WO2022239460A1 - Method for analyzing biomolecule in hair - Google Patents

Method for analyzing biomolecule in hair Download PDF

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Publication number
WO2022239460A1
WO2022239460A1 PCT/JP2022/011616 JP2022011616W WO2022239460A1 WO 2022239460 A1 WO2022239460 A1 WO 2022239460A1 JP 2022011616 W JP2022011616 W JP 2022011616W WO 2022239460 A1 WO2022239460 A1 WO 2022239460A1
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hair
biomolecules
mass spectrometry
polarity
sample
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PCT/JP2022/011616
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French (fr)
Japanese (ja)
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秀一 新間
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国立大学法人大阪大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • 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

Definitions

  • the present invention relates to a method for efficiently analyzing biomolecules in hair.
  • Hair is a hair matrix cell in the hair bulb, which is the tip of the hair root in the skin.
  • the hair outside the skin is dead cells, but the structure once formed remains largely intact. That is, hair grows by sending a proliferation signal from dermal papilla cells to hair matrix cells, and the hair matrix cells that receive the signal proliferate and keratinize, and the dermal papilla cells run around them. Since hair is active by being supplied with nutrients from capillaries, there is a possibility that biomolecules contained in the blood migrate to hair matrix cells and remain in the hair as they are.
  • hair consists of a medulla, which is a porous structure that exists in the center, a fibrous bundle that exists around it, and a structure that surrounds it, cortex that accounts for 85 to 90% of the hair
  • the cuticle consists of 4 to 10 layers of translucent scales on the surface of the cuticle, 80 to 85% of which is composed of a protein called keratin.
  • melanin pigment is produced by melanocytes (pigment-forming cells) present in the hair bulb together with hair matrix cells, and is transferred from melanocytes to hair matrix cells when the hair matrix cells undergo cell division and hair grows. Immobilized in the cortex and determines the color of the hair. Therefore, biomolecules migrated from capillaries may also be immobilized in hair cortex.
  • Non-Patent Document 1 the amount of glucose in the hair of diabetic patients is correlated with HbA1c, which is an index of the average blood sugar level in the past 2 to 3 months, and also reflects the history of past blood sugar levels. is suggested.
  • Patent Document 1 an internal standard substance is applied to a hair sample sliced in the longitudinal direction, and the surface is subjected to imaging mass spectrometry to continuously and quantitatively detect physiologically active substances in the hair. discloses a method for determining when and how much of a bioactive substance is used in hair.
  • an object of the present invention is to provide a method for efficiently analyzing biomolecules in hair.
  • the inventor of the present invention has made intensive studies to solve the above problems. As a result, the present inventors have found that the target biomolecules can be easily detected by mass spectrometry by treating the hair so as to increase the polarity of the target biomolecules, thereby completing the present invention.
  • the present invention is shown below.
  • a method for analyzing biomolecules in hair comprising: treating the hair to increase the polarity of the biomolecules; and A method comprising subjecting said treated hair to mass spectrometry.
  • the polarity of the target biomolecules contained in the hair sample increases, making them easier to detect by mass spectrometry.
  • analysis of hair samples becomes more efficient. Therefore, the present invention is very useful as it can improve the efficiency of detection of target biomolecules in hair samples.
  • FIG. 1(1) is a mass spectrum of D-glucose
  • FIG. 1(2) is a mass spectrum of gluconic acid obtained by treating D-glucose with glucose oxidase
  • FIG. 2(1) is the result of mass spectrometry (MS) of a diabetic patient's hair sample
  • FIG. 2(2) is the result of tandem mass spectrometry (MS/MS) of a diabetic patient's hair sample
  • FIG. 3(1) shows the results of mass spectrometry (MS) of hair samples from healthy individuals
  • FIG. 3(2) shows the results of tandem mass spectrometry (MS/MS) of hair samples from healthy individuals.
  • FIG. 1(1) is a mass spectrum of D-glucose
  • FIG. 1(2) is a mass spectrum of gluconic acid obtained by treating D-glucose with glucose oxidase
  • FIG. 2(1) is the result of mass spectrometry (MS) of a diabetic patient's hair sample
  • FIG. 2(2) is the result of tandem mass spectrome
  • FIG. 4(1) is the mass spectrometry imaging result of the molecular ion peak (195.03) in the diabetic hair sample
  • FIG. 4(2) is the fragment ion peak (128.96) in the diabetic hair sample
  • FIG. 4(3) is the mass spectrometry imaging result of the molecular ion peak (195.03) in the hair sample of healthy subjects
  • FIG. 4(4) is the result of It is the mass spectrometry imaging result of the fragment ion peak (128.96).
  • the method for analyzing biomolecules in hair includes the steps of treating the hair to increase the polarity of the biomolecules, and subjecting the treated hair to mass spectrometry.
  • Each step of the present invention will be described below, but the present invention is not limited to the following specific examples.
  • Step 1 Pretreatment Step of Hair Sample
  • the cuticle on the outermost surface of the hair sample is removed.
  • Implementation of this step is optional, but since biomolecules often accumulate in the medulla and cortex, it is preferable to remove the cuticle for clearer analysis.
  • the hair sample is not limited to head hair, but may be armpit hair, eyelashes, eyebrows, whiskers, pubic hair, etc.
  • pets such as dogs and cats; cows, pigs, chickens, horses, and sheep , domestic animals such as goats; and experimental animals such as mice and rats.
  • a hair sample can be collected according to the time period for which you want to know the history, based on the fact that human hair grows about 1 cm per month. For example, when the most recent data is required, a hair sample is taken from a portion near the hair root, and when past data is required, a hair sample is taken from the tip.
  • the hair sample may be washed prior to cuticle removal.
  • the hair sample may be immersed in an alcohol-based solvent such as purified water, methanol, ethanol, or isopropanol, or may be soaked in a rag or the like and then wiped with the hair sample.
  • an alcohol-based solvent such as purified water, methanol, ethanol, or isopropanol
  • a surfactant may be used or ultrasonic waves may be applied.
  • the cuticle removal method is not particularly limited, and a known method may be used.
  • a hair sample is fixed on a glass slide with an adhesive or double-sided tape, frozen, and then sliced with a microtome, or a hair sample is sandwiched between grooves and the surface of the hair is cut using a razor or other blade.
  • a method of slicing at room temperature can be mentioned. Slicing is preferably done until the medulla is exposed to maximize the exposed surface of the cortex.
  • the hair sample is treated to increase the polarity of the biomolecules to be detected.
  • they are made easier to detect by mass spectrometry.
  • Biomolecules is a general term for compounds contained in living organisms and compounds derived from living organisms. Among biomolecules, compounds related to the maintenance and regulation of biological activities and physiological functions of living organisms are called bioactive substances. Biomolecules may also include pharmaceuticals and toxic compounds, which are also referred to as biologically active substances. Biomolecules include, for example, amino acids, peptides, proteins, enzymes, sugars, lipids, vitamins, coenzymes, hormones, nucleic acids, complexes and metabolites thereof.
  • the polarity of a compound means that the centers of gravity of the positive and negative charges in the compound do not coincide and have an electric dipole moment.
  • amide groups to carboxy and amino groups can be reduced to a secondary amino group (-NH-), a secondary amino group (-NH-) can be reduced to a primary amino group (-NH 2 ), or a complex
  • ring opening include, for example, the opening of creatinine (2-imino-1-methylimidazolidin-4-one) to creatine (1-methylguanidinoacetic acid).
  • a substituent that can increase the polarity under relatively mild conditions or a substituent that can effectively increase the polarity should be selected from the substituents of the biomolecule to be detected. Just do it.
  • the method of increasing the polarity of the target biomolecule can be appropriately selected as long as it does not excessively damage the hair sample, and examples include chemical methods and biological methods.
  • a substituent when hydrolyzed or converted into a salt, an acid or a base may be used.
  • Biological methods also include methods using enzymes.
  • a hair sample may be sprayed with an aqueous solution of an enzyme that increases the polarity of biomolecules, or the hair sample may be immersed in the aqueous solution and incubated at or around the optimal temperature for the enzyme.
  • enzymes include oxidoreductases, transferases, and hydrolases. Enzymes have the advantage of being able to specifically polarize target biomolecules due to their high substrate specificity, potentially making the analysis of target biomolecules and their polarised target biomolecule derivatives easier. be.
  • reaction conditions for increasing the polarity of the substituents of biomolecules it is preferable to select conditions that sufficiently increase the polarity of the target biomolecules contained in the hair sample, while also considering damage to the biomolecules to be detected.
  • the biomolecule can be selected as appropriate according to the purpose of analysis. For example, in order to understand the health condition and disease state of a subject, as well as their history, blood concentrations of glucose, proteins, peptides, lipids, nucleic acids, neurotransmitters, sugar chains, etc. You just have to choose a substance that changes.
  • neurotransmitters include catecholamines such as dopamine and noradrenaline, and amino acids such as GABA, glutamic acid, tryptophan, kylenurine, glycine, and serotonin.
  • illegal drugs such as methamphetamine, amphetamine, MDMA, MDA, morphine, ***e, heroin, and cannabinoids may be detected for criminal investigations.
  • doping control drugs such as steroids may be detected for doping inspection.
  • Mass Spectrometry Step the hair sample in which the polarity of the target biomolecules contained in the previous step 2 has been increased is subjected to mass spectrometry, and the molecular ion peaks of the target biomolecules or the target biomolecule derivatives whose polarity has been increased, and their molecular ion peaks. Check the presence or absence of fragment peaks and their intensity.
  • the ionization method is not particularly limited, and may be electron ionization (EI), chemical ionization (CI), fast atom bombardment (FAB), electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI). , atmospheric pressure ionization (API) and the like can be used, but matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) are preferably used. can.
  • EI electron ionization
  • CI chemical ionization
  • FAB fast atom bombardment
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption ionization
  • API atmospheric pressure ionization
  • MALDI matrix-assisted laser desorption/ionization
  • AP-MALDI atmospheric pressure matrix-assisted laser desorption/ionization
  • a person skilled in the art can predict the molecular weight of a target biomolecule with increased polarity and the molecular weight of fragment ions generated in tandem mass spectrometry (MS/MS). In the obtained mass spectrum, the intensity of peaks related to target biomolecules may be measured.
  • Hair matrix cells may contain biomolecules contained in the blood when they proliferate. Since hair matrix cells are keratinized to form hair, the biomolecules contained in the hair matrix cells remain in the hair sample and are fixed, so that the history of the biomolecules contained in the blood can remain in the hair. Therefore, by cutting a hair sample into two or more pieces, processing each part, and subjecting it to mass spectrometry, the results of the tip part are more recent, and the results of the root part are closer to the present. it may be possible to
  • mass spectrometry imaging may enable continuous mass spectrometry from end to end or part of a hair sample to understand the distribution of target biomolecules in the hair sample. Such an aspect may enable a more detailed understanding of the subject's past conditions, medical history, and the like.
  • Mass spectrometry imaging involves measuring the mass spectrum of minute measurement points in a hair sample evenly, extracting only the signal of interest from the obtained mass spectrum, and performing two-dimensional imaging according to the intensity of the target biomolecule. It is a technique to visualize the distribution of
  • a matrix is applied to the surface of a fixed hair sample.
  • the matrix may be applied by a conventional method, and may be applied manually using an airbrush, but from the viewpoint of uniformity, a method of applying the matrix by vapor deposition or an automatic spraying device, or a method combining vapor deposition and spraying is preferred. , crystallizes the matrix after application.
  • the thickness of the matrix may be adjusted as appropriate, and may be, for example, 0.1 ⁇ m or more and 2 ⁇ m or less.
  • the laser irradiation interval can be, for example, 5 ⁇ m or more and 200 ⁇ m or less.
  • the molecular ion peaks of the target biomolecules or the derivatives produced from the target biomolecules by treatment of the hair sample are selected from the obtained mass spectrum, and the peaks at each laser irradiation position are selected.
  • a two-dimensional image is obtained according to the intensity of the same molecular ion peak.
  • the distribution of target biomolecules in the hair sample is visualized from the pixel intensity information of the obtained image using dedicated software for mass spectrometry imaging and image analysis software such as Image J. can do.
  • Example 1 (1) Measurement of mass spectrum of glucose N-(1-naphthyl)ethylenediamine hydrochloride (NEDC) was dissolved in a 50% methanol aqueous solution to prepare a 7 mg/mL solution. Equivalent amounts of a 1.0 mg/mL aqueous solution of D-glucose and a 7 mg/mL solution of NEDC were mixed. The resulting mixed solution (0.1 ⁇ L) was dropped on an ITO glass plate of 2.5 cm ⁇ 7.5 cm and allowed to air dry. After air-drying, the mass spectrum was measured using an imaging mass microscope (“iMScope TRIO” manufactured by Shimadzu Corporation). The results are shown in FIG. 1(1).
  • iMScope TRIO an imaging mass microscope
  • a 100 units/mL glucose oxidase solution (approximately 200 ⁇ L) was evenly sprayed onto the D-glucose-supported ITO glass plate using an airbrush, incubated at 40° C. for 30 minutes, and air-dried.
  • the NEDC solution (approximately 200 ⁇ L) was uniformly sprayed with an airbrush and air-dried, and the mass spectrum of the obtained sample was analyzed using an imaging mass microscope (“iMScope TRIO” manufactured by Shimadzu Corporation). measured by The results are shown in FIG. 1(2).
  • the mass spectral peak intensity of gluconic acid obtained by enzymatically oxidizing D-glucose was approximately 100 times higher than the mass spectral peak intensity of D-glucose itself.
  • Example 2 Hair was collected from a healthy male in his 40s and a diabetic male in his 40s, and cuticles on the surface were removed using a human hair/fiber piece slicing device (“Bio Slicer DBS501 KATANA” manufactured by Y.A.C. I.D.S Tech Co., Ltd.). .
  • a human hair/fiber piece slicing device (“Bio Slicer DBS501 KATANA” manufactured by Y.A.C. I.D.S Tech Co., Ltd.).
  • Each hair sample from which the cuticle was removed was placed on a 2.5 cm x 7.5 cm ITO glass plate, and about 200 ⁇ L of a 100 units/mL glucose oxidase solution prepared under the same conditions as in Example 1 (2) was added to each glass plate. After uniformly spraying and incubating at 40° C. for 30 minutes, it was air-dried.
  • FIG. 2 The results of mass spectrometry (MS) of hair samples from diabetic patients are shown in FIG. 2 (1), the results of tandem mass spectrometry (MS/MS) of hair samples from diabetic patients are shown in FIG.
  • FIG. 3(1) shows the results of mass spectrometry (MS)
  • FIG. 3(2) shows the results of tandem mass spectrometry (MS/MS) of a hair sample from a healthy subject.
  • the mass spectrometry imaging result of the molecular ion peak (195.03) in the hair sample of the diabetic patient is shown in FIG. 4 (2) shows the mass spectrometry imaging result of the molecular ion peak (195.03) in the hair sample of healthy subjects
  • the mass spectrometric imaging result of the fragment ion peak (128.96) in the hair sample of healthy subjects is shown in FIG. 4 (3).
  • the imaging result is shown in FIG. 4(4).
  • a warm color with a longer wavelength indicates a higher concentration of the target compound
  • a cooler color with a shorter wavelength indicates a lower concentration of the target compound.
  • the mass spectrometric imaging results in FIG. 4 show the ion peak concentrations of gluconic acid and its fragments, which naturally have a correlation with the glucose concentration.
  • the glucose concentration in hair samples from healthy subjects is clearly lower than that in hair samples from diabetic patients.
  • glucose concentrations were locally elevated in diabetic hair samples, suggesting that blood glucose levels were high during this period.
  • the present invention converts glucose in a hair sample into gluconic acid to increase its polarity, it can be sensitively detected by mass spectrometry, and even the past state of the subject can be grasped.

Abstract

The purpose of the present invention is to provide a method for efficiently analyzing a biomolecule in hair. The method for analyzing a biomolecule in hair according to the present invention is characterized by comprising: a step for increasing the polarity of the biomolecule by treating the hair; and a step for subjecting the treated hair to mass spectrometry.

Description

毛髪中の生体分子の分析方法Method for analyzing biomolecules in hair
　本発明は、毛髪中の生体分子を効率的に分析する方法に関するものである。 The present invention relates to a method for efficiently analyzing biomolecules in hair.
　毛髪は、皮膚中の毛根部の先端部である毛球中の毛母細胞が***して増殖し、更に各部位に分化した上で角化し、皮膚外に押し出されたものである。皮膚外の毛髪は死んだ細胞であるが、いったん形成された構造がほぼそのまま残されている。即ち、毛髪は、毛乳頭細胞から毛母細胞へ増殖のシグナルが出され、そのシグナルを受けた毛母細胞が増殖・角化することにより成長するものであり、毛乳頭細胞はその周辺を走る毛細血管から栄養を供給されて活動しているため、血液に含まれる生体分子が毛母細胞に移行し、そのまま毛髪中に残留する可能性がある。 Hair is a hair matrix cell in the hair bulb, which is the tip of the hair root in the skin. The hair outside the skin is dead cells, but the structure once formed remains largely intact. That is, hair grows by sending a proliferation signal from dermal papilla cells to hair matrix cells, and the hair matrix cells that receive the signal proliferate and keratinize, and the dermal papilla cells run around them. Since hair is active by being supplied with nutrients from capillaries, there is a possibility that biomolecules contained in the blood migrate to hair matrix cells and remain in the hair as they are.
　より詳しくは、毛髪は、中心に存在する多孔質構造である髄質（メデュラ）、その周りに存在する繊維状の束とそれを取り巻く構造であり、毛髪の８５～９０％を占めるコルテックス、毛髪の表面に存在して半透明のうろこ状に４～１０枚重なっているキューティクルからなり、その８０～８５％がケラチンというタンパク質からなる。例えば、メラニン色素は、毛母細胞と共に毛球部に存在するメラノサイト（色素形成細胞）で産生され、毛母細胞が細胞***して髪が成長する際にメラノサイトから毛母細胞に受け渡されてコルテックス中に固定化され、毛髪の色を決定する。よって、毛細血管から移行した生体分子も、毛髪のコルテックス中などに固定化される可能性がある。 More specifically, hair consists of a medulla, which is a porous structure that exists in the center, a fibrous bundle that exists around it, and a structure that surrounds it, cortex that accounts for 85 to 90% of the hair The cuticle consists of 4 to 10 layers of translucent scales on the surface of the cuticle, 80 to 85% of which is composed of a protein called keratin. For example, melanin pigment is produced by melanocytes (pigment-forming cells) present in the hair bulb together with hair matrix cells, and is transferred from melanocytes to hair matrix cells when the hair matrix cells undergo cell division and hair grows. Immobilized in the cortex and determines the color of the hair. Therefore, biomolecules migrated from capillaries may also be immobilized in hair cortex.
　例えば非特許文献１には、糖尿病患者の毛髪中のグルコース量は、過去２～３ヵ月の血糖値の平均値の指標となるＨｂＡ１ｃと相関があり、しかも過去の血糖値の履歴も反映することが示唆されている。 For example, in Non-Patent Document 1, the amount of glucose in the hair of diabetic patients is correlated with HbA1c, which is an index of the average blood sugar level in the past 2 to 3 months, and also reflects the history of past blood sugar levels. is suggested.
　また、特許文献１には、長軸方向にスライスした毛髪試料に内部標準物質を適用した上で表面をイメージング質量分析に付し、毛髪中の生理活性物質を連続的かつ定量的に検出することにより、毛髪中の生理活性物質の使用時期と使用量を特定する方法が開示されている。 Further, in Patent Document 1, an internal standard substance is applied to a hair sample sliced in the longitudinal direction, and the surface is subjected to imaging mass spectrometry to continuously and quantitatively detect physiologically active substances in the hair. discloses a method for determining when and how much of a bioactive substance is used in hair.
特開２０１４－５２３２２号公報JP 2014-52322 A
　上述したように、毛髪試料を質量分析に付し、過去の履歴なども明らかにする方法が提案されている。
　しかし、毛髪試料は生体試料の一つであり、多種多様な化合物が含まれており、また検出すべき生体分子の含有量が微量である場合もあり、目的の生体分子の検出が難しい場合がある。
　そこで本発明は、毛髪中の生体分子を効率的に分析する方法を提供することを目的とする。 As described above, a method has been proposed in which a hair sample is subjected to mass spectrometry to clarify its past history and the like.
However, hair samples are one of the biological samples, and contain a wide variety of compounds, and the amount of biomolecules to be detected may be very small, making it difficult to detect the target biomolecules. be.
Accordingly, an object of the present invention is to provide a method for efficiently analyzing biomolecules in hair.
　本発明者は、上記課題を解決するために鋭意研究を重ねた。その結果、目的の生体分子の極性が上がるよう毛髪を処理することにより、質量分析で目的の生体分子が検出し易くなることを見出して、本発明を完成した。
　以下、本発明を示す。 The inventor of the present invention has made intensive studies to solve the above problems. As a result, the present inventors have found that the target biomolecules can be easily detected by mass spectrometry by treating the hair so as to increase the polarity of the target biomolecules, thereby completing the present invention.
The present invention is shown below.
　［１］　毛髪中の生体分子の分析方法であって、
　前記毛髪を処理することにより、前記生体分子の極性を上げる工程、及び、
　処理した前記毛髪を質量分析に付す工程を含むことを特徴とする方法。
　［２］　前記毛髪を酵素で処理することにより、前記生体分子の極性を上げる前記［１］に記載の方法。
　［３］　質量分析イメージングにより前記毛髪における前記生体分子の分布を測定する前記［１］または［２］に記載の方法。
　［４］　前記毛髪を２以上に切断し、各部を処理し且つ質量分析に付す前記［１］または［２］に記載の方法。
　［５］　前記生体分子がグルコースである前記［１］～［４］のいずれかに記載の方法。
　［６］　前記酵素が前記生体分子を酸化するオキシダーゼである前記［２］に記載の方法。 [1] A method for analyzing biomolecules in hair, comprising:
treating the hair to increase the polarity of the biomolecules; and
A method comprising subjecting said treated hair to mass spectrometry.
[2] The method according to [1] above, wherein the hair is treated with an enzyme to increase the polarity of the biomolecules.
[3] The method according to [1] or [2], wherein the distribution of the biomolecules in the hair is measured by mass spectrometry imaging.
[4] The method according to [1] or [2] above, wherein the hair is cut into two or more pieces, and each portion is treated and subjected to mass spectrometry.
[5] The method according to any one of [1] to [4], wherein the biomolecule is glucose.
[6] The method according to [2] above, wherein the enzyme is an oxidase that oxidizes the biomolecule.
　本発明方法によれば、毛髪試料に含まれる目的の生体分子の極性が上がることにより、質量分析で検出され易くなる。その結果、毛髪試料の分析がより効率的になる。よって本発明は、毛髪試料における目的の生体分子の検出の効率を改善できるものとして、非常に有用である。 According to the method of the present invention, the polarity of the target biomolecules contained in the hair sample increases, making them easier to detect by mass spectrometry. As a result, analysis of hair samples becomes more efficient. Therefore, the present invention is very useful as it can improve the efficiency of detection of target biomolecules in hair samples.
図１（１）は、Ｄ－グルコースのマススペクトルであり、図１（２）は、Ｄ－グルコースをグルコースオキシダーゼで処理して得られたグルコン酸のマススペクトルである。FIG. 1(1) is a mass spectrum of D-glucose, and FIG. 1(2) is a mass spectrum of gluconic acid obtained by treating D-glucose with glucose oxidase. 図２（１）は、糖尿病患者の毛髪試料の質量分析（ＭＳ）の結果であり、図２（２）は、糖尿病患者の毛髪試料のタンデム質量分析（ＭＳ／ＭＳ）の結果である。FIG. 2(1) is the result of mass spectrometry (MS) of a diabetic patient's hair sample, and FIG. 2(2) is the result of tandem mass spectrometry (MS/MS) of a diabetic patient's hair sample. 図３（１）は、健常者の毛髪試料の質量分析（ＭＳ）の結果であり、図３（２）は、健常者の毛髪試料のタンデム質量分析（ＭＳ／ＭＳ）の結果である。FIG. 3(1) shows the results of mass spectrometry (MS) of hair samples from healthy individuals, and FIG. 3(2) shows the results of tandem mass spectrometry (MS/MS) of hair samples from healthy individuals. 図４（１）は、糖尿病患者の毛髪試料における分子イオンピーク（１９５．０３）の質量分析イメージング結果であり、図４（２）は、糖尿病患者の毛髪試料におけるフラグメントイオンピーク（１２８．９６）の質量分析イメージング結果であり、図４（３）は、健常者の毛髪試料における分子イオンピーク（１９５．０３）の質量分析イメージング結果であり、図４（４）は、健常者の毛髪試料におけるフラグメントイオンピーク（１２８．９６）の質量分析イメージング結果である。FIG. 4(1) is the mass spectrometry imaging result of the molecular ion peak (195.03) in the diabetic hair sample, and FIG. 4(2) is the fragment ion peak (128.96) in the diabetic hair sample. FIG. 4(3) is the mass spectrometry imaging result of the molecular ion peak (195.03) in the hair sample of healthy subjects, and FIG. 4(4) is the result of It is the mass spectrometry imaging result of the fragment ion peak (128.96).
　本発明に係る毛髪中の生体分子の分析方法は、前記毛髪を処理することにより、前記生体分子の極性を上げる工程、及び、処理した前記毛髪を質量分析に付す工程を含む。以下、本発明の各工程を説明するが、本発明は以下の具体例に限定されるものではない。 The method for analyzing biomolecules in hair according to the present invention includes the steps of treating the hair to increase the polarity of the biomolecules, and subjecting the treated hair to mass spectrometry. Each step of the present invention will be described below, but the present invention is not limited to the following specific examples.
　１．毛髪試料の事前処理工程
　本工程では、毛髪試料の最表面のキューティクルを除去する。本工程の実施は任意であるが、生体分子はメデュラやコルテックスに蓄積することが多いため、より明確な分析のためには、キューティクルを除去することが好ましい。 1. Pretreatment Step of Hair Sample In this step, the cuticle on the outermost surface of the hair sample is removed. Implementation of this step is optional, but since biomolecules often accumulate in the medulla and cortex, it is preferable to remove the cuticle for clearer analysis.
　毛髪試料は、頭髪に限定されず、腋毛、睫毛、眉毛、髭、陰毛などであってもよいし、また、ヒトの他、イヌ、ネコ等の愛玩動物；ウシ、ブタ、ニワトリ、ウマ、ヒツジ、ヤギ等の家畜；マウス、ラット等の実験動物の毛髪試料であってもよい。 The hair sample is not limited to head hair, but may be armpit hair, eyelashes, eyebrows, whiskers, pubic hair, etc. In addition to humans, pets such as dogs and cats; cows, pigs, chickens, horses, and sheep , domestic animals such as goats; and experimental animals such as mice and rats.
　例えば、被検者がヒトである場合、ヒトの毛髪は１ヵ月で約１ｃｍ伸びることを基準にして、履歴を知りたい時期に応じて毛髪試料を採取すればよい。例えば、直近のデータが必要である場合には毛根に近い部分から毛髪試料を採取し、過去のデータが必要である場合にはより先端から毛髪試料を採取する。 For example, if the subject is a human, a hair sample can be collected according to the time period for which you want to know the history, based on the fact that human hair grows about 1 cm per month. For example, when the most recent data is required, a hair sample is taken from a portion near the hair root, and when past data is required, a hair sample is taken from the tip.
　毛髪試料は、キューティクルの除去の前に洗浄してもよい。例えば、精製水、メタノール、エタノール、イソプロパノール等のアルコール系溶媒に毛髪試料を浸漬したり、これらをウェス等に染み込ませた上で毛髪試料を拭いてもよい。毛髪試料を溶媒に浸漬する場合には、更に界面活性剤を使用したり、超音波を照射してもよい。但し、分析結果への影響を考慮して、最終的には溶媒のみで洗浄した上で乾燥することが好ましい。 The hair sample may be washed prior to cuticle removal. For example, the hair sample may be immersed in an alcohol-based solvent such as purified water, methanol, ethanol, or isopropanol, or may be soaked in a rag or the like and then wiped with the hair sample. When the hair sample is immersed in a solvent, a surfactant may be used or ultrasonic waves may be applied. However, in consideration of the influence on the analysis results, it is preferable to finally wash with only the solvent and then dry.
　キューティクルの除去方法は、特に制限されず、公知方法を用いればよい。例えば、毛髪試料を接着剤や両面テープ等によりガラススライドに固定し、氷結させた後、ミクロトームによりスライスする方法や、毛髪試料を溝に挟んで固定し、剃刀などのブレードを用いて毛髪表面を常温でスライスする方法が挙げられる。スライスは、コルテックスの露出面を最大にするために、髄質が露出するまで行うことが好ましい。 The cuticle removal method is not particularly limited, and a known method may be used. For example, a hair sample is fixed on a glass slide with an adhesive or double-sided tape, frozen, and then sliced with a microtome, or a hair sample is sandwiched between grooves and the surface of the hair is cut using a razor or other blade. A method of slicing at room temperature can be mentioned. Slicing is preferably done until the medulla is exposed to maximize the exposed surface of the cortex.
　２．毛髪試料の処理工程
　本工程では、毛髪試料を処理することにより、検出対象である生体分子の極性を上げる。目的の生体分子の極性を上げることにより、質量分析で検出され易いようにする。 2. Hair Sample Treatment Step In this step, the hair sample is treated to increase the polarity of the biomolecules to be detected. By increasing the polarity of the biomolecules of interest, they are made easier to detect by mass spectrometry.
　生体分子とは、生体に含まれる化合物や生体由来の化合物の総称であり、生体分子の中でも、生体の生命活動や生理機能の維持および調節にかかわる化合物は生理活性物質と呼ばれる。また、生体分子には、医薬品や毒性をもつ化合物を含める場合もあるが、これらを生物活性物質と呼ぶこともある。生体分子としては、例えば、アミノ酸、ペプチド、タンパク質、酵素、糖、脂質、ビタミン、補酵素、ホルモン、核酸、及びこれらの複合体や代謝産物が挙げられる。 Biomolecules is a general term for compounds contained in living organisms and compounds derived from living organisms. Among biomolecules, compounds related to the maintenance and regulation of biological activities and physiological functions of living organisms are called bioactive substances. Biomolecules may also include pharmaceuticals and toxic compounds, which are also referred to as biologically active substances. Biomolecules include, for example, amino acids, peptides, proteins, enzymes, sugars, lipids, vitamins, coenzymes, hormones, nucleic acids, complexes and metabolites thereof.
　化合物の極性とは、化合物中の正負の電荷の重心が一致せず電気的双極子モーメントをもつことをいい、本工程では、毛髪処理により目的の生体分子の双極子モーメントが大きくなるようにする。例えば、目的の生体分子が有する置換基の極性を高めればよく、－ＣＨ（ＯＨ）－Ｏ－基を－Ｃ（＝Ｏ）－Ｏ－基に酸化したり、エステル基をカルボキシ基と水酸基に加水分解したり、アミド基をカルボキシ基とアミノ基に加水分解したり、－ＣＯ2Ｈ基を－ＣＯ2 -基にしたり、－ＮＨ2基を－Ｎ+Ｈ3基にしたり、第三級アミノ基（＞Ｎ－）を第二級アミノ基（－ＮＨ－）に還元したり、第二級アミノ基（－ＮＨ－）を第一級アミノ基（－ＮＨ2）に還元したり、複素環の開環などが挙げられる。複素環の開環としては、例えば、クレアチニン（２－イミノ－１－メチルイミダゾリジン－４－オン）からクレアチン（１－メチルグアニジノ酢酸）への開環が挙げられる。 The polarity of a compound means that the centers of gravity of the positive and negative charges in the compound do not coincide and have an electric dipole moment. . For example, the polarity of the substituents of the target biomolecule may be increased, such as oxidizing the -CH(OH)-O- group to -C(=O)-O- group or converting the ester group to carboxy group and hydroxyl group. hydrolysis, amide groups to carboxy and amino groups, -CO 2 H groups to -CO 2 - groups, -NH 2 groups to -N + H 3 groups, tertiary An amino group (>N-) can be reduced to a secondary amino group (-NH-), a secondary amino group (-NH-) can be reduced to a primary amino group (-NH 2 ), or a complex Examples include ring opening. Heterocyclic ring openings include, for example, the opening of creatinine (2-imino-1-methylimidazolidin-4-one) to creatine (1-methylguanidinoacetic acid).
　極性を上げるべき生体分子の置換基としては、検出すべき生体分子が有する置換基の中から、比較的穏和な条件で極性を上げられる置換基や、極性を有効に上げられる置換基を選択すればよい。 As the substituent of the biomolecule whose polarity should be increased, a substituent that can increase the polarity under relatively mild conditions or a substituent that can effectively increase the polarity should be selected from the substituents of the biomolecule to be detected. Just do it.
　目的の生体分子の極性を上げる方法は、毛髪試料に過剰なダメージを与えるものでない限り適宜選択することができ、例えば化学的方法や生物学的方法が挙げられる。例えば、置換基を加水分解したり塩にする場合などには、酸や塩基を用いればよい。また、生物学的方法としては、酵素を用いる方法が挙げられる。例えば、生体分子の極性を上げる作用を示す酵素の水溶液を毛髪試料に噴霧したり或いは毛髪試料を当該水溶液に浸漬し、酵素の至適温度またはその前後でインキュベートすればよい。酵素としては、例えば、酸化還元酵素、転移酵素、加水分解酵素などが挙げられる。酵素は基質特異性が高いため、標的生体分子の極性を特異的に上げることができ、標的生体分子及び極性が上げられた標的生体分子誘導体の分析がより容易になる可能性があるという利点がある。 　The method of increasing the polarity of the target biomolecule can be appropriately selected as long as it does not excessively damage the hair sample, and examples include chemical methods and biological methods. For example, when a substituent is hydrolyzed or converted into a salt, an acid or a base may be used. Biological methods also include methods using enzymes. For example, a hair sample may be sprayed with an aqueous solution of an enzyme that increases the polarity of biomolecules, or the hair sample may be immersed in the aqueous solution and incubated at or around the optimal temperature for the enzyme. Examples of enzymes include oxidoreductases, transferases, and hydrolases. Enzymes have the advantage of being able to specifically polarize target biomolecules due to their high substrate specificity, potentially making the analysis of target biomolecules and their polarised target biomolecule derivatives easier. be.
　生体分子の置換基の極性を上げるための反応条件としては、検出すべき生体分子のダメージも考慮しつつ、毛髪試料に含まれる標的生体分子の極性が十分に上がる条件を選択することが好ましい。 As the reaction conditions for increasing the polarity of the substituents of biomolecules, it is preferable to select conditions that sufficiently increase the polarity of the target biomolecules contained in the hair sample, while also considering damage to the biomolecules to be detected.
　生体分子は、分析の目的に応じて適宜選択すればよい。例えば、被験者の健康状態や病状、更にはその履歴の把握のためには、グルコース、タンパク質、ペプチド、脂質、核酸、神経伝達物質、糖鎖など、健康状態や病状に応じてその血中濃度が変化する物質を選択すればよい。神経伝達物質としては、ドーパミン、ノルアドレナリン等のカテコールアミン類や、ＧＡＢＡ、グルタミン酸、トリプトファン、キレヌリン、グリシン、セロトニン等のアミノ酸類が挙げられる。また、犯罪捜査などのために、メタンフェタミン、アンフェタミン、ＭＤＭＡ、ＭＤＡ、モルヒネ、コカイン、ヘロイン、カンビノイド等の違法薬物を検出対象としてもよい。更に、ドーピング検査のために、ステロイド等のドーピング規制薬物を検出対象としてもよい。 The biomolecule can be selected as appropriate according to the purpose of analysis. For example, in order to understand the health condition and disease state of a subject, as well as their history, blood concentrations of glucose, proteins, peptides, lipids, nucleic acids, neurotransmitters, sugar chains, etc. You just have to choose a substance that changes. Examples of neurotransmitters include catecholamines such as dopamine and noradrenaline, and amino acids such as GABA, glutamic acid, tryptophan, kylenurine, glycine, and serotonin. Also, illegal drugs such as methamphetamine, amphetamine, MDMA, MDA, morphine, ***e, heroin, and cannabinoids may be detected for criminal investigations. Furthermore, for doping inspection, doping control drugs such as steroids may be detected.
　３．質量分析工程
　本工程では、前工程２により含まれる標的生体分子の極性が上げられた毛髪試料を質量分析に付し、標的生体分子または極性が上げられた標的生体分子誘導体の分子イオンピークやそれらのフラグメントピークの有無、及びその強度を確認する。 3. Mass Spectrometry Step In this step, the hair sample in which the polarity of the target biomolecules contained in the previous step 2 has been increased is subjected to mass spectrometry, and the molecular ion peaks of the target biomolecules or the target biomolecule derivatives whose polarity has been increased, and their molecular ion peaks. Check the presence or absence of fragment peaks and their intensity.
　イオン化の方法は、特に制限されず、電子イオン化法（ＥＩ）、化学イオン化法（ＣＩ）、高速原子衝撃法（ＦＡＢ）、エレクトロスプレーイオン化法（ＥＳＩ）、マトリックス支援レーザー脱離イオン化法（ＭＡＬＤＩ）、大気圧イオン化法（ＡＰＩ）など、いずれの方法も用い得るが、マトリックス支援レーザー脱離イオン化法（ＭＡＬＤＩ）、及び大気圧マトリックス支援レーザー脱離イオン化法（ＡＰ－ＭＡＬＤＩ）を好適に用いることができる。なお、ＥＩを除くイオン源を用いる場合、分子イオンが検出されるためマススペクトルの解釈が容易になる。 The ionization method is not particularly limited, and may be electron ionization (EI), chemical ionization (CI), fast atom bombardment (FAB), electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI). , atmospheric pressure ionization (API) and the like can be used, but matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) are preferably used. can. When an ion source other than EI is used, molecular ions are detected, which facilitates the interpretation of the mass spectrum.
　極性が上げられた標的生体分子の分子量や、タンデム質量分析（ＭＳ／ＭＳ）において生じるフラグメントイオンの分子量は、当業者であれば予測できる。得られたマススペクトル中、標的生体分子に関係するピークの強度を測定すればよい。 A person skilled in the art can predict the molecular weight of a target biomolecule with increased polarity and the molecular weight of fragment ions generated in tandem mass spectrometry (MS/MS). In the obtained mass spectrum, the intensity of peaks related to target biomolecules may be measured.
　毛母細胞には、増殖した際に血液に含まれる生体分子が含まれている可能性がある。毛母細胞は角化して毛髪を構成するため、毛母細胞に含まれる生体分子は毛髪試料に残留し且つ固定化されることにより、血液に含まれる生体分子の履歴が毛髪に残り得る。よって、毛髪試料を２以上に切断し、各部を処理し且つ質量分析に付すことにより、先端部の結果からより過去の、根本部の結果からより現在に近い、被験者の状態や病状などを把握することが可能になり得る。 Hair matrix cells may contain biomolecules contained in the blood when they proliferate. Since hair matrix cells are keratinized to form hair, the biomolecules contained in the hair matrix cells remain in the hair sample and are fixed, so that the history of the biomolecules contained in the blood can remain in the hair. Therefore, by cutting a hair sample into two or more pieces, processing each part, and subjecting it to mass spectrometry, the results of the tip part are more recent, and the results of the root part are closer to the present. it may be possible to
　また、質量分析イメージングにより、毛髪試料の端部から端部、或いは一部を連続的な質量分析に付し、毛髪試料における標的生体分子の分布を把握することも可能になり得る。かかる態様により、被験者の過去の状態や病歴などをより詳細に把握することが可能になり得る。質量分析イメージングとは、毛髪試料において微小測定点のマススペクトルを満遍なく測定し、得られたマススペクトルから注目するシグナルのみを抽出し、その強度に応じて二次元画像化することにより、標的生体分子の分布を可視化する技術である。 In addition, mass spectrometry imaging may enable continuous mass spectrometry from end to end or part of a hair sample to understand the distribution of target biomolecules in the hair sample. Such an aspect may enable a more detailed understanding of the subject's past conditions, medical history, and the like. Mass spectrometry imaging involves measuring the mass spectrum of minute measurement points in a hair sample evenly, extracting only the signal of interest from the obtained mass spectrum, and performing two-dimensional imaging according to the intensity of the target biomolecule. It is a technique to visualize the distribution of
　マトリックス支援レーザー脱離イオン化法（ＭＡＬＤＩ）のためには、固定した毛髪試料の表面にマトリックスを塗布する。マトリックスの塗布は常法によればよく、エアブラシを用いて手動で塗布してもよいが、均一性からマトリックスを蒸着または自動スプレー噴霧装置で塗布する手法や、蒸着と噴霧を組み合わせた手法が好ましく、塗布後、マトリックスを結晶化する。マトリックスの厚さは適宜調整すればよいが、例えば、０．１μｍ以上、２μｍ以下とすることができる。 For matrix-assisted laser desorption ionization (MALDI), a matrix is applied to the surface of a fixed hair sample. The matrix may be applied by a conventional method, and may be applied manually using an airbrush, but from the viewpoint of uniformity, a method of applying the matrix by vapor deposition or an automatic spraying device, or a method combining vapor deposition and spraying is preferred. , crystallizes the matrix after application. The thickness of the matrix may be adjusted as appropriate, and may be, for example, 0.1 μm or more and 2 μm or less.
　レーザーの照射径が小さい程、照射間隔を小さくすることができ画像解像度が高くなる一方で、イオン量が減少してＳ／Ｎ比が低くなり、データの信頼性が低くなる。よって、レーザーの照射径を２μｍ以上、２０μｍ以下とし、１区画当たり５０回以上、２００回以下、レーザーを照射することが好ましい。レーザーの照射間隔は、例えば、５μｍ以上、２００μｍ以下とすることができる。 The smaller the laser irradiation diameter, the shorter the irradiation interval and the higher the image resolution. Therefore, it is preferable to set the laser irradiation diameter to 2 μm or more and 20 μm or less, and to irradiate the laser 50 times or more and 200 times or less per section. The laser irradiation interval can be, for example, 5 μm or more and 200 μm or less.
　次に、質量分析イメージング用の専用ソフトフェアを用い、得られたマススペクトルから標的生体分子、又は毛髪試料の処理により標的生体分子から生成した誘導体の分子イオンピークを選択し、各レーザー照射位置における同分子イオンピークの強度に応じて二次元画像を得る。得られた二次元画像を、質量分析イメージング用の専用ソフトウェアや、Ｉｍａｇｅ　Ｊ等の画像解析用ソフトウェアを用いて、得られた画像のピクセル強度情報などから、毛髪試料における標的生体分子の分布を可視化することができる。 Next, using dedicated software for mass spectrometry imaging, the molecular ion peaks of the target biomolecules or the derivatives produced from the target biomolecules by treatment of the hair sample are selected from the obtained mass spectrum, and the peaks at each laser irradiation position are selected. A two-dimensional image is obtained according to the intensity of the same molecular ion peak. The distribution of target biomolecules in the hair sample is visualized from the pixel intensity information of the obtained image using dedicated software for mass spectrometry imaging and image analysis software such as Image J. can do.
　本願は、２０２１年５月１３日に出願された日本国特許出願第２０２１－８１６５８号に基づく優先権の利益を主張するものである。２０２１年５月１３日に出願された日本国特許出願第２０２１－８１６５８号の明細書の全内容が、本願に参考のため援用される。 This application claims the benefit of priority based on Japanese Patent Application No. 2021-81658 filed on May 13, 2021. The entire contents of the specification of Japanese Patent Application No. 2021-81658 filed on May 13, 2021 are incorporated herein by reference.
　以下、実施例を挙げて本発明をより具体的に説明するが、本発明はもとより下記実施例によって制限を受けるものではなく、前・後記の趣旨に適合し得る範囲で適当に変更を加えて実施することも勿論可能であり、それらはいずれも本発明の技術的範囲に包含される。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be modified appropriately within the scope that can conform to the gist of the above and later descriptions. It is of course possible to implement them, and all of them are included in the technical scope of the present invention.
　実施例１
　（１）グルコースのマススペクトル測定
　Ｎ－（１－ナフチル）エチレンジアミン塩酸塩（ＮＥＤＣ）を５０％メタノール水溶液に溶解し、７ｍｇ／ｍＬ溶液を調製した。Ｄ－グルコースの１．０ｍｇ／ｍＬ水溶液と、ＮＥＤＣの７ｍｇ／ｍＬ溶液とを当量混合した。得られた混合溶液（０．１μＬ）を、２．５ｃｍ×７．５ｃｍのＩＴＯガラス板に滴下し、自然乾燥させた。自然乾燥させた後、イメージング質量顕微鏡（「ｉＭＳｃｏｐｅ　ＴＲＩＯ」島津製作所社製）を使って、マススペクトルを測定した。結果を図１（１）に示す。 Example 1
(1) Measurement of mass spectrum of glucose N-(1-naphthyl)ethylenediamine hydrochloride (NEDC) was dissolved in a 50% methanol aqueous solution to prepare a 7 mg/mL solution. Equivalent amounts of a 1.0 mg/mL aqueous solution of D-glucose and a 7 mg/mL solution of NEDC were mixed. The resulting mixed solution (0.1 μL) was dropped on an ITO glass plate of 2.5 cm×7.5 cm and allowed to air dry. After air-drying, the mass spectrum was measured using an imaging mass microscope (“iMScope TRIO” manufactured by Shimadzu Corporation). The results are shown in FIG. 1(1).
　（２）グルコン酸のマススペクトル測定
Figure JPOXMLDOC01-appb-C000001
　０．１Ｍ酢酸緩衝液を１０倍に希釈して１０ｍＭ酢酸緩衝液を調製した。また、グルコースオキシダーゼ（ＧＯＤ）（富士フィルム和光純薬社製）を超純水に溶解し、２０００ｕｎｉｔｓ／ｍＬ水溶液を調製した。グルコースオキシダーゼ水溶液（８０μＬ）、１０ｍＭ酢酸緩衝液（２０μＬ）、及び超純水を混合し、１００ｕｎｉｔｓ／ｍＬグルコースオキシダーゼ溶液を調製した。
　Ｄ－グルコースを担持した前記ＩＴＯガラス板に、エアブラシを使って１００ｕｎｉｔｓ／ｍＬグルコースオキシダーゼ溶液（約２００μＬ）を均一に噴霧し、４０℃で３０分間インキュベートした後、自然乾燥した。次いで、前記ＮＥＤＣ溶液（約２００μＬ）を、エアブラシを使って均一に噴霧し、自然乾燥させた後、得られた試料のマススペクトルを、イメージング質量顕微鏡（「ｉＭＳｃｏｐｅ　ＴＲＩＯ」島津製作所社製）を使って測定した。結果を図１（２）に示す。 (2) Mass spectrum measurement of gluconic acid
Figure JPOXMLDOC01-appb-C000001
A 10 mM acetate buffer was prepared by diluting 0.1 M acetate buffer 10 times. Further, glucose oxidase (GOD) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in ultrapure water to prepare a 2000 units/mL aqueous solution. A glucose oxidase aqueous solution (80 μL), 10 mM acetate buffer (20 μL), and ultrapure water were mixed to prepare a 100 units/mL glucose oxidase solution.
A 100 units/mL glucose oxidase solution (approximately 200 μL) was evenly sprayed onto the D-glucose-supported ITO glass plate using an airbrush, incubated at 40° C. for 30 minutes, and air-dried. Next, the NEDC solution (approximately 200 μL) was uniformly sprayed with an airbrush and air-dried, and the mass spectrum of the obtained sample was analyzed using an imaging mass microscope (“iMScope TRIO” manufactured by Shimadzu Corporation). measured by The results are shown in FIG. 1(2).
　図１に示される結果の通り、Ｄ－グルコースを酵素的に酸化して得られたグルコン酸のマススペクトルピーク強度は、Ｄ－グルコース自体のマススペクトルピーク強度に比べて、おおよそ１００倍も高かった。 As the results shown in FIG. 1, the mass spectral peak intensity of gluconic acid obtained by enzymatically oxidizing D-glucose was approximately 100 times higher than the mass spectral peak intensity of D-glucose itself. .
　実施例２
　４０代男性の健常者と、４０代男性の糖尿病患者の毛髪を採取し、ヒト毛髪・繊維片スライス装置（「Ｂｉｏ　Ｓｌｉｃｅｒ　ＤＢＳ５０１　ＫＡＴＡＮＡ」ワイエイシイダステック社製）を使って、表面のキューティクルを除去した。
　キューティクルを除去した各毛髪試料を２．５ｃｍ×７．５ｃｍのＩＴＯガラス板に載せ、実施例１（２）と同様の条件で調製した１００ｕｎｉｔｓ／ｍＬグルコースオキシダーゼ溶液を、ガラス板１枚あたり約２００μＬ均一に噴霧し、４０℃で３０分間インキュベートした後、自然乾燥した。次いで、前記ＮＥＤＣ溶液（約２００μＬ）を、エアブラシを使って均一に噴霧し、自然乾燥させた後、得られた試料のマススペクトルを、イメージング質量顕微鏡を使って測定した。糖尿病患者の毛髪試料の質量分析（ＭＳ）の結果を図２（１）に、糖尿病患者の毛髪試料のタンデム質量分析（ＭＳ／ＭＳ）の結果を図２（２）に、健常者の毛髪試料の質量分析（ＭＳ）の結果を図３（１）に、健常者の毛髪試料のタンデム質量分析（ＭＳ／ＭＳ）の結果を図３（２）に示す。 Example 2
Hair was collected from a healthy male in his 40s and a diabetic male in his 40s, and cuticles on the surface were removed using a human hair/fiber piece slicing device (“Bio Slicer DBS501 KATANA” manufactured by Y.A.C. I.D.S Tech Co., Ltd.). .
Each hair sample from which the cuticle was removed was placed on a 2.5 cm x 7.5 cm ITO glass plate, and about 200 µL of a 100 units/mL glucose oxidase solution prepared under the same conditions as in Example 1 (2) was added to each glass plate. After uniformly spraying and incubating at 40° C. for 30 minutes, it was air-dried. Next, the NEDC solution (approximately 200 μL) was uniformly sprayed using an airbrush, air-dried, and the mass spectrum of the resulting sample was measured using an imaging mass microscope. The results of mass spectrometry (MS) of hair samples from diabetic patients are shown in FIG. 2 (1), the results of tandem mass spectrometry (MS/MS) of hair samples from diabetic patients are shown in FIG. FIG. 3(1) shows the results of mass spectrometry (MS), and FIG. 3(2) shows the results of tandem mass spectrometry (MS/MS) of a hair sample from a healthy subject.
　図２および図３に示される結果の通り、糖尿病患者と健常者の両方の毛髪試料のマススペクトルにおいて、グルコン酸の分子イオンピーク（［Ｍ－Ｈ］-，１９５．０３）と、グルコン酸から４つの側鎖水酸基が脱離したフラグメントイオンピーク（１２８．９７）が明確に認められた。なお、健常者の毛髪試料におけるグルコース由来のグルコン酸の分子イオンピークの強度は、糖尿病患者のものと比べると１／１０以下ではあるが、明確に検出できているほど本発明方法は高感度である。 As the results shown in FIGS. 2 and 3 show, in the mass spectra of both diabetic and healthy hair samples, the molecular ion peak of gluconic acid ([M−H] − , 195.03) and A fragment ion peak (128.97) resulting from elimination of four side chain hydroxyl groups was clearly recognized. Although the intensity of the molecular ion peak of glucose-derived gluconic acid in a hair sample from a healthy subject is 1/10 or less compared to that in a diabetic patient, the more clearly detected, the higher the sensitivity of the method of the present invention. be.
　また、糖尿病患者の毛髪試料における分子イオンピーク（１９５．０３）の質量分析イメージング結果を図４（１）に、糖尿病患者の毛髪試料におけるフラグメントイオンピーク（１２８．９６）の質量分析イメージング結果を図４（２）に、健常者の毛髪試料における分子イオンピーク（１９５．０３）の質量分析イメージング結果を図４（３）に、健常者の毛髪試料におけるフラグメントイオンピーク（１２８．９６）の質量分析イメージング結果を図４（４）に示す。なお、図４中、波長が長い暖色ほど対象化合物の濃度が高いことが示され、波長が短い寒色ほど対象化合物の濃度が低いことが示される。また、図４の質量分析イメージング結果はグルコン酸とそのフラグメントのイオンピークの濃度を示すが、当該濃度は当然にグルコース濃度に相関性を有する。 In addition, the mass spectrometry imaging result of the molecular ion peak (195.03) in the hair sample of the diabetic patient is shown in FIG. 4 (2) shows the mass spectrometry imaging result of the molecular ion peak (195.03) in the hair sample of healthy subjects, and the mass spectrometric imaging result of the fragment ion peak (128.96) in the hair sample of healthy subjects is shown in FIG. 4 (3). The imaging result is shown in FIG. 4(4). In FIG. 4, a warm color with a longer wavelength indicates a higher concentration of the target compound, and a cooler color with a shorter wavelength indicates a lower concentration of the target compound. Also, the mass spectrometric imaging results in FIG. 4 show the ion peak concentrations of gluconic acid and its fragments, which naturally have a correlation with the glucose concentration.
　図４に示される結果の通り、健常者の毛髪試料中のグルコース濃度は、糖尿病患者の毛髪試料中のグルコース濃度に比べて明らかに低いことが分かる。また、糖尿病患者の毛髪試料においてはグルコース濃度が局所的に高まっており、この時期に血糖値が高かったことが示唆されている。
　この様に、本発明により毛髪試料中のグルコースをグルコン酸に変換してその極性を高めれば、質量分析で鋭敏に検出され、被験者の過去の状態まで把握できることが明らかとなった。 As can be seen from the results shown in FIG. 4, the glucose concentration in hair samples from healthy subjects is clearly lower than that in hair samples from diabetic patients. In addition, glucose concentrations were locally elevated in diabetic hair samples, suggesting that blood glucose levels were high during this period.
Thus, it was clarified that if the present invention converts glucose in a hair sample into gluconic acid to increase its polarity, it can be sensitively detected by mass spectrometry, and even the past state of the subject can be grasped.

Claims (6)

  1. 　毛髪中の生体分子の分析方法であって、
    　前記毛髪を処理することにより、前記生体分子の極性を上げる工程、及び、
    　処理した前記毛髪を質量分析に付す工程を含むことを特徴とする方法。     A method for analyzing biomolecules in hair, comprising:
    treating the hair to increase the polarity of the biomolecules; and
    A method comprising subjecting said treated hair to mass spectrometry.
  2. 　前記毛髪を酵素で処理することにより、前記生体分子の極性を上げる請求項１に記載の方法。 The method according to claim 1, wherein the polarity of the biomolecules is increased by treating the hair with an enzyme.
  3. 　質量分析イメージングにより前記毛髪における前記生体分子の分布を測定する請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the distribution of said biomolecules in said hair is measured by mass spectrometry imaging.
  4. 　前記毛髪を２以上に切断し、各部を処理し且つ質量分析に付す請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the hair is cut into two or more pieces, and each section is processed and subjected to mass spectrometry.
  5. 　前記生体分子がグルコースである請求項１～４のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the biomolecule is glucose.
  6. 　前記酵素が前記生体分子を酸化するオキシダーゼである請求項２に記載の方法。 The method according to claim 2, wherein said enzyme is an oxidase that oxidizes said biomolecule.
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JP2009014433A (en) * 2007-07-03 2009-01-22 Yokohama City Univ Method for analyzing modification of bio-molecule in solid support
JP2012233925A (en) * 2012-09-05 2012-11-29 Canon Inc Mass spectroscope, ionizing agent for mass spectrometry, and mass spectrometric method
JP2014052322A (en) * 2012-09-10 2014-03-20 Osaka Medical College Analysis method of physiologically active substance contained in hair
WO2021054386A1 (en) * 2019-09-20 2021-03-25 株式会社あすか製薬メディカル Method for analyzing steroid hormones contained in animal hair sample, stress evaluation method and method for analyzing cause of hair loss

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Publication number Priority date Publication date Assignee Title
JP2007529001A (en) * 2003-07-15 2007-10-18 エクストレーム，シモン Apparatus and method for sample analysis using complex sample processing and sample holding apparatus
JP2009014433A (en) * 2007-07-03 2009-01-22 Yokohama City Univ Method for analyzing modification of bio-molecule in solid support
JP2012233925A (en) * 2012-09-05 2012-11-29 Canon Inc Mass spectroscope, ionizing agent for mass spectrometry, and mass spectrometric method
JP2014052322A (en) * 2012-09-10 2014-03-20 Osaka Medical College Analysis method of physiologically active substance contained in hair
WO2021054386A1 (en) * 2019-09-20 2021-03-25 株式会社あすか製薬メディカル Method for analyzing steroid hormones contained in animal hair sample, stress evaluation method and method for analyzing cause of hair loss

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