PyII-0, PyII-6 and PyII-12 described above.
- examples of the combination include those containing two or more compounds represented by the formula (I) having a mass difference of 2 or more or salts thereof.
- a compound represented by the formula (I) not containing a carbon atom having a mass number of 13, a compound represented by the formula (I) having two carbon atoms having a mass number of 13, and a mass number of 13 A compound represented by the formula (I) having 4 carbon atoms, a compound represented by the formula (I) having 6 carbon atoms having a mass number of 13 and a formula (I having 8 carbon atoms having a mass number of 13) ), A compound represented by formula (I) having 10 carbon atoms having a mass number of 13, and a compound represented by formula (I) having 12 carbon atoms having a mass number of 13 And those containing two or more compounds selected from or a salt thereof.
- each of the combinations exemplified above has preferably 3 or more, more preferably 4 or more, compounds represented by the formula (I) or salts thereof. Even more preferably, 5 or more, particularly preferably 6 or more, and most preferably 7 is included.
- the kit of the present invention contains one or more reaction buffers, washing solutions, or other components necessary or suitable for use in combination with the isotope labeled compound of the present invention, in addition to the combination of the isotopically labeled compound of the present invention. You can leave.
- the kit of the present invention also optionally includes instructions for its use.
- the kit of the present invention may further contain an unreacted component removal reagent (washing reagent), a restriction enzyme, a purification column, a purification solvent, and the like.
- the present invention also provides a method for quantitatively analyzing a target substance containing amino groups in two or more biological samples using a mass spectrometer, which utilizes two or more of the aforementioned isotope-labeled compounds of the present invention ( Hereinafter, the quantitative analysis method of the present invention is also provided.
- the target substance include amino group-containing non-peptide compounds, amino group-containing peptide compounds, and proteins.
- the quantitative analysis method of the present invention can be carried out according to essentially the same steps regardless of the type of target substance.
- the protein quantitative analysis method (hereinafter also referred to as the protein analysis method of the present invention) is usually subjected to specific processing such as digestion with restriction enzymes and identification of proteins from peptide fragments, and will be described separately later.
- the quantitative analysis of so-called peptide compounds generally having about 2 to about 10 amino acids can be appropriately performed by those skilled in the art based on the description of the protein analysis method described later. Not performed.
- a quantitative analysis method for an amino group-containing non-peptide compound (hereinafter also referred to as a non-peptide compound analysis method of the present invention) will be described.
- an amino group-containing non-peptide compound or an amino group-containing non-peptide compound means any compound having one or more amino groups in the molecule and having no peptide bond in the molecule.
- the amino group means ammonia, a primary amine (that is, a compound in which one hydrogen atom of ammonia is optionally substituted with any hydrocarbon group) or a secondary amine (that is, , A monovalent functional group obtained by removing hydrogen from a compound obtained by substituting two hydrogen atoms of ammonia with the same or different arbitrary hydrocarbon groups which may be optionally substituted.
- the amino group-containing non-peptide compound is NH 3 , NH 2 R, or NHRR ′ (wherein R and R ′ are the same or different and each represents an optionally substituted hydrocarbon group.
- a non-peptidic compound having the chemical formula it is considered that the compound having the chemical formula of NHRR ′ does not react with the labeling reagent used in the method of the present invention or has extremely low reactivity. Therefore, the amino group-containing non-peptide compound to be subjected to the method of the present invention is usually NH 2 R (wherein R represents hydrogen or an optionally substituted hydrocarbon group). It is a non-peptidic compound having a chemical formula.
- the molecular weight of the amino group-containing non-peptide compound to be measured is not particularly limited as long as it enables the method of the present invention, but is generally a low molecular weight compound.
- the specific molecular weight is 17 to 1000, preferably 17 to 700, and more preferably 17 to 500.
- Examples of amino group-containing non-peptide compounds to be measured include bioactive amines, amino acids, drugs, stimulants, narcotics, non-volatile spoilage amines, and compounds having amino groups in their metabolites. . In one analysis, a plurality of types of amino group-containing non-peptide compounds can be measured.
- amino group-containing non-peptide compounds include, but are not limited to, bioactive amines that act on the nervous system (eg, L-dopa, norepinephrine (noradrenaline), dopamine, tryptamine, serotonin, putomain, histamine).
- bioactive amines that act on the nervous system (eg, L-dopa, norepinephrine (noradrenaline), dopamine, tryptamine, serotonin, putomain, histamine).
- the number of biological samples subjected to analysis is not particularly limited. However, when analyzing more samples than the number of types of the isotope-labeled compounds of the present invention having a mass difference that can be prepared (for example, seven isotope-labeled compounds having a mass difference of 2 from each other) After dividing into groups, analysis is performed for each group using internal standard samples. The internal standard sample will be described later.
- the biological sample to be analyzed can be collected from any source and tissue according to the purpose of analysis by a method known per se.
- examples of the biological sample include various body fluids of mammals (eg, humans, monkeys, cows, horses, pigs, sheep, goats, dogs, cats, rabbits, hamsters, guinea pigs, mice, rats, etc.).
- cells / tissues eg: Brain, spinal cord, eyeball, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung
- the target non-peptide compound in each biological sample from the collected sample using an appropriate means such as solid phase extraction.
- concentration can be performed, for example, by the following procedure. That is, in each collected sample, in order to separate the target non-peptide compound and the protein, protein removal treatment is performed by an appropriate means such as acid extraction.
- the target non-peptide compound in the deproteinized liquid sample is then captured using a cation exchange resin that selectively captures it. Subsequently, the acidic or neutral low molecular weight substance nonspecifically adsorbed on the resin is washed with alcohol. Further, the remaining resin-bound cation is eluted with hydrochloric acid or the like, and then hydrochloric acid is removed under reduced pressure. In this way, a biological sample to be analyzed can be prepared.
- an internal standard sample is also prepared as one of samples used for analysis.
- the internal standard sample is a sample that serves as a reference for the amount of the target non-peptide compound contained in the sample.
- the internal standard sample can be prepared, for example, by dissolving a commercially available target non-peptide compound in HCl at a predetermined concentration (eg, 0.05M). Further, the concentration of the target non-peptide compound present in the internal standard sample is not particularly limited, but in general, it is preferably close to the assumed concentration of the compound in other samples subjected to analysis.
- the internal standard usually contains the target non-peptide compound at a known concentration. Thereby, it is possible to determine the absolute amount of the target compound present in the sample.
- an amino group-containing non-peptide compound that is not present in any sample subjected to analysis in order to determine the mixing ratio between samples in the subsequent step (3) May be added to all the samples as a compound serving as an index of the mixing ratio so as to have a known concentration.
- the compound that serves as an index of the mixing ratio include dihydroxybenzylamine (DHBA).
- the amount of the compound serving as an index of the mixing ratio is not particularly limited as long as it does not hinder subsequent mass spectrometry.
- the concentration of the compound serving as an index of the mixing ratio in each sample is preferably 0.2 to 10 pmol, preferably Is added in an amount of 0.5 to 5 pmol, more preferably 1 to 3 pmol.
- a compound serving as an index of the mixing ratio is added so that the same concentration is obtained in any of the samples to be analyzed.
- each sample prepared in step (1) is labeled with a target amino group-containing non-peptide compound using a different isotope-labeled compound of the present invention.
- Amino group-containing non-peptide compounds are basic and have a relatively high pKa value.
- the amino group takes the ionic form of —NH 3 +.
- H + is released and —NH 2 Take the structure.
- the isotope-labeled compounds of the present invention are known to be more reactive towards —NH 2 .
- the pH of the reaction solution may be appropriately adjusted according to the pKa value of the target non-peptide compound.
- the maximum pH range that can be protected with a borate buffer solution is 10.0. It may be adopted.
- the specific composition of the reaction solution is, for example, as follows: 0.05 M borate buffer (pH 10.0) 10 ⁇ L, amino group-containing non-peptide compound 0.05 M hydrochloric acid solution (10 mM) 1 ⁇ L, distilled water, 100 mMPyII Compound 1 ⁇ L, total volume 20 ⁇ L.
- the reaction is carried out, for example, at 50 ° C. for several minutes to 2 hours, and 1 ⁇ L of 1M hydrochloric acid is added to stop the reaction.
- Examples of the combination of labeling compounds used include those containing two or more compounds represented by the formula (I) having a mass difference of 2 or more or salts thereof.
- a compound represented by the formula (I) not containing a carbon atom having a mass number of 13, a compound represented by the formula (I) having two carbon atoms having a mass number of 13, and a mass number of 13 A compound represented by the formula (I) having 4 carbon atoms, a compound represented by the formula (I) having 6 carbon atoms having a mass number of 13 and a formula (I having 8 carbon atoms having a mass number of 13) ), A compound represented by formula (I) having 10 carbon atoms having a mass number of 13, and a compound represented by formula (I) having 12 carbon atoms having a mass number of 13 And those containing two or more compounds selected from or a salt thereof.
- each of the combinations exemplified above has preferably 3 or more, more preferably 4 or more, compounds represented by the formula (I) or salts thereof. Even more preferably, 5 or more, particularly preferably 6 or more, and most preferably 7 is included.
- non-peptide compound containing amino groups To label the non-peptide compound containing amino groups.
- the same target non-peptide compound from different samples is labeled with compounds having different masses by isotope labeling, resulting in a mass difference from each other.
- each amino group-containing non-peptide compound-containing sample subjected to labeling in step (2) is mixed.
- the amount collected from each sample during preparation of the mixed solution is preferably equal, but is not limited thereto.
- excess labeling reagent present in the mixed solution may be removed. Excess labeling reagent can be removed by organic solvent extraction using ethyl acetate or the like, or cation exchange resin. Furthermore, it is preferable to concentrate the mixed solution before subjecting it to mass spectrometry.
- Concentration of the mixed solution is performed by adding the mixed solution to an equilibrated cation exchange resin (H + type), thoroughly washing with water, and then eluting the reaction product with 1% aqueous ammonia or 0.1 M hydrochloric acid.
- the eluate can be obtained by concentrating to an appropriate amount with a vacuum centrifugal concentrator.
- the mixture is subjected to mass spectrometry, and for target substances having a mass difference from each other by the label, the abundance ratio of the target substances in the mixture is determined based on the ratio of their peak intensities in the mass spectrum.
- the mixture obtained according to the above procedure is subjected to mass spectrometry.
- Any mass spectrometry system can be used in the present invention, but in order to enable highly sensitive quantification, a nano liquid chromatography mass spectrometry (nano LC / MS) system (for example, NanoFrontier eLD (Hitachi High Technologies) Etc.)).
- a mass spectrum can be acquired by performing mass spectrometry according to a conventional method.
- ionization mode is nano ESI (positive ionization)
- spray voltage is 1600 V
- detector voltage is 1950 V
- counter nitrogen gas amount is 0.8 L / min
- scan range is 100-500 m / z
- the recording time for mass spectrometry is 50 minutes.
- the same amino group-containing non-peptide compounds from different samples have different masses, so the same amino group-containing non-peptide compounds from different samples appear as separate peaks in the mass spectrum. . By determining the intensity ratio of these separated peaks, the abundance ratio of those peptides in the mixture is obtained.
- the determination of the abundance ratio in this step is usually the peak between the target non-peptide compound derived from each sample other than the internal standard sample and the same compound that differs only in mass from the internal standard sample. This is done by determining the intensity ratio.
- the compound that is an index of the mixing ratio having a mass difference due to the labeling in the step (2) Determine the ratio of their peak intensities in the mass spectrum.
- the determined ratio corresponds to the abundance ratio of those compounds in the mixture. Therefore, mixing between samples in the step (3) is further performed based on the concentration of the compound serving as an index of the mixing ratio added to each sample and the existing ratio of the compound serving as an index of the mixing ratio in the mixture. The ratio can be determined.
- the quantitative ratio of the target non-peptide compound among the samples subjected to the preparation of the mixture can be determined.
- the number, origin, and collection method of the samples to be analyzed are as described above in the non-peptide compound analysis method of the present invention. It is preferable to perform protein extraction from the collected sample by a method known per se. Specifically, for example, a protein extraction process can be performed using a solution called a cell lysate (CLS).
- CLS cell lysate
- the composition of CLS is 7M urea, 3M thiourea, 2% CHAPS, 0.2M borate buffer, pH 9.6.
- an internal standard sample is also prepared as one of samples used for analysis.
- the internal standard sample is a sample that serves as a reference for the amount of each protein contained in the sample.
- the sample to be analyzed is divided into two or more groups, and the analysis is performed for each group using the same internal standard sample, so that all samples to be analyzed can be compared.
- the internal standard sample preferably contains any protein present in the sample to be analyzed.
- the internal standard sample can be prepared, for example, by mixing all the protein-containing samples to be analyzed prepared as described above. In that case, after preparing a starting sample with the same total protein content from each sample prepared as described above, an internal standard sample is prepared by mixing the starting samples (eg, mixing in equal amounts). Can also be mentioned as an example of its preparation method. A method for quantitative analysis of protein using an internal standard sample is also described in Patent Document 1.
- each sample prepared in step (1) is labeled with a protein using a different isotope-labeled compound of the present invention.
- a treatment for reducing and alkylating SH groups of all proteins in a sample to be analyzed is usually performed before labeling.
- the treatment may be carried out according to a conventional method.
- dithiothreitol can be used for reduction and iodoacetamide can be used for alkylation.
- the labeling reaction is carried out under basic conditions in an appropriate solvent (for example, CLS as described above, provided that the final concentration after addition of the labeled compound is 6.5 M urea, 2.5 M thiourea, 1.7% CHAPS or more.
- the labeled compound is added to the protein-containing sample dissolved in the reaction solution, and the reaction is carried out at 50 ° C. for 2 hours.
- the amount of the labeling compound can be 100 times the amount of lysine residue in the protein present in the sample.
- any compounds are preferable.
- examples of such combinations include a compound represented by the formula (I) not containing a carbon atom having a mass number of 13, a compound represented by the formula (I) having 6 carbon atoms having a mass number of 13, and a mass number.
- examples include compounds represented by the formula (I) having 12 13 carbon atoms, or salts thereof.
- Specific examples of preferred combinations include those containing PyII-0, PyII-6 and PyII-12 described above.
- each protein-containing sample provided for labeling in step (2) is mixed.
- Unreacted labeled compound is usually removed by gel filtration or a protein precipitation reagent, and the labeled protein is collected and concentrated.
- the flow until the peptide is subjected to mass spectrometry is as follows.
- the labeled peptide and the unlabeled peptide released from the protein separated by the operation (a) may be directly subjected to mass spectrometry by MALDI-TOF / MS without peptide separation operation.
- ESI / MS / MS analysis can be performed after the peptides are separated by liquid chromatography.
- the peptide released by the operation of (b) is subjected to a two-dimensional liquid chromatographic system, for example, one-dimensional separation is performed on an SCX column, and the eluted component is separated on a second reversed-phase resin column, and ESI / MS / MS
- the molecular weight of the peptide used for the measurement of the MS spectrum in the next step is not particularly limited, but is, for example, 500 to 3000, and preferably 1000 to 2500. Therefore, this step preferably comprises isolating a peptide having a molecular weight in the above range from the digested product of the protein.
- the peptide obtained according to the above procedure is subjected to mass spectrometry.
- Any mass spectrometric system can be used in the present invention.
- a nano liquid chromatography mass spectrometric (nano LC / MS) system eg, NanoFrontierNeLD (Hitachi High Technologies) Etc.
- a mass spectrum can be acquired by performing mass spectrometry according to a conventional method.
- determination of the abundance ratio in this step is usually the ratio of peak intensity between peptides derived from each sample other than the internal standard sample and the same peptide that differs only in mass from the internal standard sample. It is done by seeking.
- step (5) referring to the mass spectrum of step (5), select the peptide whose amino acid sequence should be specified, and qualify the amino acid sequence of the peptide by the MS / MS spectrum of the product ion generated from the peptide By identifying the corresponding protein from the known DNA sequence based on the amino acid sequence of the peptide, the protein from which the peptide is derived can be specified.
- the abundance ratio obtained in step (5) for the peptide is equal to the abundance ratio of the protein from which the peptide is derived in the mixture, the abundance ratio and the sample mixture ratio in step (3) From this it is possible to determine the quantitative ratio of the protein between the samples subjected to the preparation of the mixture.
- a compound represented by the formula (wherein R represents an optionally substituted hydrocarbon group) (excluding a compound having a peptide bond) or a salt thereof is provided.
- the compound or a salt thereof has one or more carbon atoms having a mass number of 13 at positions other than R in the formula (IV) (eg, 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13).
- the compound represented by the formula (IV) or a salt thereof including those not isotopically labeled is also referred to as a labeled product of the present invention.
- the labeled product of the present invention can be used, for example, for the preparation of an internal standard sample in the non-peptide compound analysis method of the present invention.
- the labeled product of the present invention may be produced, for example, by labeling a given amino group-containing non-peptide compound as defined above with an isotopically labeled compound of the present invention. That is, any compound obtained by labeling the amino group-containing non-peptide compound defined above with the labeling compound is included in the labeling product of the present invention. In that case, the 13 C position in the labeled product of the present invention corresponds to the 13 C position in the isotopically labeled compound of the present invention.
- R in formula (IV) represents NH 2 R (wherein R represents hydrogen or an optionally substituted hydrocarbon group, as described above in the method for analyzing non-peptide compounds of the present invention).
- the salt may be any salt, for example, those exemplified as salts of the compound of formula (I) (eg, trifluoromethanesulfonate, hexafluorophosphate, tetrafluoroborate, perchlorate) Trimethylsilyl trifluoromethanesulfonate, methanesulfonate, etc.), and nitrates, hydrochlorides, sulfates, and the like.
- Electron Impact Mass Spectrometry (EI-MS) Equipment JMS-AX505W (2-2) Identification / Identification of Compounds by GC-MS Measurement Data supporting the change in molecular weight associated with 13 C labeling by measuring GC-MS for undeuterated samples and 13 C-labeled reagents This was done by confirming that ⁇ NMR measurement by using the identification D 2 O of compound A sample was dissolved, was confirmed from the observed peak information. Protons adjacent to 13 C-labeled carbon can be largely coupled to confirm the 13 C-labeled site.
- FIG. 1 shows an EI-MS spectrum of each PyII compound
- FIG. 2 shows a 1 H-NMR (270 MHz, D 2 O) spectrum of each PyII compound.
- FIG. 3 shows the elution curves of the BSA-derived peptide and the Tfn-derived peptide that were fractionated 10 times on a cation exchange (SCX) column.
- SCX cation exchange
- FIG. 4 shows an example of nano LC / MS / MS analysis of the two fractions. Although many peptide-derived ions were detected, a BSA-derived peptide having a molecular weight of 1819.1314 appeared at an elution time of 37.1 minutes. Mass analysis of this peptide is the bottom left side of FIG.
- Mass spectrometry peaks of peptides labeled with PyII-0, PyII-6, and PyII-12 were observed at 605.37, 607.38, and 609.38, respectively.
- This M / z value was exactly one third of the expected mass, indicating that this peptide was detected as a trivalent ion.
- This ion gave an intensity ratio of 1: 2.5: 4.
- the peptide derived from Tfn appeared in fraction 9 at 28.7 minutes, and it was detected as a trivalent ion in the same manner as M / z, which was one third of 1759.9453, and the intensity ratio was 1: 0625: 0.25. It was.
- the mass spectrum has a complicated shape including isotope peaks.
- the monoisotopic peak is made up of all the elements of the peptide having the smallest mass, and the first isotope peak is the spectrum of the peptide with one carbon at 13 C.
- a Py reagent with a mass difference of 4 a Py4 monoisotopic peak overlaps with the fourth isotope peak, and the interference cannot be ignored.
- the mass difference is 6
- the interference is completely negligible.
- the number of identified proteins (p ⁇ 0.05) was 146, more than 126 with cICAT reagent. This may reflect the difference that PyII is Lys-modified but cICAT is Cys-modified. The number of proteins that could be quantified with PyII reagent was 33, 23% of the total. From the above, it was confirmed that the PyII introduction rate into the Lys residue was high.
- Cy5-labeled protein dissolved in CLS was labeled with 10 mM PyII at 50 ° C. for 3 hours.
- An equal amount of Cy3 labeled protein not labeled with PyII was mixed with PyII labeled protein, two-dimensional electrophoresis separation was performed, and two-color fluorescence was detected with a fluorescence scanner.
- the result of superimposing both fluorescences is the right diagram of FIG. Protein green fluorescent spots were distributed over the entire surface of the gel. The movement of the red fluorescent spot is recognized in the molecular weight increasing direction (upward) in the vicinity of each spot.
- the dopamine-PyII compound in the sample was purified using a column (MonoSpin PBA, GL Science) having phenylboric acid (PBA) as a functional group. Bind the sample to PBA, wash with acetonitrile, then 100 mM phosphate K buffer pH 8.0, then 5% acetonitrile solution with 0.5% trifluoroacetic acid, then elute with 30% acetonitrile solution with 0.5% trifluoroacetic acid did.
- a column Monitoring of phenylboric acid
- the eluate was concentrated by a vacuum centrifugal concentrator, filtered through a filter having a pore size of 0.22 ⁇ m (DURAPORE PVDF 0.22 ⁇ m, MILLIPORE), and analyzed by a nano LC / MS system.
- the results are shown in FIG. From the mass 326.1 to 338.2, it was confirmed that seven types of compounds were produced with a mass difference of about 2.
- Calibration curves of standard dopamine 1250 fmol, 625 fmol, 313 fmol and 0 fmol were prepared.
- Standard dopamine at 1250 fmol was reacted with PyII-12, 625 fmol with PyII-10, 313 fmol with PyII-8, and 0 fmol with PyII-0.
- 500 fmol 3,4-dihydroxybenzylamine (DHBA) was used as an internal standard.
- 5% perchloric acid was added to a standard dopamine solution (containing 500 fmol of DHBA) to make a total volume of 10 ⁇ l. Thereafter, the PyII compound and dopamine in the sample were reacted according to the method of Example 9, and then each reaction solution was mixed.
- Example 10 Measurement of rat brain dopamine content using PyII reagent
- the rat head was irradiated with microwaves (5 kW, 1.7 seconds) (microwave applicator, Muromachi Machine) and fixed. After brain removal, a 30 ⁇ m thick brain tissue section of the region containing the striatum was prepared with a freezing microtome (CM3050S, Leica), and 1 mm square on each side, 30 ⁇ m thick with a laser microdissection (ASLMD, Leica) Obtained brain tissue. The volume of each section taken corresponds to 30 nl.
- microwaves 5 kW, 1.7 seconds
- Muromachi Machine microwave applicator, Muromachi Machine
- FIG. 11 shows the calculated amount of dopamine in each part of the brain based on the obtained mass spectrometry spectrum result and the calibration curve of FIG.
- the black squares indicate the measured brain tissue pieces corresponding to 30 nl, and the numbers in them indicate the dopamine content contained in each brain tissue piece in fmol.
- the results showed that the dopamine content was low in the cerebral cortex (123 and 317 fmol) and high in the striatum (996-1866 fmol). This is the same level as the conventional knowledge of measuring dopamine. From these results, it was confirmed that the dopamine content in 30 nl of brain tissue can be measured by the analysis method of the present invention using PyII reagent.
- Example 11 Analysis example of amine and amino acid by PyII reagent
- the mass measurement unit settings are as follows.
- the ionization mode is nano ESI (positive ionization)
- the spray voltage is 1600 V
- the detector voltage is 1950 V
- the counter nitrogen gas amount is 0.8 L / min
- the scan range is 100-500 m / z
- the recording time of mass spectrometry is 50 minutes.
- an ion chromatograph showing a mass M / z value is shown in FIG. 12, and a mass spectrum of the peak portion in FIG. 12 is shown in FIG.
- a single chromatographic peak and a mass peak consistent with the theoretical value were identified.
- the isotope-labeled compound of the present invention can provide a plurality of compounds having a large mass difference (eg, 6 or more) with respect to each other, an isotope peak can be used for any molecular weight peptide in a comprehensive expression difference analysis of proteins. Can solve the problem of mutual interference.
- there is no conventional stable isotope labeling reagent that can provide three types of compounds with a mass difference of 6, which contributes to a significant improvement in the quantitative analysis of proteins.
- the isotope-labeled compound of the present invention also makes it possible to simultaneously analyze amino group-containing non-peptide compounds contained in a plurality of samples (eg, 7 types) with high sensitivity.
- a system capable of automatically performing high-sensitivity analysis at a femtomole level for 24 hours can be established by automation using a trap column.
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Abstract
Description
そのため、より高い感度を実現するために、蛍光標識の使用が提案されており、それにより、約10倍以上の高感度化が実現されている。
更に、それでも検出できない低濃度のアミノ酸に対しては、液体クロマト分離した後に質量分析して検出する方法が採用されている。この場合、分離したアミノ酸をイオン化し易くするために、アミノ基と反応する試薬を用いてアミノ酸を誘導体化してから、それを液体クロマト分離および質量分析に供する方法が採用されてきた。しかしながら、この方法論に従った場合でも、従来の技術では高感度分析には限界があった。
本発明者等は更に、該化合物をアミンやアミノ酸等のアミノ基含有非ペプチド化合物の分析に適用することを試みた。アミノ基含有非ペプチド化合物の分析のためには、標識化合物間に2の質量差があればピーク間の干渉を回避できるので、互いに2の質量差を有して、なるべく多種類の同位体標識化合物を用意できることが分析効率の点で有益である。そこで更に合成実験を重ねた結果、上記PyII-0に対して質量差2、4、8または10を有する化合物(後述のPyII-2、PyII-4、PyII-8、PyII-10)を高純度で合成することに成功した。これらの同位体標識化合物を用いて分析を行うことで、生体試料中のアミノ基含有非ペプチド化合物を高感度に検出し得るのみならず、複数試料を同時に定量して、各試料中に含まれるアミノ基含有非ペプチド化合物を網羅的に分析できることを見出した。また、アミノ基含有非ペプチド化合物のPy誘導体は、ナノ液体クロマトグラフィーシステムにおいて必須である、分析カラムへの導入前に一旦導入されるトラップカラム(ODSカラム)への結合性が低いため、オートサンプラーを用いた高感度自動分析に繋げることは容易でないことが本発明者等により確認されていた。しかし、PyII化合物は、Py化合物と比べて環の側鎖の疎水性が高く、疎水性吸着能の向上に寄与するため、トラップカラムとしてODSカラムを利用して、アミノ基含有非ペプチド化合物の高感度分析を自動化する可能性を拓くことが分かった。
本発明者等は上記の知見に基づき更に検討を進めた結果、本発明を完成するに至った。
[1]式(I):
[2]式(I)において質量数13の炭素を1つ以上有する、上記[1]記載の化合物またはその塩。
[3]式(II):
[4]同位体標識により互いに異なる質量を有する2つ以上の式(I):
[5]質量差が6以上である2つ以上の式(I)で表される化合物またはそれらの塩を含み、かつ標的物質がタンパク質である、上記[4]記載のキット。
[6]質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
またはそれらの塩を含む、上記[5]記載のキット。
[7]式(III):
[8]質量差が2以上である2つ以上の式(I)で表される化合物またはそれらの塩を含み、かつ標的物質がアミノ基含有非ペプチド化合物である、上記[4]記載のキット。
[9]質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を2個有する式(I)で表される化合物、
質量数13の炭素原子を4個有する式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、
質量数13の炭素原子を8個有する式(I)で表される化合物、
質量数13の炭素原子を10個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
からなる群から選択される2つ以上の化合物またはそれらの塩を含む、上記[8]記載のキット。
[10]式(II):
[11]質量分析計を用いて2以上の生体試料中のアミノ基を含有する標的物質を定量分析するための方法であって、
(1)分析に供する2以上の生体試料を調製する工程、
(2)試料間で標的物質に質量差を与えるように、同位体標識により互いに異なる質量を有する2つ以上の式(I):
(3)標識に供された全ての試料から混合物を調製する工程、
(4)混合物を質量分析に供し、該標識により互いに質量差を有する標的物質について、質量スペクトルにおけるそれらのピーク強度の比に基づいて、該混合物中でのそれらの標的物質の存在比を求め、得られた存在比と工程(3)における試料の混合比とから、混合物の調製に供された試料間での該標的物質の量比を決定する工程
を含む、方法。
[12]標的物質が、アミノ基を含有する非ペプチド化合物である、上記[11]記載の方法。
[13]標識化合物が、質量差が2以上である2つ以上の式(I)で表される化合物またはそれらの塩を含む、上記[12]記載の方法。
[14]標識化合物が、
質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を2個有する式(I)で表される化合物、
質量数13の炭素原子を4個有する式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、
質量数13の炭素原子を8個有する式(I)で表される化合物、
質量数13の炭素原子を10個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
からなる群から選択される2つ以上の化合物またはそれらの塩を含む、上記[13]記載の方法。
[15]標識化合物が、式(II):
[16]工程(1)において調製された試料の一つが、既知濃度の標的物質を含有する内部標準試料であり、かつ、
工程(4)における存在比の決定が、内部標準試料以外の各試料由来の標的物質と、内部標準試料由来の標的物質とのピーク強度の比を求めることを含む、
上記[11]記載の方法。
[17]質量分析計を用いて2以上の生体試料中のタンパク質を定量分析するための方法であって、
(1)分析に供する2以上の生体試料を調製する工程、
(2)試料間で同一タンパク質に質量差を与えるように、同位体標識により互いに異なる質量を有する2つ以上の式(I):
(3)標識に供された全ての試料から混合物を調製する工程、
(4)混合物中のタンパク質を制限酵素により消化してペプチドを得る工程、
(5)得られたペプチドを質量分析に供し、該標識により互いに質量差を有するペプチドについて、質量スペクトルにおけるそれらのピーク強度の比に基づいて、該混合物中でのそれらのペプチドの存在比を決定する工程、および、
(6)存在比を決定されたペプチドについて、それが由来するタンパク質を特定し、該存在比および工程(3)における試料の混合比から、混合物の調製に供された試料間での該タンパク質の量比を決定する工程
を含む、方法。
[18]標識化合物が、質量差が6以上である2つ以上の式(I)で表される化合物またはそれらの塩を含む、上記[17]記載の方法。
[19]標識化合物が、
質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
またはそれらの塩を含み、3つの生体試料中のタンパク質を定量分析する、上記[18]記載の方法。
[20]標識化合物が、下記式(III):
[21]工程(1)において調製された試料の一つが、他の全ての調製された試料を混合して得られた内部標準試料であり、かつ、
工程(5)における存在比の決定が、内部標準試料以外の各試料由来のペプチドと、内部標準試料由来のペプチドとのピーク強度の比を求めることを含む、
上記[17]記載の方法。
[22]無水酸存在下で、3-エチル-3-ペンタノール又は3-エチル-2-ペンテンと無水プロピオン酸とを縮合し、式(I):
[23]式(IV):
本発明の同位体標識化合物はまた、複数試料(例、7種)中に含有されるアミノ基含有非ペプチド化合物を一斉に高感度で分析することを可能とする。更に、トラップカラムを用いた自動化により、フェムトモルレベルの高感度分析を24時間自動で行うことができる体制を構築し得る。
本発明は、下記式(I):
無水酸としては、トリフルオロメタンスルホン酸、ヘキサフルオロリン酸、テトラフルオロホウ酸、過塩素酸、トリフルオロメタンスルホン酸トリメチルシリル、メタンスルホン酸等が挙げられ、中でも塩の結晶性がよく比較的安全性が高いトリフルオロメタンスルホン酸が好ましい。
反応温度は、通常40~100℃、好ましくは50~65℃であり、反応時間は通常10分~24時間、好ましくは1~1.5時間である。
原料化合物の量としては、3-エチル-2-ペンタノールを用いる場合、3-エチル-2-ペンタノールに対して無水プロピオン酸を通常3~20モル当量、好ましくは約4モル当量である。一方、3-エチル-2-ペンテンを用いると、無水プロピオン酸の消費量を低減させることが可能であり、具体的には、3-エチル-2-ペンテンに対して無水プロピオン酸は通常3~10モル当量、好ましくは約3モル当量とすることができる。トリフルオロメタンスルホン酸等の無水酸は、3-エチル-2-ペンタノールまたは3-エチル-2-ペンテンに対して、通常0.8~1.2モル当量で添加される。
上記縮合反応後の反応液を自体公知の方法により濃縮、中和、抽出等を行った後、再結晶することにより所望の化合物を得ることができる。
上述の本発明の同位体標識化合物であって、同位体標識により質量数が異なる以外は同一の化合物を2つ以上組み合わせて、質量分析計を用いた生体試料中のアミノ基を含有する標的物質の定量用キット(以下、本発明のキットともいう)とすることができる。標的物質としては、アミノ基を含有する限り任意の化合物であってよく、典型的には、アミノ基含有非ペプチド化合物(例、アミン、アミノ酸等)、アミノ基含有ペプチド化合物、およびタンパク質である。
標的物質がタンパク質の場合、質量スペクトルにおいて同位体ピーク間の干渉を回避できる点で、いずれの化合物間についても質量差が6以上となる組み合わせが好ましい。そのような組み合わせとしては例えば、質量数13の炭素原子を含まない式(I)で表される化合物、質量数13の炭素原子を6個有する式(I)で表される化合物、および質量数13の炭素原子を12個有する式(I)で表される化合物、またはそれらの塩を含むものが挙げられる。好ましい組み合わせの具体例としては、上記のPyII-0、PyII-6およびPyII-12を含むものが挙げられる。
一方、標的物質がアミノ基含有非ペプチド化合物の場合、いずれの化合物間についても質量差が2以上であれば、同位体ピーク間の干渉を回避できる。そのため、該組み合わせとしては、質量差が2以上である2つ以上の式(I)で表される化合物またはそれらの塩を含むものが挙げられる。そのような組み合わせとしては例えば、質量数13の炭素原子を含まない式(I)で表される化合物、質量数13の炭素原子を2個有する式(I)で表される化合物、質量数13の炭素原子を4個有する式(I)で表される化合物、質量数13の炭素原子を6個有する式(I)で表される化合物、質量数13の炭素原子を8個有する式(I)で表される化合物、質量数13の炭素原子を10個有する式(I)で表される化合物、および質量数13の炭素原子を12個有する式(I)で表される化合物からなる群から選択される2つ以上の化合物、またはそれらの塩を含むものが挙げられる。好ましい組み合わせの具体例としては、上記のPyII-0、PyII-2、PyII-4、PyII-6、PyII-8、PyII-10およびPyII-12のうちの2つ以上を含むものが挙げられる。また、より多くの試料を一括で処理できるという観点から、上記の各例示した組み合わせには、式(I)で表される化合物またはその塩が、好ましくは3つ以上、より好ましくは4つ以上、更により好ましくは5つ以上、特により好ましくは6つ以上、最も好ましくは7つ含まれる。
本発明はまた、上述の本発明の同位体標識化合物の2つ以上を利用する、質量分析計を用いて2以上の生体試料中のアミノ基を含有する標的物質を定量分析するための方法(以下、本発明の定量分析方法ともいう)を提供する。標的物質としては、アミノ基含有非ペプチド化合物、アミノ基含有ペプチド化合物、およびタンパク質等が挙げられる。本発明の定量分析方法は、標的物質の種類によらず、本質的には同様のステップに従って実行できる。但し、タンパク質の定量分析方法(以下、本発明のタンパク質分析方法ともいう)については、制限酵素による消化、ペプチド断片からのタンパク質の同定等、特有の処理が通常為されるため、後に別途説明する。また、アミノ酸の個数が一般に2個から十数個程度までであるいわゆるペプチド化合物の定量分析については、後述するタンパク質の分析方法の説明に基づいて当業者は適宜実施し得るため、別段の説明は行わない。以下、アミノ基含有非ペプチド化合物の定量分析方法(以下、本発明の非ペプチド化合物分析方法ともいう)について説明する。
本明細書においてアミノ基含有非ペプチド化合物またはアミノ基を含有する非ペプチド化合物とは、分子内に1つ以上のアミノ基を有し、かつ、分子内にペプチド結合を有しない任意の化合物を意味し、ここでアミノ基とは、アンモニア、第一級アミン(即ち、アンモニアの水素原子を任意に置換されていてもよい任意の炭化水素基で1つ置換した化合物)または第二級アミン(即ち、アンモニアの水素原子を、それぞれ同一または異なる任意に置換されていてもよい任意の炭化水素基で、2つ置換した化合物)から水素を除去した1価の官能基をいう。従って、アミノ基含有非ペプチド化合物は、NH3、NH2R、またはNHRR’(式中、RおよびR’はそれぞれ同一または異なって、任意に置換されていてもよい任意の炭化水素基をそれぞれ示す。)の化学式を有する非ペプチド性の化合物である。但し、NHRR’の化学式を有する化合物については、本発明の方法で用いられる標識試薬とは反応しないか、もしくは極めて低反応性であると考えられる。それ故、本発明の方法の対象となるアミノ基含有非ペプチド化合物は、通常、NH2R(式中、Rは水素または任意に置換されていてもよい任意の炭化水素基を示す。)の化学式を有する非ペプチド性の化合物である。
測定の対象とするアミノ基含有非ペプチド化合物の分子量は、本発明の方法を可能とする限り特に限定されないが、一般的には低分子量の化合物である。具体的な分子量としては17~1000であり、好ましくは17~700、より好ましくは17~500である。測定の対象とするアミノ基含有非ペプチド化合物としては、例えば、生理活性アミン、アミノ酸、薬物、覚醒剤、麻薬、不揮発性腐敗アミン、および、それらの代謝物でアミノ基を保有する化合物等が挙げられる。一度の分析において、複数種類のアミノ基含有非ペプチド化合物を測定の対象とすることもできる。
内部標準試料の調製は例えば、標的非ペプチド化合物の市販品を所定の濃度(例、0.05M)のHClに溶解することによって行うことができる。また、内部標準試料中に存在する、標的非ペプチド化合物の濃度は特に制限されないが、一般には、分析に供される他の試料中での該化合物の想定される濃度と近いことが好ましい。内部標準試料は、通常、標的非ペプチド化合物を既知の濃度で含有する。それにより、試料中に存在する標的化合物の絶対量を決定することが可能となる。
LC条件は分離用カラムとしてモノリス型のMonoCap for FastFlow (0.05 mm IDx150mmL, GLサイエンス社)、トラップカラムとしてC18-Monolithトラップカラム(0.05mm IDx150mm L GLサイエンス社)を用い、流量200 nl/分、移動層としてA)ギ酸/水/アセトニトリル(0.1:98:2)、B) ギ酸/水/アセトニトリル(0.1:2:98)からなるグラジエント溶出を行う(すなわち、A/B=98/2(0分)-2/98(50分)-2/98(50.1-70分)-98/2(70.1-90分))。質量測定部設定として、イオン化モードはナノESI(正イオン化)、スプレー電圧は1600 V、検出器電圧は1950 V、カウンター窒素ガス量は0.8 L/分、スキャン範囲は100-500 m/zとし、質量分析の記録時間は50分とする。
以下、本発明のタンパク質分析方法について、各工程を説明する。
上記目的のために、内部標準試料は、分析される試料中に存在するあらゆるタンパク質を含むことが好ましい。そのために、内部標準試料は、例えば、上述のようにして調製された分析される全てのタンパク質含有試料を混合することにより調製されたものであり得る。その場合、上述のようにして調製された各試料から総タンパク質含有量が等しい出発試料をそれぞれ調製した後、該出発試料を混合(例、等量混合)することにより内部標準試料を調製することも、その調製法の例として挙げることができる。
内部標準試料を用いるタンパク質の定量分析方法については、上記特許文献1にも説明されている。
(a)該混合物を1次元ゲル分離、2次元ゲル分離または適当なクロマトメディアで大まかに分離する等したタンパク質をタンパク質分解酵素で加水分解し、ペプチドを遊離する;
(b)(a)のように前以て含有タンパク質を相互分離のためにゲル分離やクロマト展開することなく、該混合物を直接タンパク質分解酵素で分解する、
のいずれかに従う。タンパク質の分解は自体公知の手順に従って行うことができる。一次選択のトリプシン以外に、ArgペプチダーゼやGluペプチダーゼ等を二次選択として用いることができるが、Lysエンドペプチダーゼは用いない。
(a)の操作により分離したタンパク質から遊離した標識ペプチドおよび非標識ペプチドについては、ペプチドの分離操作無しで直接MALDI-TOF/MSにより質量分析する場合もある。また、液体クロマトグラフによりペプチドを分離後、ESI/MS/MS分析することもできる。
(b)の操作により遊離したペプチドは、2次元液体クロマトグラフシステムによって、例えば、1次元分離をSCXカラムで行い、溶出した成分を第2の逆相樹脂カラムにより分離し、ESI/MS/MSに導入することで、標識ペプチドの相対強度とそのアミノ酸配列情報を一度の分析で獲得する。
ここで、次工程のMSスペクトルの測定に用いるペプチドの分子量は特に限定されないが、例えば500~3000であり、好ましくは1000~2500である。従って、本工程は、好ましくは、タンパク質の消化産物から、上記範囲の分子量を有するペプチドを単離することを含む。
なお、工程(2)における標識において互いに質量差6以上の標識化合物を用いた場合には通常必要でないが、そうでない場合は、ピーク強度の対比において、例えば特開2005-181011に教示されているようにして、天然に存在する同位体元素に起因するペプチドの同位体ピークとの重なりを除去して量比の補正をする点に留意する。
ここで該ペプチドについて工程(5)で得られた存在比は、該ペプチドが由来するタンパク質の前記混合物中での存在比に等しいため、該存在比と、工程(3)における試料の混合比とから、混合物の調製に供された試料間での該タンパク質の量比を決定することができる。
化合物の同定および重水素化率の算出はNMR又はMSを測定することで行った。
(2-1)装置
・NMR分析
日本電子株式会社製 フーリエ変換核磁気共鳴(NMR)装置
JNM-ECS400
・化学純度分析
GLサイエンス株式会社製 高速液体クロマトグラフィー(HPLC)装置
GL-7400シリーズ
・EI-MS分析
日本電子株式会社製 電子衝撃質量分析(EI-MS)装置
JMS-AX505W
(2-2)同定
・GC-MS測定による化合物の同定
重水素化されていない試料と13C標識された試薬についてGC-MSを測定して、13C標識に伴う分子量の変化を支持するデータが得られていることを確認することで行った。
・NMR測定による化合物の同定
D2Oを用いて試料を溶解し、観測されたピーク情報より確認を行った。13C標識された炭素に隣接するプロトンは大きくカップリングすることにより、13C標識部位の確認ができる。
(2-3)13C濃縮率の算出方法
重水素化されていない試料と13C標識された試薬について、同条件でGC-MSを測定して、得られたフラグメントのピーク強度比より算出した。
(2-4)化学純度分析
HPLC分析を行い、得られたピーク面積比より化学純度を算出した。
(3)試薬
東京化成工業株式会社製:一般試薬
大陽日酸株式会社製またはIsotec製:13C標識原料
化学純度:99.6%
1H-NMR(270MHz、D2O):δ:1.06(3H、t、J=7.7Hz)、1.27(6H、t、J=7.4Hz)、2.30(6H、s)、2.85(2H、q、J=7.7Hz)、3.07(4H、q、J=7.5Hz)
EI-MS:m/z for C13H21O+(M-CF3SO3)calculated 193.16、found 193.2
化学純度:99.6%
13C濃縮度:99.3atom%13C
1H-NMR(270MHz、D2O):δ:1.07(3H、t、J=7.7Hz)、1.28(6H、td、J=7.4Hz、5.1Hz)、2.31(6H、d、J=5.3Hz)、2.81(2H、q、J=7.7Hz)、3.08(4H、dt、J=14.2Hz、6.3Hz)
EI-MS:m/z for 13C2C11H21O+(M-CF3SO3)calculated 195.29、found 195.2
化学純度:99.9%
13C濃縮度:99.1atom%13C
1H-NMR(270MHz、D2O):δ:1.06(3H、ddd、J=127.8Hz、)、1.28(6H、t、J=7.3Hz)、2.31(6H、dd、J=129.8Hz、3.7Hz)、2.86(2H、dt、J=19.9Hz、6.8Hz)、3.08(4H、q、J=7.4Hz)
EI-MS:m/z for 13C4C9H21O+(M-CF3SO3)calculated 197.28、found 197.2
化学純度:98.8%
13C濃縮度:99.5atom%13C
1H-NMR(270MHz、D2O):δ:1.06(3H、ddt、J=128.5Hz、12.8Hz、4.7Hz)、1.27(6H,t,J=7.4Hz)、2.30(6H、dd、129.6Hz、5.9Hz)、2.85(2H、dm、J=129.6Hz)、3.07(4H、ddd、J=14.8Hz、7.3Hz、2.2Hz)
EI-MS:m/z for 13C6C7H21O+(M-CF3SO3)calculated 199.26、found 199.2
化学純度:99.8%
13C濃縮度:98.4atom%13C
1H-NMR(270MHz、D2O):δ:1.06(3H、ddd、J=127.8Hz、11.4Hz,7.3Hz)、1.27(6H,dd,J=12.2Hz、7.1Hz)、2.30(6H、dt、J=129.9Hz、5.1Hz)、2.85(2H、dm、J=133.4Hz)、3.07(4H、t、J=6.3Hz)
EI-MS:m/z for 13C8C5H21O+(M-CF3SO3)calculated 201.25、found 201.2
化学純度:97.7%
13C濃縮度:98.6atom%13C
1H-NMR(270MHz、D2O):δ:1.06(3H、ddd、J=127.8Hz、11.4Hz,7.3Hz)、1.27(6H、ddt、J=129.9Hz、8.9Hz、3.3Hz)、2.30(6H、dt、J=130.1Hz、4.1Hz)、2.85(2H、dt、J=133.0Hz、7.1Hz)、3.07(4H、dm、J=131.0Hz)
EI-MS:m/z for 13C10C3H21O+(M-CF3SO3)calculated 203.23、found 203.2
化学純度:99.3%
13C濃縮度:99.5atom%13C
1H-NMR(270MHz、D2O):δ:1.06(3H、dtd、J=129.7Hz、7.6Hz、4.2Hz)、1.26(6H,dtt,J=129.5Hz、8.0Hz、3.4Hz)、2.29(6H、dt、J=129.8Hz、5.5Hz)、2.84(2H、dm、J=132.2Hz)、3.06(4H、dm、J=130.9Hz)
EI-MS:m/z for 13C12C1H21O+(M-CF3SO3)calculated 205.22、found 205.2
モデルタンパク質であるウシ血清アルブミン(BSA)およびヒト血清トランスフェリン(Tfn)をそれぞれPyII-0、PyII-6、PyII-12で標識し、全ての試料を下記の混合比で混合した。
SCX分画の条件は下記の通りである:
カラム : Poly Sulfoethyl A 4.6x100mm
溶離液 : A=10mM KH2PO4(pH2.8) 25%ACN
B=10mM KH2PO4(pH2.8) 25%ACN 1M KCl
システム : HPLC ELITE Lachrom(日立)
サンプルループ : 2mL、流速: 1mL/min
Detect : 220nm 、8min~32minまで1min/Tube採取
ヒト血漿タンパク質(健常者および動脈硬化患者)をPyII試薬で標識した。健常者由来のタンパク質をPyII-0およびPyII-12で、動脈硬化患者由来のタンパク質をPyII-6で標識し、図6に示すプロトコルに従って分析した。また、上記検体についての分析結果を、既に実施済みのcICAT解析結果と比較してデータの妥当性を評価した。
以上から、Lys残基へのPyII導入率は高いことが確認された。
この実験にはヒト由来細胞株のHeLa細胞を1試料あたり106個用い、沈殿した細胞塊に100μLのCLSを加えて溶解し氷上に30分置いた後で15,000回転で20分間遠心して上清に全細胞タンパク質を回収した。これを2群準備し、両者のタンパク質量を等しくし、一方には緑色の蛍光色素のCy3を、もう一方には赤色のCy5を全タンパク質について平均2%位結合するように蛍光量を調節して結合させた。どのタンパク質のリジン残基も平均して2%程度が蛍光色素で標識されたことになる。両者を等量混合して2D-DIGE法で展開し、蛍光色素の分布をレーザー蛍光スキャナーで検出すると、多くの蛍光スポットが透明ゲル板上に検出された。Cy3とCy5は異なった蛍光特性をもつので、Cy3の蛍光とCy5蛍光を区別して見ることができた。両者の蛍光を重ねることも出来た。同じタンパク質をCy3とCy5で蛍光標識し混合して2次元電気泳動分離すると同じ位置にスポットを作り、蛍光が重ね合わさったスポットは混合色である黄色になった(図8の左図の右下の四角内のスポット)。Cy5で標識した方には更にPyII試薬による標識を行なった。即ち、CLS(pH9.6)に溶解したCy5標識タンパク質に対して10mMPyIIにより50℃で3時間標識した。PyII標識タンパク質に等量のPyII標識しないCy3標識タンパク質を混合し2次元電気泳動分離し、蛍光スキャナーで2色蛍光を検出した。両蛍光を重ね合わせた結果が図8の右図である。タンパク質の緑蛍光スポットはゲル一面に分布していた。各スポット近傍の分子量増大方向(上方向)に赤蛍光スポットの移動が認められる。しかも弱い蛍光(タンパク質の含量が低い)スポットでも近傍に赤蛍光が見られることから、PyII標識により分子量が増大したことを反映する結果と見られる。
以上から、PyII標識は、現在の標識法では細胞内蛋白質の多くを標識できる能力を持っていることが示された。
次に、官能基にフェニルホウ酸(PBA)を持つカラム(MonoSpin PBA、GLサイエンス)を用いて試料中のドーパミン-PyII化合物の精製を行った。試料をPBAに結合させ、アセトニトリル、次に100 mM リン酸K緩衝液pH 8.0、さらに0.5 %トリフルオロ酢酸含有5 %アセトニトリル溶液で洗浄してから、0.5 %トリフルオロ酢酸含有30 %アセトニトリル溶液で溶出した。溶出液を減圧遠心濃縮機により濃縮し、孔径0.22 μmのフィルター(DURAPORE PVDF 0.22μm、MILLIPORE)で濾過し、ナノLC/MSシステムにより分析を行った。
その結果を図9に示す。質量326.1から338.2に質量差約2で7種の化合物が生成しているのが確認された。
得られた結果をドーパミンとDHBAの比を縦軸として作図すると直線関係が得られた(図10)。従って、DHBAを内部標準としてPyIIによりドーパミンが定量できることが判明した。
LC条件は分離用カラムとしてモノリス型のMonoCap for FastFlow (0.05 mm IDx150mmL,GLサイエンス社)、トラップカラムとしてC18-Monolithトラップカラム(0.05mm IDx150mm L, GLサイエンス社)を用い、流量200 nl/分、移動層としてA)ギ酸/水/アセトニトリル(0.1:98:2)、B) ギ酸/水/アセトニトリル(0.1:2:98)からなるグラジエント溶出を行った(すなわち、A/B=98/2(0分)-2/98(50分)-2/98(50.1-70分)-98/2(70.1-90分))。
質量測定部設定は以下の通りである。イオン化モードはナノESI(正イオン化)、スプレー電圧は1600 V、検出器電圧は1950 V、カウンター窒素ガス量は0.8 L/分、スキャン範囲は100-500 m/zとし、質量分析の記録時間は50分とした。
今回測定した各々のPyII誘導体について、質量M/z値の示すイオンクロマトグラフを図12に、図12のピーク部分の質量スペクトルを図13に示している。測定した8種それぞれについて、単独のクロマトピークおよび理論値と一致した質量ピークが同定された。
本発明の同位体標識化合物はまた、複数試料(例、7種)中に含有されるアミノ基含有非ペプチド化合物を一斉に高感度で分析することを可能とする。更に、トラップカラムを用いた自動化により、フェムトモルレベルの高感度分析を24時間自動で行うことができる体制を構築し得る。
Claims (23)
- 式(I)において質量数13の炭素を1つ以上有する、請求項1記載の化合物またはその塩。
- 質量差が6以上である2つ以上の式(I)で表される化合物またはそれらの塩を含み、かつ標的物質がタンパク質である、請求項4記載のキット。
- 質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
またはそれらの塩を含む、請求項5記載のキット。 - 質量差が2以上である2つ以上の式(I)で表される化合物またはそれらの塩を含み、かつ標的物質がアミノ基含有非ペプチド化合物である、請求項4記載のキット。
- 質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を2個有する式(I)で表される化合物、
質量数13の炭素原子を4個有する式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、
質量数13の炭素原子を8個有する式(I)で表される化合物、
質量数13の炭素原子を10個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
からなる群から選択される2つ以上の化合物またはそれらの塩を含む、請求項8記載のキット。 - 質量分析計を用いて2以上の生体試料中のアミノ基を含有する標的物質を定量分析するための方法であって、
(1)分析に供する2以上の生体試料を調製する工程、
(2)試料間で標的物質に質量差を与えるように、同位体標識により互いに異なる質量を有する2つ以上の式(I):
で表される化合物またはそれらの塩を標識化合物として用いて調製した試料中の標的物質を標識する工程、
(3)標識に供された全ての試料から混合物を調製する工程、
(4)混合物を質量分析に供し、該標識により互いに質量差を有する標的物質について、質量スペクトルにおけるそれらのピーク強度の比に基づいて、該混合物中でのそれらの標的物質の存在比を求め、得られた存在比と工程(3)における試料の混合比とから、混合物の調製に供された試料間での該標的物質の量比を決定する工程
を含む、方法。 - 標的物質が、アミノ基を含有する非ペプチド化合物である、請求項11記載の方法。
- 標識化合物が、質量差が2以上である2つ以上の式(I)で表される化合物またはそれらの塩を含む、請求項12記載の方法。
- 標識化合物が、
質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を2個有する式(I)で表される化合物、
質量数13の炭素原子を4個有する式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、
質量数13の炭素原子を8個有する式(I)で表される化合物、
質量数13の炭素原子を10個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
からなる群から選択される2つ以上の化合物またはそれらの塩を含む、請求項13記載の方法。 - 工程(1)において調製された試料の一つが、既知濃度の標的物質を含有する内部標準試料であり、かつ、
工程(4)における存在比の決定が、内部標準試料以外の各試料由来の標的物質と、内部標準試料由来の標的物質とのピーク強度の比を求めることを含む、
請求項11記載の方法。 - 質量分析計を用いて2以上の生体試料中のタンパク質を定量分析するための方法であって、
(1)分析に供する2以上の生体試料を調製する工程、
(2)試料間で同一タンパク質に質量差を与えるように、同位体標識により互いに異なる質量を有する2つ以上の式(I):
で表される化合物またはそれらの塩を標識化合物として用いて調製した試料中のタンパク質を標識する工程、
(3)標識に供された全ての試料から混合物を調製する工程、
(4)混合物中のタンパク質を制限酵素により消化してペプチドを得る工程、
(5)得られたペプチドを質量分析に供し、該標識により互いに質量差を有するペプチドについて、質量スペクトルにおけるそれらのピーク強度の比に基づいて、該混合物中でのそれらのペプチドの存在比を決定する工程、および、
(6)存在比を決定されたペプチドについて、それが由来するタンパク質を特定し、該存在比および工程(3)における試料の混合比から、混合物の調製に供された試料間での該タンパク質の量比を決定する工程
を含む、方法。 - 標識化合物が、質量差が6以上である2つ以上の式(I)で表される化合物またはそれらの塩を含む、請求項17記載の方法。
- 標識化合物が、
質量数13の炭素原子を含まない式(I)で表される化合物、
質量数13の炭素原子を6個有する式(I)で表される化合物、および
質量数13の炭素原子を12個有する式(I)で表される化合物
またはそれらの塩を含み、3つの生体試料中のタンパク質を定量分析する、請求項18記載の方法。 - 工程(1)において調製された試料の一つが、他の全ての調製された試料を混合して得られた内部標準試料であり、かつ、
工程(5)における存在比の決定が、内部標準試料以外の各試料由来のペプチドと、内部標準試料由来のペプチドとのピーク強度の比を求めることを含む、
請求項17記載の方法。
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JP2015184253A (ja) * | 2014-03-26 | 2015-10-22 | 株式会社島津製作所 | 化合物分析方法、化合物分析装置、及び化合物分析用プログラム |
JP2021516350A (ja) * | 2018-03-07 | 2021-07-01 | アンドレン パー | 脱離又はレーザー・アブレーション・イオン化の為の反応性マトリックスとその使用 |
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CN111024874A (zh) * | 2019-12-30 | 2020-04-17 | 上海百趣生物医学科技有限公司 | 液相色谱质谱联用法定量检测儿茶酚胺及其代谢物的方法 |
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JP2015068762A (ja) * | 2013-09-30 | 2015-04-13 | 大陽日酸株式会社 | タンパク質又はペプチドの解析方法、及び、ピリリウム化ペプチド精製濃縮キット |
JP2015184253A (ja) * | 2014-03-26 | 2015-10-22 | 株式会社島津製作所 | 化合物分析方法、化合物分析装置、及び化合物分析用プログラム |
JP2021516350A (ja) * | 2018-03-07 | 2021-07-01 | アンドレン パー | 脱離又はレーザー・アブレーション・イオン化の為の反応性マトリックスとその使用 |
JP7360135B2 (ja) | 2018-03-07 | 2023-10-12 | アンドレン パー | 質量分析において試料のイオン化に用いられるマトリックスとしての使用、および、質量分析において試料のイオン化に用いられるマトリックス |
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