JP2019211217A - Nucleotide quantification method - Google Patents

Abstract

To provide a method for simultaneously quantifying two or more nucleotides without using an ion pair reagent.SOLUTION: A quantification method of two or more nucleotides in a sample using liquid-chromatography mass-spectrometry includes: a step a of extracting a mixture of nucleotides from the sample; a step b of adding two or more nucleotides labeled with an isotope as an internal standard to the nucleotide mixture, and preparing a measurement sample; a step c of separating each nucleotide from the measurement sample by liquid chromatography using a normal phase column; a step d of identifying each separated nucleotide by mass spectrometry; and a step e of quantifying each nucleotide by determining the ratio of the chromatography peak area of each identified nucleotide to the chromatography peak area of the internal standard.SELECTED DRAWING: None

Description

  The present invention relates to a method for quantifying nucleotides.

  Nucleoside triphosphates are the basic biological processes of cells, including DNA and RNA constituents, phosphate donors, energy carriers and coenzymes for enzymatic reactions, and also involved in cell signaling. And plays an important role (Non-Patent Documents 1 and 2). In order to keep the quantitative balance of these cells strictly, clever control such as negative feedback of nucleotide biosynthesis (Non-patent Document 1) and selective production of deoxyribonucleotides by ribonucleotide reductase (Non-patent Document 3) Has a system. Abnormal quantitative balance of nucleoside triphosphates includes gene transcription inhibition (Non-patent document 4), protein translation inhibition (Non-patent document 5), and DNA mutagenesis that can lead to carcinogenesis (Non-patent documents 6 and 7). It has a significant effect on cells. In fact, it has been clarified that the cause of several genetic diseases such as Lesch-Nyhan syndrome, mitochondral depletion syndrome, and mitochondral neurogastrintestinal encephalopathy is due to a defect in a gene involved in nucleotide synthesis (Non-patent Document 8). On the other hand, nucleoside triphosphate imbalance is also an attractive target for drug discovery. Antimetabolites typified by 5-fluorouracil and hydroxyurea kill cancer cells by inhibiting nucleotide synthesis, and this type of anticancer drug has been vigorously developed in recent years. Patent Document 9). Furthermore, various nucleosides and nucleotide analogues (such as 5-fluorouracil) are being developed as anticancer agents and antiviral agents, but these action targets themselves are abnormal nucleoside triphosphate balances, As a side effect, abnormal balance may be caused (Non-patent Document 10).

  A method for the simultaneous determination of intracellular nucleoside triphosphates by liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been reported. For example, in Non-Patent Document 11, simultaneous quantification of GTP, CTP, UTP, dATP, dCTP, and dTTP is performed. In Non-Patent Documents 12 and 13, eight types of nucleoside triphosphates including ATP and dGTP are simultaneously quantified.

  On the other hand, Patent Document 1 describes a method of measuring a protein by mass spectrometry by adding a metabolically isotope-labeled biomolecule as an internal standard substance.

Japanese Patent No. 4714584

Lane AN, Fan TW (2015) Nucleic Acids Res 43 (4): 2466-2485 Spiegel AM (1988) Vitam Horm 44: 47-101 Torrents E (2014) Front Cell Infect Microbiol 4:52 Papadopoulou C, Guilbaud G, Schiavone D, Sale JE (2015) Cell Rep 13 (11): 2491-2503 Iglesias-Gato D, Martin-Marcos P, Santos MA, Hinnebusch AG, Tamame M (2011) Genetics 187 (1): 105-122 Song S, Wheeler LJ, Mathews CK (2003) J Biol Chem 278 (45): 43893-43896 Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach DS, Kerem B (2011) Cell 145 (3): 435-446 Fasullo M, Endres L (2015) Int J Mol Sci 16 (5): 9431-9449 Murase M, Iwamura H, Komatsu K, Saito M, Maekawa T, Nakamura T, Yokokawa T, Shimada Y (2016) Pharmacol Res Perspect 4 (1): e00206 Jordheim LP, Durantel D, Zoulim F, Dumontet C (2013) Nat Rev Drug Discov 12 (6): 447-464 Chen P, Liu Z, Liu S, Xie Z, Aimiuwu J, Pang J, Klisovic R, Blum W, Grever MR, Marcucci G, Chan KK (2009) Pharm Res 26 (6): 1504-1515 Zhang W, Tan S, Paintsil E, Dutschman GE, Gullen EA, Chu E, Cheng YC (2011) Biochem Pharmacol 82 (4): 411-417 Kamceva T, Bjanes T, Svardal A, Riedel B, Schjott J, Eide T (2015) J Chromatogr B Analyt Technol Biomed Life Sci 1001: 212-220

  In Non-Patent Document 11, GTP, CTP, UTP, dATP, dCTP, and dTTP are simultaneously quantified, but ATP and dGTP are not separated in either HPLC or MS. Even when Non-Patent Documents 12 and 13 are included, these simultaneous quantification methods have the following problems. The first problem is that a cell matrix is used to correct the matrix effect of the cell extract or that a standard addition method is used. When a cell matrix is used, it is necessary to remove nucleotides in the cell extract, which is troublesome for the preparation of a standard curve sample. In addition, the removal of nucleotides may cause artifacts in the matrix. In addition, since the matrix varies depending on the cell line, a cell matrix must be prepared for each cell line to be analyzed. When using the standard addition method, a calibration curve must be prepared for each sample, which is an obstacle to analysis throughput. The second problem is that the analysis takes too much time, specifically, it takes 40 to 100 minutes or more to analyze one sample. The third problem is that the ion pair reagent used in the methods of Non-Patent Documents 11 to 13 tends to remain in the LC (Liquid Chromatography) line, so that it is necessary for the analysis of the measurement object that does not require it. There is a problem that adverse effects such as suppression, retention time, and change in peak shape are adversely affected.

  In Patent Document 1, there is no example in which biomolecules other than proteins are measured, and there is no specific description about simultaneous quantification of a plurality of nucleotides.

  In testing, treatment, and drug discovery, it is desired to develop a simultaneous quantitative method with high accuracy of intracellular nucleotides. An object of the present invention is to provide a method for simultaneously quantifying two or more nucleotides without using an ion pair reagent.

  As a result of intensive studies to solve the above problems, the present inventors have identified a step of separating each nucleotide from a measurement sample by liquid chromatography using a normal phase column, and identifying each separated nucleotide by mass spectrometry. It was found that two or more kinds of nucleotides can be quantified at the same time without using an ion pair reagent depending on the process. The present invention has been completed based on the above findings.

That is, according to the present invention, the following inventions are provided.
<1> A method for quantifying two or more nucleotides in a sample by liquid chromatography-mass spectrometry,
Extracting a nucleotide mixture from the sample, a.
Adding the two or more nucleotides labeled with an isotope to the nucleotide mixture as an internal standard substance to prepare a measurement sample; and b
A step c of separating each nucleotide from the measurement sample by liquid chromatography using a normal phase column;
Identifying each separated nucleotide by mass spectrometry, step d;
Determining the ratio of the chromatogram peak area of each identified nucleotide and the chromatogram peak area of the internal standard substance to quantify each nucleotide; e.
Including methods.

<2> a step of measuring the number of cells contained in the sample;
Calculating the number of nucleotide molecules per cell from nucleotide quantification results;
The method according to <1>, further comprising:
<3> The method according to <1> or <2>, wherein the sample is selected from the group consisting of a tissue, a cell, and a biological fluid.
<4> The method according to any one of <1> to <3>, wherein the nucleotide is nucleoside triphosphate or deoxynucleoside triphosphate.
<5> The method according to any one of <1> to <4>, wherein the isotope is selected from the group consisting of ² H, ¹³ C, ¹⁵ N, ¹⁷ O, and ¹⁸ O.

<6> The method according to any one of <1> to <5>, wherein the liquid chromatography is hydrophilic interaction type chromatography.
<7> The method according to any one of <1> to <6>, wherein the liquid chromatography is hydrophilic interaction type chromatography using a separation column having a stationary phase modified with a sulfobetaine group.
<8> The method according to any one of <1> to <7>, wherein the material of the inner wall of the normal phase column is polyetheretherketone.

<9> The method according to any one of <1> to <8>, wherein in the step d identified by mass spectrometry, multiple reaction monitoring is performed in a positive ion mode.
<10> The method according to any one of <1> to <9>, wherein in step e, a calibration curve for each nucleotide is prepared in advance, and each nucleotide is quantified based on the prepared calibration curve.
<11> In step e, a sample containing a standard substance for each nucleotide to be analyzed and a sample containing each nucleotide labeled with an isotope as an internal standard substance is used as a calibration curve preparation sample. <1> to <10> The method as described in any one of.

<12> The method according to any one of <1> to <11>, wherein, in the step e, a sample for preparing a calibration curve is prepared using a solution containing a volatile ammonium salt and acetonitrile.
<13> The liquid chromatography-mass spectrometry is an analysis method using a liquid chromatograph-tandem mass spectrometer called LC-MS / MS, and any one of <1> to <12> the method of.
<14> The method according to any one of <1> to <13>, wherein the mobile phase used in liquid chromatography comprises any one selected from the group consisting of ammonium bicarbonate, ammonium carbonate, and ammonium acetate.

<15> The method according to any one of <1> to <14>, wherein the mobile phase used for liquid chromatography has a pH of 8 to 11.
<16> The method according to any one of <1> to <15>, wherein the salt concentration of the mobile phase used for liquid chromatography is 10 to 20 mmol / L.
<17> The method according to any one of <1> to <16>, wherein an absolute amount of each nucleotide can be measured.

  According to the method of the present invention, two or more kinds of nucleotides can be simultaneously quantified without using an ion pair reagent.

FIG. 1 is an MS / MS chromatogram of a substance to be measured in an extract from Molm-13 cells and its internal standard (IS). Peaks indicating the test substance or IS are indicated by arrows. FIG. 2 is a graph showing the inhibition of cell proliferation of Molm-13 cells treated with 200 μmol / L or 2000 μmol / L thymidine for 24 hours. Mean ± SD (Standard Deviation), n = 3, *** p <0.001 vs. vehicle (t test). FIG. 3 is a graph showing changes in dNTP and NTP levels in Molm-13 cells treated with 200 μmol / L or 2000 μmol / L thymidine for 24 hours. Mean ± SD, n = 3, * p <0.05, ** p <0.01, *** p <0.001 vs. vehicle (t test). FIG. 4 is a diagram showing a presumed mechanism of changes in dNTP levels in Molm-13 cells induced by treatment with 2000 μmol / L thymidine for 24 hours. RNR, ribonucleotide reductase; dT, thymidine. The width of the arrow indicating “increase” reflects the amount of change in dNTP induced by thymidine treatment. FIG. 5 is a flowchart showing an example of a protocol for calculating the number of nucleotides per cell.

Hereinafter, the present invention will be described in detail.
In the present specification, “to” indicates a range including numerical values described before and after that as a minimum value and a maximum value, respectively.
The nucleotide quantification method of the present invention includes the following steps and is a method of quantifying two or more nucleotides in a sample by liquid chromatography-mass spectrometry.
Step a: Extracting a nucleotide mixture from a sample Step b: Adding two or more kinds of nucleotides labeled with isotopes as an internal standard to the nucleotide mixture to prepare a measurement sample c: Using a normal phase column Step d: Separating each nucleotide from the measurement sample by liquid chromatography, d: Identifying each separated nucleotide by mass spectrometry e: Chromatogram peak area of each identified nucleotide and chromatogram peak area of the internal standard substance Each nucleotide is quantified.

<Explanation of terms>
(Quantitative)
In the present invention, “quantification” is a concept including absolute quantification and relative quantification. In the present invention, “absolute quantification” is a quantification method for the purpose of obtaining a measurement result as an amount or concentration, and “relative quantification” refers to a quantification method other than absolute quantification.

(Internal standard substance)
In the present invention, “internal standard substance” refers to a substance to which a certain amount can be added to a sample when the substance to be measured is quantified by mass spectrometry. In the present invention, it is preferable to add a certain amount of the internal standard substance in the sample in order to correct the variation in the quantitative value due to the error in the experimental operation after extraction of the nucleotide mixture and the influence of the matrix in the sample.

(sample)
The sample in this invention is not specifically limited, Arbitrary samples can be used if it is a sample containing a nucleotide. As the sample, tissue, biological fluid, cells, or a sample obtained by processing them can be used, but it is not particularly limited. The origin of the sample is not particularly limited, and for example, samples derived from mammals such as humans and mice can be used.

  Examples of tissues include the brain, each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, hypothalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, Liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, duodenum, small intestine, large intestine, blood vessel, heart, thymus, spleen, submandibular gland, parotid gland, sublingual gland, peripheral blood, prostate, testicle Ovary, placenta, uterus, bone, joint, skeletal muscle, etc.

  Examples of the biological fluid include blood (including plasma and serum), urine, feces, saliva, tear fluid, infiltrating fluid (including ascites and tissue fluid), and the like.

  The cell may be a cell collected from a living body or a cultured cell established. The type of cell is not particularly limited, and any cell such as a normal cell and a cancer cell can be used.

(nucleotide)
Examples of nucleotides to be measured include the following 27 types. In the present invention, nucleotides do not include DNA or RNA.

Nucleoside triphosphates: 9 types ATP (adenosine triphosphate), GTP (guanosine triphosphate),
CTP (cytidine triphosphate), UTP (uridine triphosphate),
dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate),
dCTP (deoxycytidine triphosphate), dTTP (thymidine triphosphate), dUTP (deoxyuridine triphosphate)

Nucleoside diphosphates; 9 species ADP (adenosine diphosphate), GDP (guanosine diphosphate),
CDP (cytidine diphosphate), UDP (uridine diphosphate),
dADP (deoxyadenosine diphosphate), dGDP (deoxyguanosine diphosphate),
dCDP (deoxycytidine diphosphate), dTDP (thymidine diphosphate), dUDP (deoxyuridine diphosphate)

Nucleoside monophosphate; 9 species AMP (adenosine monophosphate), GMP (guanosine monophosphate),
CMP (cytidine monophosphate), UMP (uridine monophosphate),
dAMP (deoxyadenosine monophosphate), dGMP (deoxyguanosine monophosphate),
dCMP (deoxycytidine monophosphate), dTMP (thymidine monophosphate), dUTP (deoxyuridine monophosphate)

  In the present invention, simultaneous quantification of 2 or more and 27 or less nucleotides can be performed. The lower limit of the types of nucleotides to be simultaneously quantified is not particularly limited as long as it is 2 or more, but 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more Good. The upper limit of the types of nucleotides to be simultaneously quantified is not particularly limited as long as it is 27 or less, but may be 20 or less, 18 or less, 16 or less, 14 or less, 12 or less, or 10 or less.

Specific examples of the present invention include simultaneous determination of nine types of the above-described nucleoside triphosphates, simultaneous determination of nine types of the above-described nucleoside diphosphates, and simultaneous determination of nine types of the above-described nucleoside monophosphates. Quantification can be mentioned.
It is particularly preferable to simultaneously quantify the above-described eight types of nucleoside triphosphates, excluding deoxyuridine triphosphate, which is a direct substrate for DNA and RNA synthesis.

<Step a: Extraction of nucleotide>
In this step, a nucleotide mixture is extracted from the sample. As an extraction method, a method conventionally used for extracting this type of substance can be appropriately employed. For example, a method for crushing and extracting a sample includes, but is not limited to, an ultrasonic crushing device, a method using a cell lysate, or a freeze-thaw method.

  An example of nucleotide extraction is described below. First, the cells are preferably pelleted by centrifugation and the supernatant is removed. Further, the pellet is preferably resuspended with PBS (phosphate buffered saline), and this operation is preferably repeated several times. Thereafter, the supernatant is removed and alcohol (for example, methanol) is added to the precipitated sample and stirred. Thereby, a metabolite can be extracted from the sample. It is preferable to measure the number of cells by separating a part of the suspension that is appropriately resuspended immediately before, during, or at the end of this extraction operation. This number of cells can be measured in order to calculate the number of nucleotide molecules per cell in step f (FIG. 5).

<Step b: Addition of internal labeling substance>
In this step, two or more types of nucleotides labeled with isotopes are added as internal standard substances to the mixture of nucleotides (metabolites) to prepare a measurement sample. As the nucleotide isotopes, those usually used for this kind of analysis are preferably employed as appropriate. Stable isotopes include ² H, ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, and the like. As the substance, it is preferable to add an isotope corresponding to the nucleotide expected to be contained in the metabolite. For example, [ ¹⁵ N ₅ , ¹³ C ₁₀ ] ATP for ATP, [ ¹⁵ N ₅ , ¹³ C ₁₀ ] GTP for GTP, and [ ¹⁵ N ₃ , ¹³ C ₉ ] CTP for CTP, For _{^{UTP [15 N 2, 13 C}} 9] UTP, for the _{^{dATP [15 N 5, 13 C}} 10] dATP, against _{^{dGTP [15 N 5, 13 C}} 10] dGTP, the dCTP On the other hand, [ ¹⁵ N ₃ , ¹³ C ₉ ] dCTP and [ ¹⁵ N ₂ , ¹³ C ₁₀ ] dTTP are mentioned for dTTP.

  The sample is then preferably vortexed vigorously with the addition of water. It is preferable to centrifuge, collect the supernatant, and evaporate to dryness.

  The amount of the internal standard substance added is not particularly limited, but is preferably 1 pmol or more and 100 nmol or less, more preferably 10 pmol or more and 10 nmol or less, and further preferably 100 pmol or more and 1 nmol or less.

  The sample obtained above can be separated by liquid chromatography (LC) described later.

<Step c: Separation by liquid chromatography>
Liquid chromatography (LC) can be appropriately selected and used by those skilled in the art, but is preferably HPLC (High Performance Liquid Chromatography). The HPLC apparatus includes a separation column, a sample introduction unit, and a pump that sends a mobile phase to the separation column. The HPLC apparatus may include other elements such as an autosampler, a heater, a detector for detecting separated components, a degassing apparatus, a column oven, a data processing apparatus, and the like. Examples of the detector include a UV detector, a fluorescence detector, a suggestive refractive index detector, an evaporative light scattering detector, a charged particle detector, an electrochemical detector, and an electrical conductivity detector. For example, the detector can be connected between a column and an ion source (ionization section).

  In the present invention, the separation mode of liquid chromatography (LC) employs normal phase liquid chromatography (NPLC) using a normal phase column, among which HILIC (hydrophilic interaction chromatography). : Hydrophilic Interaction Chromatography).

  In NPLC, the order of elution is usually the order of low polarity compounds and high polarity compounds. HILIC is included in NPLC. In particular, referring to the unique features of HILIC, HILIC is a retention mode based on a combination of a highly polar stationary phase and a hydrophobic mobile phase, which is mostly an organic solvent, and is suitable for separation of highly polar compounds. Furthermore, in HILIC, a highly polar stationary phase is used, and the higher the polarity, the stronger the retention, and various interactions such as electrostatic interaction and hydrophilic partition are involved.

  In the present invention, the material of the inner wall (wetted part) of the normal phase column is preferably a metal (for example, stainless steel), polyetheretherketone (peak: PEEK), and particularly preferably polyetheretherketone. In the case of a metal column, a column in which the unintentional adsorption of nucleotides into the column is suppressed by irreversible adsorption to the inner wall of the matrix is preferable.

  As the mobile phase used for liquid chromatography (LC), mobile phase A in an aqueous solution, which is preferably basic, and mobile phase B containing an organic solvent can be used.

  The pH value of the mobile phase A is preferably 7 to 11, more preferably 8 to 11, further preferably 8.4 to 10.4, and particularly preferably 9.0 to 9.8. . The salt concentration of the mobile phase A is preferably 1 to 300 mmol / L, more preferably 5 to 200 mmol / L. In the measurement by LC-MS, the salt concentration is usually lowered (for example, 5 to 20 mmol / L, preferably 10 to 20 mmol / L) in order to obtain high sensitivity. Examples of the compound species used as the mobile phase A used in LC in the present invention include ammonium acetate, ammonium formate, phosphoric acid, ammonia, ammonium bicarbonate, ammonium carbonate, etc. Among them, volatile ammonium salts are preferable, Particularly preferred are ammonium bicarbonate, ammonium carbonate or ammonium acetate.

  The mobile phase A may contain an alkali. Examples of the alkali include 28% by mass ammonia water. The concentration of the alkali may be adjusted as appropriate. For example, 28% by mass of ammonia water is preferably 0 to 0.5% by volume, and more preferably 0 to 0.2% by volume.

  Examples of the organic solvent used for the mobile phase B include acetonitrile, methanol, 2-propanol, hexane, ethyl acetate, tetrahydrofuran, ethanol and the like, preferably acetonitrile or methanol, and more preferably acetonitrile.

  When acetonitrile is used as the mobile phase B, the concentration of acetonitrile in the mobile phase is preferably 0 to 95% by volume, more preferably 0 to 90% by volume, and still more preferably 0 to 85% by volume. is there. The elution may be performed by gradient elution in which the ratio of the mobile phase A and the mobile phase B is changed over time, or by isocratic that fixes the ratio.

The conditions for the separation column used for liquid chromatography are not particularly limited, and can be appropriately selected according to various conditions. As the separation column, those applicable to normal phase partition chromatography can be used as appropriate. As the column filler (stationary phase), inorganic fillers such as silica gel, alumina gel, zirconia gel, or styrene are used. -Synthetic polymer gels such as divinylbenzene copolymer and polymethacrylate, and resin (polymer) fillers such as natural polymer gels can be applied. The silica gel is preferably surface-modified, and examples thereof include NH ₂ silica gel, CN silica gel, and diol silica gel. Furthermore, in HILIC, silica gel or a polymer is applied as a column filler, and a neutral amide group, a charge-type sulfoethyl group, a zwitterionic sulfobetaine group (* -LN ⁺ R _{2) is} formed on the surface thereof. ^{_{-L n -SO 3 -: R =}} CH 3, L = CH 2, n = 1~6 ( preferably 3), * is a predetermined connection between the inlet portion, but the filler and L to filler A diol group, a hydroxyl group, an amino group, or a phosphorylcholine residue may be modified and introduced on the surface, and in the present invention, a column filler modified with a sulfobetaine group Separation columns with (stationary phase) are preferred.

  When preparing a mobile phase (separation solution) by mixing two or more solutions, the concentration of volatile ammonium salt and acetonitrile in the two or more solutions is determined by the volatilization in the mobile phase after mixing. It can set suitably according to a mixing ratio so that the density | concentration of water-soluble ammonium salt and acetonitrile may become a suitable density | concentration. For example, when ammonium bicarbonate is used as the volatile ammonium salt, the concentration of ammonium bicarbonate and acetonitrile in the two or more solutions is preferably the concentration of ammonium bicarbonate and acetonitrile in the mobile phase after mixing. What is necessary is just to set suitably according to a mixing ratio so that it may become a density | concentration.

The flow rate can be appropriately selected according to various conditions such as the inner diameter of the separation column. The mobile phase flow rate may or may not be constant throughout the separation process. For example, the flow rate of the mobile phase can be appropriately selected within the range of 0.1 to 1.5 mL / min in accordance with the electrospray ionization method (ESI), and preferably 0.2 to 1.0 mL / min.
In addition, the column temperature in liquid chromatography can be appropriately selected by those skilled in the art according to the analysis target and the specifications of the analytical column to be used. For example, it may be 40 to 70 ° C., specifically 40 ° C.

  In the present invention, the liquid chromatograph (LC) is preferably integrated with the mass spectrometer (MS), and the liquid chromatograph-mass spectrometer (LC-) is associated with the next step d. MS) is preferably used. Specifically, an analysis method using a liquid chromatograph-tandem mass spectrometer called LC-MS / MS is preferable.

  In LC-MS / MS, the following processing is generally performed. A sample separated by liquid chromatography is ionized through an interface (ion source). The generated ions are separated by a first mass spectrometer (MS) to dissociate and fragment ions having a specific mass. The ions are detected with a second mass spectrometer. In the LC-MS / MS method, since only ions having a specific mass can be selected and fragmented, it is possible to suitably cope with quantitative analysis of trace components.

<Step d: Quantitative measurement by MS>
In this step, the mass of the nucleotide separated by the above liquid chromatography is identified by mass spectrometry.
As the mass spectrometer, a known mass spectrometer can be used, but a mass spectrometer that can be connected in series to an LC apparatus is particularly preferable because it can be used easily. One mass spectrometer may be used, or two or more mass spectrometers may be used. Two or more mass spectrometers can be used connected in series. That is, the LC-MS system may be, for example, an LC-MS / MS or an LC-MS / MS / MS that includes two or more mass spectrometers connected in series.

  Mass spectrometers include magnetic sector sector mass spectrometers, quadrupole mass spectrometers (QMS), time-of-flight mass spectrometers (TOF-MS) , Ion trap mass spectrometer, Fourier transform mass spectrometer and the like. In the present invention, a tandem quadrupole mass spectrometer is particularly preferable.

  As ionization methods in a mass spectrometer, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), fast atom bombardment (FAB), photoionization (APPI), electron ionization (EI), chemical Examples include ionization method (CI method), field desorption method (FD method), matrix-assisted laser desorption ionization method (MALDI method), and sonic ionization method (SSI), and electrospray ionization method (ESI) is particularly preferable. .

  The tandem quadrupole mass spectrometer is a mass spectrometer including an ion source, a quadrupole (Q1), a collision cell (Q2), a quadrupole (Q3), and a detector. The measurement object is ionized by an ion source, precursor ions are generated, and mass-separated by the quadrupole (Q1) according to the mass-to-charge ratio (m / z). Next, product ions are generated by collision with an inert gas such as nitrogen or argon in the collision cell (Q2) (collision-induced dissociation: CID). The mass is again separated by the quadrupole (Q3) according to the mass-to-charge ratio (m / z) of the product ions and detected by the detector.

MS / MS is preferably a method of monitoring product ions obtained by collision-induced dissociation of precursor ions. This includes multiple reaction monitoring (MRM) or selective reaction monitoring (SRM). In the present invention, multiple reaction monitoring (MRM) is preferred.
SRM can perform highly sensitive quantification with high selectivity by using m / z of specific product ions obtained by product ion scanning. Here, a precursor is selected in Q1, CID is performed in the collision cell, and a specific product ion is selected and detected in Q3.

  MRM selects a specific precursor ion in Q1 for various ions ionized by an ionization probe, breaks that ion in a collision cell (CID), and selects a specific ion from among the broken ions (product ions) in Q3 It is a method of detecting. This method has an advantage that a plurality of channels can be set by one measurement, and is a preferable monitoring method for simultaneous quantification. For example, it is a commercial product, and it is possible to set up to 512 events × 32 channels per analysis.

  On the other hand, the analyzer is a part that separates ions introduced under vacuum. The ion source also generates positive ions and negative ions, which are molecule-related ions, and neutral molecules that are not charged. However, it is impossible in principle to simultaneously capture both positive and negative ions. Therefore, in order to monitor positive and negative ions at the same time, measurement must be performed by switching between positive and negative with time. In the present invention, it is preferable to perform multiple reaction monitoring in the positive ion mode.

  The detection unit is a site that detects and sensitizes ions selected by the analysis unit with an electron multiplier or a microchannel plate. The data processing unit is a part that creates a mass spectrum from the obtained data.

Detailed setting conditions of LC-MS / MS are based on the example shown in the examples. It is preferable to separate the isocratic and linear gradient elution components at each retention time and to link the mass spectrometer. The MS / MS analysis is preferably performed in a multiple reaction monitoring (MRM) mode under the following conditions.
Atomization gas, nitrogen, ion source temperature, 10 to 300 ° C. (preferably 50 to 200 ° C.), desolvation temperature, 200 to 800 ° C. (preferably 300 to 600 ° C.), desolvation gas flow rate, 800 to 1500 L / H (preferably 1000 to 1500 L / h), capillary voltage, 0.5 to 10 kV (preferably 1 to 5 kV), cone voltage, 1 to 50 V (preferably 10 to 50 V), cone gas flow rate, 10 to 300 L / h (preferably 50 to 200 L / h), collision gas, argon

<Process e>
In this step, each nucleotide is quantified by determining the ratio between the chromatogram peak area of each identified nucleotide and the chromatogram peak area of the internal standard substance.

  Specifically, an example of performing absolute quantitative measurement of ATP is given below. It is assumed that an ATP peak is obtained by LC-MS / MS by the measurement in steps c and d. Confirm that the position of this peak coincides with the peak of the internal standard labeled with an isotope to form a pair. It is assumed that the peak of the internal standard substance is that of the isotope ATP. If so, the detected peak can be identified as that of ATP. The types of other nucleotides are specified in the same manner. The chromatogram peak area of each identified nucleotide is measured. If this area ratio is determined among the detected nucleotides, relative quantification is possible.

  In the present invention, it is preferable to prepare a calibration curve for each nucleotide in advance and quantify each nucleotide based on the created calibration curve. Thereby, for example, the absolute amount can be measured from the ratio of the previously detected ATP and the chromatogram peak area of the isotope ATP by comparing with the calibration curve of ATP. Similarly, absolute quantification of each nucleotide is possible. As a sample for preparing a calibration curve, it is preferable to use a sample containing the above-mentioned nucleotides labeled with a standard substance of each nucleotide to be analyzed and an isotope as an internal standard substance.

  Moreover, it is preferable to prepare a sample for preparing a calibration curve using a solution containing a volatile ammonium salt and acetonitrile. As the ammonium salt, ammonium bicarbonate, ammonium carbonate or ammonium acetate is preferred. The concentration of the ammonium salt is preferably 1 mmol / L or more and 300 mmol / L or less, more preferably 5 mmol / L or more and 50 mmol / L or less, and further preferably 10 mmol / L or more and 20 mmol / L or less. The concentration of acetonitrile is preferably 0% to 95% by volume, more preferably 0% to 90% by volume, and still more preferably 0% to 85% by volume. The concentration of nucleotide and isotope-labeled nucleotide (internal standard) is not particularly limited, and may be adjusted according to the concentration of nucleotide in the extract. As an example, for deoxynucleoside triphosphate (dNTP) It is preferable to prepare in the range of 1 nmol / L to 10,000 nmol / L, and to prepare nucleoside triphosphate (NTP) in the range of 100 nmol / L to 1,000,000 nmol / L.

  Thereby, according to this invention, in order to correct | amend the matrix effect of a cell extract, the complicated operation of preparing and using a separate cell matrix or using a standard addition method is not required. Moreover, the measurement which is understood that there are many unstable and unnecessary operations, such as measuring the activity of the protein in the extract on the premise of the known enzyme activity, is not required.

<Process f>
In the method of the present invention, other steps may be included. For example, it is preferable to include (step f1) a step of measuring the number of cells contained in the sample and (step f2) a step of calculating the number of nucleotide molecules per cell from the nucleotide quantification result. This is conceptually described as shown in FIG. In this protocol, for example, the number of cells is measured at the stage of collecting and processing the cells (step a). On the other hand, the remaining cells extract metabolites therefrom and add an internal standard substance (step b). Thereafter, the supernatant is evaporated to dryness and redissolved to form a sample suitable for measurement. This is subjected to LC-MS / MS and the absolute amount of each nucleotide is measured.

  Therefore, the number of nucleotide molecules per cell can be calculated from the data on the number of cells acquired first and the data on the absolute amount (mol) of each nucleotide (metabolite). The calculation is determined according to the following formula.

<Application of nucleotide quantification method of the present invention>

  The present invention provides an LC-MS / MS based method for the absolute quantification of dNTPs and NTPs that is accurate, accurate and efficient. The time required for one measurement is about 15 to 25 minutes only for the LC-MS / MS process. If a metabolite extraction step is included, depending on the number of types of nucleotides, the time required to complete the absolute quantification is about 80 to 100 minutes when eight types of quantification are performed. Furthermore, the process up to obtaining the number of molecules per cell is efficient because the number of cells can be measured simultaneously with the quantification of nucleotides. Even in this case, the time required from the extraction of the metabolite to the number of molecules per cell is about 90 to 110 minutes.

  The quantification method of the present invention includes a step of adding an internal standard substance, which is an isotope-labeled nucleotide, a step of extracting and fractionating nucleotides from each sample, a step of measuring with a mass spectrometer, and a mass From the results obtained by the analyzer, the nucleotide unlabeled peak (peak of each nucleotide not labeled with an isotope phase-separated from the sample extract) and the labeled peak (isotope-labeled each nucleotide as an internal standard substance) And a step of quantifying nucleotides by obtaining an intensity ratio with respect to the peak of ().

  Further, it is preferable to use a program for reading and calculating the chart according to the present invention, and it is preferable to execute each step of the quantification or analysis method according to the present invention on a computer. The program according to the present invention can be installed or downloaded to a computer through various recording media such as a CD-ROM, a magnetic disk, and a semiconductor memory.

  As mentioned above, nucleotides such as nucleoside triphosphates are constituents of DNA and RNA, phosphate group donors, energy carriers, coenzymes for enzyme reactions, and are also involved in cell signal transmission. Plays an important role in modern biological processes. Abnormal quantitative balance of nucleoside triphosphates has a significant effect on cells and is responsible for several genetic disorders, such as Lesch-Nyhan syndrome, mitochondrial depletion syndrome, mitochondrial neurogastrointestinal encephalomyopathy, and the genes responsible for nucleotide synthesis, It has been revealed that

  In addition, as described above, measurement of nucleoside triphosphate balance abnormality is also useful in drug discovery. For example, antimetabolites such as 5-fluorouracil and hydroxyurea kill cancer cells by inhibiting nucleotide synthesis, and this type of anticancer drug has been vigorously developed in recent years. . Furthermore, various nucleosides and nucleotide analogs (5-fluorouracil is also a kind) are being developed as anticancer agents and antiviral agents. These action targets themselves are nucleoside triphosphate balance abnormalities and cause side effects as a side effect.

  Therefore, the nucleotide quantification method of the present invention is useful not only for studies of genetics and cell biology, but also for analysis of mechanisms related to patient diseases, tests for drugs and genotoxic substances, and evaluation of drug efficacy and side effects.

  According to the present invention, for example, quantitative analysis (examination) of a genome replication requirement amount and a nucleotide pool can be performed. Abnormal quantitative balance of cellular nucleotide pools suggests that genetic diseases may be lurking. To date, little is known about the quantitative relationship between changes in the amount of nucleotide pool and cell cycle arrest. According to the present invention, the amount of nucleotides contained in a specimen can be measured as an absolute amount, not a relative amount. By utilizing this result, it is possible to know in detail and accurately the abnormality in the amount of nucleotides, for example, by comparing with the amount necessary for replication of the genome in human cells. Thereby, there is a possibility that genetic diseases and cancer pathologies can be discovered earlier.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not construed as being limited thereto.

<Materials and methods>
(material)
RPMI 1640 medium, penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Life Technologies, Calif., Life Technologies, California, USA. dATP was purchased from GE Healthcare, Illinois, USA (GE Healthcare, Illinois, USA). dGTP, dCTP and dTTP were purchased from Thermo Fisher Scientific, Massachusetts, USA (Thermo Fisher Scientific, Massachusetts, USA). [ ¹⁵ N ₅ , ¹³ C ₁₀ ] ATP, [ ¹⁵ N ₅ , ¹³ C ₁₀ ] GTP, [ ¹⁵ N ₃ , ¹³ C ₉ ] CTP, [ ¹⁵ N ₂ , ¹³ C ₉ ] UTP, [ ¹⁵ N ₅ , ¹³ C ₁₀ ] dATP, [ ¹⁵ N ₅ , ¹³ C ₁₀ ] dGTP, [ ¹⁵ N ₃ , ¹³ C ₉ ] dCTP, and [ ¹⁵ N ₂ , ¹³ C ₁₀ ] dTTP are from Sigma-Aldrich, Missouri, USA (Sigma) -Purchased from Aldrich, Missouri, USA). Other chemicals were purchased from FUJIFILM Wako Pure Chemical (Wako) (Osaka Prefecture, Japan). The stock solution of thymidine was dissolved in ultrapure water.

(Cell culture)
Human acute myeloid leukemia Molm-13 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin in a 5% CO ₂ incubator humidified at 37 ° C. did.

(Extraction of metabolites)
Molm-13 cells (1 × 10 ⁶ cells in 2 ml of medium) were treated with vehicle [2% (v / v) ultrapure water] or thymidine for 24 hours. The chemical solution was added directly to the medium. The cell suspension was transferred to a 15 mL tube and 150 μL of the suspension was stored for cell counting to determine cell viability. The remaining cell suspension was pelleted by centrifugation at 1,000 rpm for 5 minutes using a centrifuge LC-200 (manufactured by Tommy, Tokyo, Japan), and the supernatant was removed by decantation. After adding 10 mL of ice-cold PBS (phosphate buffered saline), the cell pellet was resuspended and centrifuged at 1,000 rpm for 5 minutes using a centrifuge MX-301 (manufactured by Tommy). And pelletized again. After removing the supernatant by decantation, 1 mL of ice-cold PBS was added to the cell pellet and resuspended. 100 μL of the suspension was saved for cell counting and the number of cells used for metabolite extraction was determined. Cell counting was performed using a Coulter Counter Z2 (Beckman Coulter, California, USA: Beckman Coulter, CA, USA). 1 mL of the remaining suspension was transferred to a 1.5 mL tube, pelleted by centrifugation at 2,000 × g for 2 minutes at 4 ° C., and 900 μL of supernatant was removed. To extract metabolites, 500 μL of methanol was added to the remaining sample and vortexed vigorously.

(Addition of internal standard substance)
Furthermore, 10 μL of the internal standard mixture (10 μmol / L [ ¹⁵ N ₅ , ¹³ C ₁₀ ] ATP, 10 μmol / L [ ¹⁵ N ₅ , ¹³ C ₁₀ ] GTP, 10 μmol / L [ ¹⁵ N ₃ , ¹³ C ₉ ] CTP, 10 μmol / L [ ¹⁵ N ₂ , ¹³ C ₉ ] UTP, 10 μmol / L [ ¹⁵ N ₅ , ¹³ C ₁₀ ] dATP, 10 μmol / L [ ¹⁵ N ₅ , ¹³ C ₁₀ ] dGTP, 10 μmol / L [ ¹⁵ N ₃ , ¹³ C ₉ ] dCTP, and 10 μmol / L [ ¹⁵ N ₂ , ¹³ C ₁₀ ] dTTP) and 190 μL of ultrapure water were added and vigorously vortexed. After centrifugation at 10,000 × g for 15 minutes at 4 ° C., 700 μL of the supernatant was transferred to a new 1.5 mL test tube, and a centrifugal evaporator CVE-3100 (Tokyo Rika Kikai Co., Ltd., Tokyo, Japan) was used. And evaporated to dryness at 37 ° C. The dried sample was stored at −20 ° C. until use.

(LC-MS / MS)
The dried sample was redissolved in 100 μL of a mixture of 10 mmol / L ammonium bicarbonate and acetonitrile [35 (v): 65 (v)]. After centrifugation at 16,000 × g for 5 minutes, 70 μL of the supernatant was transferred to a vial. Mass spectrometric analysis was performed using a Xevo TQ-S micro (Waters) equipped with an H-classBio system (Waters, Manchester, UK). 2 μL of each sample was separated on a SeQuant® ZIC-pHILIC HPLC column (5 μm 4.6 × 150 mm column (Merck Milpore, Darmstadt, Germany)) at a flow rate of 0.5 mL / min, followed by ((Solvent A, 10 mmol / L ammonium bicarbonate and 0.05 vol% aqueous ammonia; solvent B, acetonitrile): 0-5 min, 65% B-40% B linear gradient; 5-7 min , Linear gradient to 0% B; 7-9 minutes, isocratic at 0% B; 9-9.1 minutes, linear gradient to 65% B; 9.1-15 minutes, 65% B In isocratic).

Multiple reaction monitoring (MRM) was performed in positive ion mode using nitrogen as the atomizing gas. The experimental conditions were set as follows:
Ion source temperature, 150 ° C .;
Solvent removal temperature, 550 ° C .;
Desolvent gas flow rate, 1200 L / hour;
Capillary voltage, 2.0 kV;
Cone gas flow rate, 110 L / hr;
The collision gas is argon.

The conditions of MRM (Multiple Reaction Monitoring) transition are as follows (Q1> Q3):
[Cone voltage (V), collision energy (eV)] ATP, 508.1> 136.0 (20, 35); GTP, 524.1> 152.0 (20, 25); CTP, 484.1> 112.0 (20, 20); UTP, 485.0> 96.9 (20, 25); dATP, 492.1> 136.0 (20, 20); dGTP, 508.1> 152.0 (20, 20); dCTP, 468.0> 112.0 (20, 15); dTTP, 483.0> 81.0 (50, 15) [ ¹⁵ N ₅ , ¹³ C ₁₀ ] ATP, 523.1> 146.0 (20, 35); [ ¹⁵ N ₅ , ¹³ C ₁₀ ] GTP, 539.1> 162.1 (20, 25); [ ¹⁵ N ₃ , ¹³ C ₉ ] CTP, 496.1> 119.0 (20, 20); [ ¹⁵ N ₂ , ¹³ C ₉ ] UTP, 496.0> 101.9 (20, 25); [ ¹⁵ N ₅ , ¹³ C ₁₀ ] dATP, 507.1> 146.0 (20, 20) [ ¹⁵ N ₅ , ¹³ C ₁₀ ] dGTP, 523.1> 162.1 (20, 20); [ ¹⁵ N ₃ , ¹³ C ₉ ] dCTP, 480.1> 119.0 (20, 15); [ ¹⁵ N ₂ , ¹³ C ₁₀ ] dTTP, 495.1> 86.0 (50, 15).

  The amount of each metabolite was quantified by calculating the peak area ratio between the target metabolite and its isotopically labeled internal standard. A calibration curve was obtained by using a standard metabolite spiked with an isotopically labeled internal standard.

<Result>
(Method development)
Eight standard metabolites (dATP, dGTP, dCTP, dTTP, ATP, GTP, CTP, and UTP) and their respective isotope-labeled metabolites were used as internal standards. The use of an isotopically labeled standard is one of the advantages of the method of the present invention. This is because it has the same separation, ionization and fragment pattern and can achieve high precision and accuracy (Pitt JJ (2009) Clin Biochem Rev 30 (1): 19-34). The MRM measurement conditions for these metabolites were manually optimized. It has been reported that the dNTP level of a cell is about 2 orders of magnitude smaller than the NTP level of a cell (nonpatent literature 1). Therefore, calibration curves were obtained in different concentration ranges between dNTP and NTP. As a result, the calibration curve was linear over the entire range of 3 to 1000 nmol / L for dNTP and over the entire range of 300 to 100,000 nmol / L for NTP. The correlation coefficients are 0.9983 for dATP, 0.9977 for dGTP, 0.9990 for dCTP, 0.9981 for dTTP, 0.9995 for ATP, 0.9994 for GTP, 0.9997 for CTP, and 0 for UTP. .9996. Next, accuracy and accuracy were calculated at three levels for each metabolite. Accuracy and accuracy have been obtained based on 8 replicates, specifically 0.6 to 5.4% CV (Coefficient of Variation) and − for all metabolites. The range was 3.1 to 2.5% Bias (bias) (Table 1). These data show that the method of the present invention has acceptable accuracy and accuracy.

(Use of extracts from cells)
It was tested whether the method of the present invention could be applied to biological samples. Immediately after extraction of metabolites from Molm-13 cells with methanol, internal standard substances labeled with dNTPs and NTP isotopes were added to the extract to reduce variability between samples generated in subsequent processing. It can be corrected. After evaporating to dryness and redissolving, detection of dNTP and NTP was attempted using the method of the present invention. As shown in FIG. 1, clear peaks representing dNTPs and NTPs were observed with the same retention times as the respective internal standards. From these data, it can be seen that according to the method of the present invention, dNTP and NTP can be specifically detected from a cell sample.

(Quantitative analysis of cell samples)
Next, the quantitative test of the absolute amount of dNTP and NTP in the extract from Molm-13 cells was performed. In this example, thymidine was used as an inducer of abnormal nucleotide pool balance. Molm-13 cells were treated with thymidine at two concentrations. Low concentration (200 μmol / L) did not significantly affect cell growth, but high concentration (2000 μmol / L) inhibited proliferation by 60% compared to control vehicle (FIG. 2). After the treatment, the number of cells used in the metabolic extract described in the above <Materials and methods> and the calculated value of the number of molecules of each metabolite in Molm-13 cells were determined according to the following formula.

Under vehicle conditions, the amount of dNTP in the cell is 4.63 × 10 ⁶ for dATP, 1.72 × 10 ^{6 for} dGTP, 4.22 × 10 ^{6 for} dCTP, and 1.40 × 10 ⁷ for dTTP. there were. Thymidine increased dTTP in cells in a dose-dependent manner. After treatment with 2000 μmol / L thymidine, dTTP in the cells showed the most significant increase of 12.9 times (number of molecules / 1.81 × 10 ^{8 in} cells). Conversely, thymidine reduced the level of dCTP in cells in a dose-dependent manner (2000 μmol / L thymidine molecule number / cell 2.02 × 10 ⁶ ). The level of dGTP in the cells was not affected by 200 μmol / L thymidine, but 2000 μmol / L thymidine increased the level of dGTP by 4.6 times (number of molecules / 7.83 × 10 ⁶ cells). The level of dATP decreased 0.69 times after treatment with 200 μmol / L thymidine (number of molecules / cell 3.19 × 10 ⁶ ), but 1.41 times after treatment with 2000 μmol / L thymidine. It increased to (number of molecules / cell 6.55 × 10 ⁶ ). The amount of NTP in the cell is one digit or more of dNTP, the number of molecules is 2.29 × 10 ^{9 for} ATP, 5.05 × 10 ^{8 for} GTP, and for dCTP The number of molecules was 2.07 × 10 ⁸ , and in the case of UTP, the number of molecules was 5.20 × 10 ⁸ . CTP levels increased dose-dependently with thymidine treatment and increased 1.56 times (number of molecules / 3.22 × 10 ^{8 in} cells). GTP and UTP levels did not change with 200 μmol / L thymidine treatment, but 2000 μmol / L thymidine increased these levels (1.21 fold for GTP and 2.02 fold for UTP). ATP levels in the cells did not change under the conditions employed in this example.

  As described above, a simple and reliable method for simultaneous absolute quantification of nucleotides such as nucleoside triphosphates using stable isotopes, HILIC columns and LC-MS / MS could be established. By using the method of the present invention, quantitative changes in intracellular nucleoside triphosphates caused by thymidine exposure can be captured quantitatively.

(Nucleotide pool disturbance mechanism by thymidine)
In the present invention, the following mechanism is presumed as a nucleotide pool disturbance mechanism by thymidine, but is not limited thereto.
Thymidine is converted to dTTP in cells via the salvage pathway of pyrimidine nucleotides. Excess thymidine exposure causes excessive production of intracellular dTTP, resulting in an unbalanced nucleotide pool. This activates the cell cycle checkpoint and stops cell growth. In the field of cell biology, this principle is utilized for cell cycle synchronization (known as double thymidine block). However, the exact mechanism by which excess thymidine disrupts the nucleotide pool balance is largely unknown. In this example, exposure of Molm-13 cells to 2000 μmol / L thymidine significantly inhibited cell growth (FIG. 2), increased levels of not only dTTP but also dGTP and dATP, and decreased only dCTP levels. (Figure 3). Taking the ratio of dNTP levels in the cells of the thymidine treated group and the vehicle group, dTTP (12.9 times)> dGTP (4.6 times)> dATP (1.41 times)> dCTP ( 0.48 times). This result can be explained from the point of production control of deoxyribonucleotides by ribonucleotide reductase (RNR) (FIG. 4). RNR reduces NDP to produce dNDP, and the reaction is further phosphorylated in the cell to become dNTP (dTTP is generated from dUDP through a multi-step reaction). The activity and substrate selectivity of RNR are allosterically controlled. When Molm-13 cells are exposed to excess thymidine, dTTP is induced, and when this is excessive, the RNR bound to dTTP selectively reduces GDP to produce dGDP. This is then phosphorylated to produce dGTP. As a result, increasing intracellular dGTP promotes dGTP binding to the RNR, which selectively reduces ADP to produce dADP. This is then phosphorylated in the cell to produce dATP. Then, dATP binds to the activity regulatory site of RNR and inhibits the RNR reaction. Thus, as a result of consuming dNTP along with cell growth, it is considered that only dCTP in the cell is depleted and cell growth is stopped. The method of the present invention is useful for elucidating the mechanisms of genotoxic substances and anticancer agents that disrupt nucleotide metabolism.

(Quantitative relationship between genome replication requirement and pool)
The quantitative relationship between the genome replication requirement and the pool can be estimated as follows, but is not limited thereto.
When a quantitative balance abnormality occurs in the cell nucleotide pool, the cell cycle checkpoint is activated and the cell cycle stops at the G0 / G1 phase. However, little is known about the quantitative relationship between changes in pool volume and cell cycle arrest. The amount of dNTP required for one replication of the genome in human cells that are diploid is estimated as follows.

The size of the human genome is 3.26 × 10 ⁹ bp based on the Genome Reference Consortium Human Build 38 patch release 12. X is the ratio of each base constituting the human genome, which is 29.3% (A), 20.7% (G), 20.0% (C), and 30.0% (T), respectively. From the above formula, the amount of dNTP required for one replication of the genome in a diploid human cell is 3.82 × 10 ⁹ (dATP) and 2.70 × 10 ⁹ (dGTP), respectively. 2.61 × 10 ⁹ (dCTP) and 3.91 × 10 ⁹ (dTTP). Table 2 shows the result of comparing these values with the quantitative value of dNTP in Molm-13 cells obtained in this example. In vehicle conditions, intracellular dNTPs are very low, 0.064-0.358% of the amount required for diploid genome doubling. One reason is probably to prevent mutations in the genome from unnecessary DNA replication reactions in order to preserve the integrity of the cell's genome. Thymidine decreased only dCTP, but even when intracellular dCTP decreased from 0.162% to 0.109% of the diploid genome, cell proliferation was not affected (FIGS. 2 and 3). ). However, when the high dose thymidine treatment reduced intracellular dCTP to 0.077% of the diploid genome, a marked growth inhibitory effect was seen. This means that, in order to activate the cell cycle checkpoint of Molm-13 cells, intracellular dCTP is reduced to 0.077% of the diploid genome (number of molecules / 2.02 × 10 ^{6 in} cells). It suggests that there is a need. On the other hand, it should be noted that the quantitative value of the nucleotide pool in this example is an average value of the amount of intracellular nucleotides in each condition. Even so, the method of the present invention is useful for quantitative analysis in molecular biology and other related fields.

  According to the present invention, absolute quantification of deoxynucleoside triphosphate (dNTP) and nucleoside triphosphate (NTP) using LC-MS / MS was realized. According to the present invention, dNTP and NTP can be accurately and accurately quantified in the range of 3 to 1,000 nmol / L for dNTP and 300 to 100,000 nmol / L for NTP, respectively. . The method of the present invention can be applied to human cell samples. The method of the present invention has a short processing time (15 minutes / one time (run)), a wide adaptive range, and a high throughput. Therefore, the method of the present invention is not only for genetic and molecular biological studies, but also for studying the mechanism of action of genotoxic compounds and drugs, screening for anticancer drugs, clinical research, and other fields. It can be applied as a simple tool.

Claims (17)

A method for quantifying two or more nucleotides in a sample by liquid chromatography-mass spectrometry,
Extracting a nucleotide mixture from the sample, a.
Adding the two or more kinds of nucleotides labeled with an isotope to the nucleotide mixture as an internal standard substance to prepare a measurement sample; and b
A step c of separating each nucleotide from the measurement sample by liquid chromatography using a normal phase column;
Identifying each separated nucleotide by mass spectrometry, step d;
Determining the ratio of the chromatogram peak area of each identified nucleotide and the chromatogram peak area of the internal standard substance to quantify each nucleotide; e.
Including methods. Measuring the number of cells contained in the sample;
Calculating the number of nucleotide molecules per cell from nucleotide quantification results;
The method of claim 1, further comprising:   The method according to claim 1 or 2, wherein the sample is selected from the group consisting of tissue, cell and biological fluid.   The method according to any one of claims 1 to 3, wherein the nucleotide is nucleoside triphosphate or deoxynucleoside triphosphate. The method according to any one of claims 1 to 4, wherein the isotope is selected from the group consisting of ² H, ¹³ C, ¹⁵ N, ¹⁷ O and ¹⁸ O.   The method according to any one of claims 1 to 5, wherein the liquid chromatography is hydrophilic interaction chromatography.   The method according to any one of claims 1 to 6, wherein the liquid chromatography is hydrophilic interaction chromatography using a separation column having a stationary phase modified with a sulfobetaine group.   The method according to any one of claims 1 to 7, wherein the material of the inner wall of the normal phase column is polyetheretherketone.   The method according to any one of claims 1 to 8, wherein multiple reaction monitoring is performed in a positive ion mode in the step d of identifying by mass spectrometry.   The method according to any one of claims 1 to 9, wherein in step e, a calibration curve for each nucleotide is prepared in advance, and each nucleotide is quantified based on the prepared calibration curve.   11. The sample according to claim 1, wherein in step e, a sample containing a standard substance for each nucleotide to be analyzed and an isotope labeled with an isotope as an internal standard substance is used as a sample for preparing a calibration curve. The method described in 1.   The method according to any one of claims 1 to 11, wherein in step e, a sample for preparing a calibration curve is prepared using a solution containing a volatile ammonium salt and acetonitrile.   The method according to any one of claims 1 to 12, wherein the liquid chromatography-mass spectrometry is an analysis method using a liquid chromatograph-tandem mass spectrometer called LC-MS / MS.   The method according to any one of claims 1 to 13, wherein the mobile phase used for liquid chromatography comprises any selected from the group consisting of ammonium bicarbonate, ammonium carbonate and ammonium acetate.   The method according to any one of claims 1 to 14, wherein the pH of the mobile phase used for liquid chromatography is 8 to 11.   The method according to any one of claims 1 to 15, wherein the salt concentration of the mobile phase used for liquid chromatography is 10 to 20 mmol / L.   The method according to any one of claims 1 to 16, wherein the absolute amount of each nucleotide can be measured.