JP7390229B2 - Method for analyzing biological samples and evaluating mitochondrial function - Google Patents
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Description
本発明は、生体試料中のＳ－（２－スクシニル）システイン（２ＳＣ）を液体クロマトグラフィー－質量分析法を用いて定量する生体試料の分析方法に関する。 The present invention relates to a biological sample analysis method for quantifying S-(2-succinyl)cysteine (2SC) in a biological sample using liquid chromatography-mass spectrometry.
従来、糖尿病等の生活習慣病に起因して動脈硬化等の血管障害が発症することが知られている。血管障害は様々な加齢関連疾患の原因となるため、世界的に血管障害のマーカーとなる物質が探索されている。
本発明者らは、高血糖状態で培養した３Ｔ３－Ｌ１脂肪細胞において、Ｓ－（２－スクシニル）システイン（２ＳＣ）が増加することを明らかにしている（非特許文献２）。また、本発明者らは、１型及び２型糖尿病マウスは、健常なマウスに比して、血清中の２ＳＣの量が顕著に多いことも明らかにしている（非特許文献１参照）。
上述のように糖尿病は血管障害の原因となり得ることから、２ＳＣは血管障害の発症や進展に関与していることが示唆されている。
It has been known that lifestyle-related diseases such as diabetes cause vascular disorders such as arteriosclerosis. Since vascular disorders are the cause of various age-related diseases, substances that can serve as markers for vascular disorders are being searched for worldwide.
The present inventors have revealed that S-(2-succinyl)cysteine (2SC) increases in 3T3-L1 adipocytes cultured under hyperglycemic conditions (Non-Patent Document 2). The present inventors have also revealed that type 1 and type 2 diabetic mice have a significantly higher amount of 2SC in their serum than healthy mice (see Non-Patent Document 1).
As mentioned above, diabetes can cause vascular disorders, and it has been suggested that 2SC is involved in the onset and progression of vascular disorders.
また、２ＳＣは、フマル酸によってタンパク質が修飾されることにより製造されるものである。フマル酸は、ミトコンドリアで行われる代謝経路であるＴＣＡ（Ｔｒｉｃａｒｂｏｘｙｌｉｃ Ａｃｉｄ Ｃｙｃｌｅ）回路の中間体である。したがって、２ＳＣは、ミトコンドリアの機能異常にも関与していることも示唆されている。 Furthermore, 2SC is produced by modifying a protein with fumaric acid. Fumaric acid is an intermediate in the TCA (Tricarboxylic Acid Cycle) cycle, which is a metabolic pathway carried out in mitochondria. Therefore, it has been suggested that 2SC is also involved in mitochondrial dysfunction.
上述のように、２ＳＣは血管障害やミトコンドリア機能異常に関与していることが示唆されているため、血管障害やミトコンドリア機能異常を評価するためのマーカーや、血管障害やミトコンドリア機能異常を治療するための創薬ターゲットとして２ＳＣを利用することが期待されている。例えば２ＳＣを創薬ターゲットとして利用するためには、血中や生体組織中など、様々な場所に存在する２ＳＣを定量し、生体内において２ＳＣ量が増加するメカニズムを解明することが必要となる。生体試料中に含まれる２ＳＣを定量する方法としては、例えば特許文献１及び２が提案されている。 As mentioned above, 2SC has been suggested to be involved in vascular disorders and mitochondrial dysfunction, so it is necessary to develop markers for evaluating vascular disorders and mitochondrial dysfunction, and for treating vascular disorders and mitochondrial dysfunction. It is expected that 2SC will be used as a drug discovery target. For example, in order to utilize 2SC as a drug discovery target, it is necessary to quantify 2SC present in various locations such as blood and living tissues, and to elucidate the mechanism by which the amount of 2SC increases in vivo. As a method for quantifying 2SC contained in a biological sample, Patent Documents 1 and 2, for example, have been proposed.
特許文献１及び２には、液体クロマトグラフィー－質量分析により生体試料中の２ＳＣを定量することが記載されている。
特許文献１においては、液体クロマトグラフィー－質量分析に用いられる試料は以下のようにして調製される。まず、生体試料を、塩酸を用いて加水分解する。次に、加水分解後の生体試料に、強酸性陽イオン交換樹脂を充填したカラムを非酸性条件下で通過させる。前記カラムを通過した試料が、特許文献１において液体クロマトグラフィー－質量分析に用いられる試料である。
特許文献２においては、液体クロマトグラフィー－質量分析に用いられる試料は以下のようにして調製される。まず、生体試料を、限外濾過膜を用いて濾過処理する。次に、濾過処理後の生体試料に対して、例えばヒドリド還元剤を用いて還元処理を行う。前記還元処理後の試料が、特許文献２において液体クロマトグラフィー－質量分析に用いられる試料である。
Patent Documents 1 and 2 describe quantifying 2SC in a biological sample by liquid chromatography-mass spectrometry.
In Patent Document 1, a sample used for liquid chromatography-mass spectrometry is prepared as follows. First, a biological sample is hydrolyzed using hydrochloric acid. Next, the hydrolyzed biological sample is passed through a column packed with a strongly acidic cation exchange resin under non-acidic conditions. The sample that has passed through the column is the sample used in liquid chromatography-mass spectrometry in Patent Document 1.
In Patent Document 2, a sample used for liquid chromatography-mass spectrometry is prepared as follows. First, a biological sample is filtered using an ultrafiltration membrane. Next, the biological sample after the filtration treatment is subjected to a reduction treatment using, for example, a hydride reducing agent. The sample after the reduction treatment is the sample used for liquid chromatography-mass spectrometry in Patent Document 2.
特許文献１及び２の方法では、脂質の含有量が低い血液試料（血清、血漿等）中の２ＳＣを定量することは可能である一方、高脂血症等の脂質の含有量が多い血液試料や生体組織中の２ＳＣを精度よく定量することは困難である。そのような血液試料や生体組織には脂質等の夾雑物が多く含まれており、特許文献１及び２のように試料を調製しても、該夾雑物を十分に除去することはできないからである。このように、特許文献１及び２の方法では、幅広い生体試料中の２ＳＣを精度よく定量することは困難であった。 With the methods of Patent Documents 1 and 2, while it is possible to quantify 2SC in blood samples with low lipid content (serum, plasma, etc.), it is possible to quantify 2SC in blood samples with high lipid content such as hyperlipidemia. It is difficult to accurately quantify 2SC in biological tissues. Such blood samples and biological tissues contain many impurities such as lipids, and even if samples are prepared as in Patent Documents 1 and 2, these impurities cannot be sufficiently removed. be. Thus, with the methods of Patent Documents 1 and 2, it is difficult to accurately quantify 2SC in a wide range of biological samples.
したがって本発明は、幅広い生体試料中の２ＳＣを精度よく定量することができる生体試料の分析方法を提供することに関する。 Therefore, the present invention relates to providing a biological sample analysis method that can accurately quantify 2SC in a wide range of biological samples.
本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、生体試料に対して、脱脂処理を行う脱脂工程、タンパク質を沈殿させ、２ＳＣ化したタンパク質を含む沈殿物を回収する沈殿工程、塩酸による加水分解を行う加水分解工程をこの順で含む前処理工程を行い、該前処理工程後の試料に対して液体クロマトグラフィー－質量分析を行うことにより、生体試料中の２ＳＣを精度よく定量できることを知見した。 As a result of intensive research to solve the above problems, the present inventors have discovered a delipidation process in which a biological sample is defatted, and a precipitation process in which proteins are precipitated and a precipitate containing 2SC protein is collected. 2SC in biological samples can be accurately determined by performing a pretreatment process including hydrolysis with hydrochloric acid in this order, and performing liquid chromatography-mass spectrometry on the sample after the pretreatment process. We found that it can be quantified.
本発明は、上記の知見に基づいてなされたもので、下記の（１）～（６）の生体試料の分析方法、並びに（７）及び（８）のミトコンドリアの機能の評価方法を提供するものである。
（１）生体試料中のＳ－（２－スクシニル）システイン（２ＳＣ）を液体クロマトグラフィー－質量分析法を用いて定量する生体試料の分析方法であって、分析する前記生体試料に対して、脱脂処理を行う脱脂工程、タンパク質又はその断片を沈殿させ、２ＳＣ化したタンパク質又はその断片を含む沈殿物を回収する沈殿工程、塩酸による加水分解を行う加水分解工程をこの順で含む前処理工程を行うことを特徴とする、生体試料の分析方法。
（２）前記前処理工程は、前記加水分解工程後に、疎水性相互作用を用いた分離方法により２ＳＣの精製を行う分離精製工程を含む、（１）に記載の生体試料の分析方法。
（３）前記前処理工程は、前記加水分解工程後に、タンパク質及びリン脂質の除去を行う除去工程を含む、（１）又は（２）に記載の生体試料の分析方法。
（４）前記前処理工程は、精密濾過膜又は限外濾過膜で濾過処理を行う濾過工程を含む、（１）～（３）の何れか１に記載の生体試料の分析方法。
（５）前記濾過工程において前記限外濾過膜で濾過処理を行う場合、該限外濾過膜の分画分子量が１００００以下である、（４）に記載の生体試料の分析方法。
（６）前記液体クロマトグラフィー－質量分析法が液体クロマトグラフィー－タンデム型質量分析法である、（１）～（５）の何れか１に記載の生体試料の分析方法。
（７）（１）～（６）の何れか１に記載の生体試料の分析方法により、被験者から採取した前記生体試料中の２ＳＣを定量し、
前記生体試料中の２ＳＣの含有量に基づいて前記被験者のミトコンドリアの機能を評価する、ミトコンドリアの機能の評価方法。
（８）標準試料中の２ＳＣの含有量と比較して前記生体試料中の２ＳＣの含有量が有意に高い場合に、前記被験者のミトコンドリアの機能に障害があると予測又は判断する、（７）に記載のミトコンドリアの機能の評価方法。
The present invention was made based on the above findings, and provides the following methods for analyzing biological samples (1) to (6), and methods for evaluating mitochondrial function as described in (7) and (8) below. It is.
(1) A biological sample analysis method for quantifying S-(2-succinyl)cysteine (2SC) in a biological sample using liquid chromatography-mass spectrometry, in which the biological sample to be analyzed is delipidated. A pretreatment step is performed, which includes, in this order, a delipidation step for treatment, a precipitation step for precipitating the protein or its fragments and recovering the precipitate containing the 2SC-converted protein or its fragments, and a hydrolysis step for hydrolysis with hydrochloric acid. A biological sample analysis method characterized by:
(2) The biological sample analysis method according to (1), wherein the pretreatment step includes a separation and purification step of purifying 2SC by a separation method using hydrophobic interaction after the hydrolysis step.
(3) The biological sample analysis method according to (1) or (2), wherein the pretreatment step includes a removal step of removing proteins and phospholipids after the hydrolysis step.
(4) The method for analyzing a biological sample according to any one of (1) to (3), wherein the pretreatment step includes a filtration step of performing filtration with a microfiltration membrane or an ultrafiltration membrane.
(5) The method for analyzing a biological sample according to (4), wherein when the filtration process is performed using the ultrafiltration membrane in the filtration step, the ultrafiltration membrane has a molecular weight cutoff of 10,000 or less.
(6) The method for analyzing a biological sample according to any one of (1) to (5), wherein the liquid chromatography-mass spectrometry method is a liquid chromatography-tandem mass spectrometry method.
(7) quantifying 2SC in the biological sample collected from the subject by the biological sample analysis method described in any one of (1) to (6);
A method for evaluating mitochondrial function, comprising evaluating the mitochondrial function of the subject based on the content of 2SC in the biological sample.
(8) When the content of 2SC in the biological sample is significantly higher than the content of 2SC in the standard sample, it is predicted or determined that the mitochondrial function of the subject is impaired. (7) The method for evaluating mitochondrial function described in .
本発明の生体試料の分析方法によれば、幅広い種類の生体試料中の２ＳＣを精度よく定量することができる。また、本発明は、該分析方法により定量された、生体試料中の２ＳＣの含有量に基づき、ミトコンドリアの機能を評価することもできる。 According to the biological sample analysis method of the present invention, 2SC in a wide variety of biological samples can be quantified with high accuracy. Furthermore, the present invention can also evaluate mitochondrial function based on the content of 2SC in a biological sample determined by the analysis method.
以下、本発明をその好ましい実施態様に基づいて説明する。
本発明の生体試料の分析方法は、生体試料中のＳ－（２－スクシニル）システイン（以下、２ＳＣともいう）を液体クロマトグラフィー－質量分析法を用いて定量する。２ＳＣは、フマル酸がタンパク質中のシステイン残基側鎖と反応して生じ、生体中では、タンパク質若しくはペプチドと結合した非遊離体、又はタンパク質及びペプチドの何れとも結合していない遊離体として存在する。本発明において定量する２ＳＣは、非遊離体の２ＳＣである。
The present invention will be explained below based on its preferred embodiments.
In the biological sample analysis method of the present invention, S-(2-succinyl)cysteine (hereinafter also referred to as 2SC) in a biological sample is quantified using liquid chromatography-mass spectrometry. 2SC is produced when fumaric acid reacts with cysteine residue side chains in proteins, and exists in living organisms as a non-free form bound to proteins or peptides, or as a free form bound to neither proteins nor peptides. . The 2SC quantified in the present invention is non-free 2SC.
タンパク質と結合した非遊離体の２ＳＣは、タンパク質中のシステイン残基がフマル酸により修飾されることにより形成された２ＳＣである。ペプチドと結合した非遊離体の２ＳＣは、ペプチド中のシステイン残基がフマル酸により修飾されていることにより生じたものである。ペプチドと結合した非遊離体の２ＳＣは、ペプチド中のシステイン残基がフマル酸により修飾されることにより生じたものと、２ＳＣが形成されたタンパク質のペプチド鎖が切断され、断片化・低分子量化したものとの両者を含む。以下、２ＳＣが形成されたタンパク質を２ＳＣ化したタンパク質と言う。 Non-free 2SC bound to a protein is 2SC formed by modifying a cysteine residue in a protein with fumaric acid. The non-free form of 2SC bound to the peptide is produced by modifying the cysteine residue in the peptide with fumaric acid. Non-free 2SC bound to a peptide is generated by modification of cysteine residues in the peptide with fumaric acid, and 2SC is generated by cleavage of the peptide chain of the protein in which 2SC is formed, resulting in fragmentation and lower molecular weight. This includes both. Hereinafter, a protein in which 2SC has been formed will be referred to as a 2SC-formed protein.
本発明の生体試料の分析方法は、分析する生体試料に対して、脱脂工程、沈殿工程、加水分解工程をこの順で含む前処理工程を行う。以下、本発明に係る前処理工程における各工程について説明する。 In the biological sample analysis method of the present invention, a biological sample to be analyzed is subjected to a pretreatment process including a delipidation process, a precipitation process, and a hydrolysis process in this order. Each step in the pretreatment step according to the present invention will be explained below.
［脱脂工程］
脱脂工程においては、生体試料に対して脱脂処理を行う。本発明に係る脱脂処理の対象物である生体試料は、健常体から採取されたものであっても、疾病罹患患者のような非健常体から採取されたものでも良い。また生体試料としては、生体から採取されたあらゆる細胞、組織、及び体液、例えば、皮膚、筋肉、骨、毛髪、爪、脂肪組織、脳神経系、心臓及び血管等の循環器系、肺、肝臓、脾臓、膵臓、腎臓、消化器系、胸腺、血管、リンパ管、血液試料（全血、血清、血漿、リンパ液、唾液、尿、精液、腹水、喀痰等、並びにそれらの培養物が挙げられる。このうち、低侵襲性の観点から血液試料（全血、血清、血漿）、リンパ液、尿、皮膚、末梢血管、筋肉、脂肪組織、が好ましく、血清、血漿がより好ましい。
[Degreasing process]
In the degreasing step, the biological sample is degreased. The biological sample to be subjected to the degreasing treatment according to the present invention may be collected from a healthy body or from an unhealthy body such as a patient suffering from a disease. Biological samples include all cells, tissues, and body fluids collected from living organisms, such as skin, muscles, bones, hair, nails, adipose tissue, cranial nervous system, circulatory system such as heart and blood vessels, lungs, liver, Spleen, pancreas, kidney, digestive system, thymus, blood vessels, lymph vessels, blood samples (whole blood, serum, plasma, lymph, saliva, urine, semen, ascites, sputum, etc., and cultures thereof. Among these, blood samples (whole blood, serum, plasma), lymph fluid, urine, skin, peripheral blood vessels, muscle, and adipose tissue are preferred from the viewpoint of minimal invasiveness, and serum and plasma are more preferred.
本発明に係る脱脂工程の主たる目的は、分析対象である生体試料から脂質を除去することにある。脱脂工程において行う脱脂処理は、例えば、生体試料に対して有機溶媒を加え撹拌した後に遠心分離し、遠心分離後の沈殿物を回収することにより行うことができる。遠心分離後の沈殿物は、必要に応じて、乾燥させた後蒸留水等に溶解又は懸濁してもよい。
有機溶媒としては、例えば、ヘキサン、クロロホルム、メタノール、エタノール、アセトン、シクロヘキサン、トルエン、キシレン、イソプロパノール、ブタノール、アセトニトリル等が挙げられ、これらの中から１種を単独で又は２種以上を混合して使用することができる。
The main purpose of the delipidation process according to the present invention is to remove lipids from the biological sample to be analyzed. The degreasing treatment carried out in the degreasing process can be carried out, for example, by adding an organic solvent to a biological sample, stirring it, centrifuging it, and collecting the precipitate after centrifugation. The precipitate after centrifugation may be dried and then dissolved or suspended in distilled water or the like, if necessary.
Examples of the organic solvent include hexane, chloroform, methanol, ethanol, acetone, cyclohexane, toluene, xylene, isopropanol, butanol, acetonitrile, etc., and one type of these may be used alone or two or more types may be mixed together. can be used.
［沈殿工程］
沈殿工程においては、脱脂工程後の試料中のタンパク質又はその断片を沈殿させ、２ＳＣ化したタンパク質又はその断片を含む沈殿物を回収する。タンパク質の断片とは、タンパク質のペプチド鎖が切断され、断片化・低分子量化したものである。
[Precipitation process]
In the precipitation step, proteins or fragments thereof in the sample after the defatting step are precipitated, and a precipitate containing the 2SC-converted proteins or fragments thereof is recovered. A protein fragment is one in which the peptide chain of a protein is cleaved, resulting in fragmentation and lower molecular weight.
本発明に係る沈殿工程の主たる目的は、２ＳＣ化したタンパク質又はその断片を回収し、夾雑物を除去することにある。タンパク質を沈殿させる方法としては、例えば、脱脂工程後の試料に沈殿剤を加える方法を用いることができる。沈殿剤としては、塩、アルコール、有機溶媒、酸、水溶性ポリマー等が挙げられる。塩としては、硫酸アンモニウム、酢酸アンモニウム等が挙げられる。アルコールとしては、エタノール、プロパノール、メタノール等が挙げられる。有機溶媒としては、アセトン、クロロホルム、アセトニトリル等が挙げられる。酸としては、トリクロロ酢酸、塩酸、トリフルオロ酢酸等が挙げられる。水溶性ポリマーとしては、ポリエチレングリコール、デキストラン等が挙げられる。回収した沈殿物は、必要に応じて乾燥させてもよい。また、乾燥させた後蒸留水等に溶解してもよい。 The main purpose of the precipitation step according to the present invention is to recover 2SC-formed proteins or fragments thereof and to remove impurities. As a method for precipitating proteins, for example, a method of adding a precipitant to a sample after a defatting step can be used. Examples of the precipitating agent include salts, alcohols, organic solvents, acids, and water-soluble polymers. Examples of the salt include ammonium sulfate and ammonium acetate. Examples of alcohol include ethanol, propanol, methanol, and the like. Examples of organic solvents include acetone, chloroform, acetonitrile, and the like. Examples of acids include trichloroacetic acid, hydrochloric acid, trifluoroacetic acid, and the like. Examples of water-soluble polymers include polyethylene glycol, dextran, and the like. The collected precipitate may be dried if necessary. Alternatively, it may be dissolved in distilled water or the like after drying.
沈殿剤として、例えばトリクロロ酢酸を用いた場合、試料中のタンパク質が変性する。仮に、トリクロロ酢酸を用いて沈殿工程を行い、試料中のタンパク質が変性した後に、脱脂工程を行った場合、脱脂工程において生体試料が十分に脱脂されない恐れがある。特に、生体試料として、脂肪含有量が多い組織を用いた場合には、生体試料が十分に脱脂されないことにより、該生体試料中の２ＳＣを精度よく定量することができない恐れがある。本発明のように、脱脂工程後に沈殿工程を行うことにより、生体試料を十分に脱脂することができるようになり、該生体試料中の２ＳＣを精度よく定量することができるようになる。 For example, when trichloroacetic acid is used as a precipitant, proteins in the sample are denatured. If a precipitation step is performed using trichloroacetic acid to denature proteins in the sample, and then a delipidation step is performed, there is a possibility that the biological sample may not be sufficiently delipidated in the delipidation step. In particular, when a tissue with a high fat content is used as a biological sample, there is a possibility that 2SC in the biological sample cannot be accurately quantified because the biological sample is not sufficiently delipidated. By performing the precipitation step after the delipidation step as in the present invention, it becomes possible to sufficiently delipidate the biological sample, and it becomes possible to accurately quantify 2SC in the biological sample.
［加水分解工程］
加水分解工程においては、塩酸による加水分解を行う。本発明に係る加水分解工程の主たる目的は、沈殿工程において回収した２ＳＣ化したタンパク質又はその断片を加水分解し、２ＳＣを遊離させることにある。加水分解工程においては、例えば、沈殿工程において回収した沈殿物に対し、塩酸を添加し、必要に応じて振盪又は撹拌した後、静置し、該沈殿物を加水分解することができる。さらに、加水分解中に反応液を加温すると好ましい。また必要に応じて、途中で試料を攪拌して、反応容器の壁に付着した試料を再度反応液に混合してもよい。上記加水分解工程は、液相において行われる。すなわち、液体の状態の反応液中で試料の加水分解反応を進行させる。加水分解に使用される塩酸の量、反応時間および温度の条件は、前記沈殿物を十分に溶解できる条件且つ反応液が液相となる条件であればよく、使用する生体試料の種類に応じて決定すればよい。例えば血液試料を用いる場合、沈殿工程により得られた沈殿物を蒸留水に溶解した試料１００μＬに対して塩酸が５～８Ｎであればよい。加水分解工程の一例においては、沈殿工程により得られた沈殿物を蒸留水に溶解した試料１００μＬに対して、２０～２９質量％塩酸溶液を１ｍＬ～４ｍＬを添加し、攪拌した後、６５～１００℃で６～２４時間、試料を加水分解させればよい。
加水分解工程後の試料は、必要に応じて遠心又は濾過されて沈殿物を除去した後、エバポレーター等により乾固処理され、酸を除去される。乾固処理した試料は、次の工程を行うまで室温保存しておくことができる。乾固処理した試料は、適切な溶媒等に溶解又は分散させた後に、次の工程に用いる。
[Hydrolysis process]
In the hydrolysis step, hydrolysis is performed using hydrochloric acid. The main purpose of the hydrolysis step according to the present invention is to hydrolyze the 2SC-converted protein or its fragment recovered in the precipitation step to liberate 2SC. In the hydrolysis step, for example, hydrochloric acid is added to the precipitate collected in the precipitation step, shaken or stirred as necessary, and then allowed to stand to hydrolyze the precipitate. Furthermore, it is preferable to heat the reaction solution during hydrolysis. Further, if necessary, the sample may be stirred midway through, and the sample adhering to the wall of the reaction vessel may be mixed into the reaction liquid again. The above hydrolysis step is carried out in the liquid phase. That is, the hydrolysis reaction of the sample is allowed to proceed in the reaction liquid in a liquid state. The amount of hydrochloric acid used for hydrolysis, reaction time, and temperature conditions may be such that the precipitate can be sufficiently dissolved and the reaction solution will be in a liquid phase, and may vary depending on the type of biological sample used. All you have to do is decide. For example, when using a blood sample, it is sufficient that the amount of hydrochloric acid is 5 to 8N per 100 μL of the sample obtained by dissolving the precipitate obtained in the precipitation step in distilled water. In an example of the hydrolysis step, 1 mL to 4 mL of a 20 to 29 mass% hydrochloric acid solution is added to 100 μL of a sample obtained by dissolving the precipitate obtained in the precipitation step in distilled water, and after stirring, 65 to 100 The sample may be hydrolyzed for 6 to 24 hours at ℃.
The sample after the hydrolysis step is centrifuged or filtered as necessary to remove precipitates, and then dried using an evaporator or the like to remove acids. The dried sample can be stored at room temperature until the next step. The dried sample is used in the next step after being dissolved or dispersed in an appropriate solvent.
加水分解工程後の試料は、そのまま液体クロマトグラフィー－質量分析法による分析に用いることができるが、前処理工程は、加水分解工程後に精製工程を含んでいることが好ましい。精製工程としては、タンパク質及びリン脂質の除去を行う除去工程、疎水性相互作用を用いた分離方法により２ＳＣの精製を行う分離精製工程、及び精密濾過膜又は限外濾過膜で濾過処理を行う濾過工程が挙げられる。除去工程、分離精製工程及び濾過工程は、この順で行うことが好ましい。精製工程としては、除去工程、分離精製工程及び濾過工程の全ての工程を行ってもよいし、除去工程、分離精製工程及び濾過工程のうち、何れか１つ又は２つの工程を行ってもよい。除去工程を行った場合は、濾過工程を行うことが好ましい。 The sample after the hydrolysis step can be used as it is for analysis by liquid chromatography-mass spectrometry, but the pretreatment step preferably includes a purification step after the hydrolysis step. The purification process includes a removal process in which proteins and phospholipids are removed, a separation and purification process in which 2SC is purified by a separation method using hydrophobic interactions, and a filtration process in which filtration is performed using a microfiltration membrane or an ultrafiltration membrane. One example is the process. It is preferable that the removal step, separation and purification step, and filtration step are performed in this order. As the purification process, all of the removal process, separation and purification process, and filtration process may be performed, or any one or two of the removal process, separation and purification process, and filtration process may be performed. . When the removal step is performed, it is preferable to perform the filtration step.
除去工程の主たる目的は、加水分解工程後の試料から夾雑物であるタンパク質及びリン脂質を除去することにある。前処理工程が除去工程を含んでいることにより、試料中の夾雑物を減らすことができるため、より精度よく２ＳＣを定量することができるようになる。以下、この点について詳述する。 The main purpose of the removal step is to remove contaminants such as proteins and phospholipids from the sample after the hydrolysis step. By including the removal step in the pretreatment step, it is possible to reduce impurities in the sample, thereby making it possible to quantify 2SC with higher accuracy. This point will be explained in detail below.
分析に用いられる試料中にタンパク質やリン脂質が含まれている場合、該試料のイオン化が抑制され、該試料を液体クロマトグラフィー－質量分析法を用いて分析した際に検出感度が低下し、精度のよい結果を得ることが困難となる恐れがある。前処理工程が除去工程を含むことにより、２ＳＣのイオン化が抑制されにくくなる。これにより、試料を液体クロマトグラフィー－質量分析法を用いて分析した際に２ＳＣをより精度よく定量することができるようになる。
生体試料として生体組織を用いる場合は、除去工程を行ってもよいし行わなくてもよいが、生体試料として全血、血清、血漿を用いる場合は、除去工程を行うことが特に好ましい。
If the sample used for analysis contains proteins or phospholipids, the ionization of the sample will be suppressed, resulting in a decrease in detection sensitivity and accuracy when the sample is analyzed using liquid chromatography-mass spectrometry. It may be difficult to obtain good results. By including the removal step in the pretreatment step, ionization of 2SC becomes less likely to be suppressed. This makes it possible to more accurately quantify 2SC when a sample is analyzed using liquid chromatography-mass spectrometry.
When using biological tissue as a biological sample, the removal step may or may not be performed, but when using whole blood, serum, or plasma as the biological sample, it is particularly preferable to perform the removal step.
除去工程は、例えば、タンパク質やリン脂質を除去することができるフィルターを用いて試料を濾過することにより行うことができる。そのようなフィルターとしては、市販品を適宜用いることができ、例えば、アジレント・テクノロジー株式会社製のＣａｐｔｉｖａ ＮＤ Ｌｉｐｉｄが挙げられる。 The removal step can be performed, for example, by filtering the sample using a filter that can remove proteins and phospholipids. As such a filter, a commercially available product can be used as appropriate, and for example, Captiva ND Lipid manufactured by Agilent Technologies, Inc. can be mentioned.
［分離精製工程］
分離精製工程の主たる目的は、加水分解工程後の試料から、脂肪酸等の疎水性化合物を除去することにある。前処理工程が分離精製工程を含んでいることにより、イオン化抑制と、分析装置及びカラムの汚染を軽減することができるため、より精度よく２ＳＣを定量することができるようになる。
また、生体試料として培養細胞を用いた場合に、該培養細胞から得られた分析用の試料中に、該培養細胞を培養するための培地に含まれる色素が混入することがある。前処理工程が分離精製工程を含んでいることにより、前記の色素を除去することもできる。
[Separation and purification process]
The main purpose of the separation and purification process is to remove hydrophobic compounds such as fatty acids from the sample after the hydrolysis process. By including the separation and purification step in the pretreatment step, ionization can be suppressed and contamination of the analyzer and column can be reduced, so that 2SC can be quantified with higher accuracy.
Furthermore, when cultured cells are used as biological samples, dyes contained in the medium for culturing the cultured cells may be mixed into the sample for analysis obtained from the cultured cells. By including the separation and purification step in the pretreatment step, the dye described above can also be removed.
分離精製工程における前記の分離方法としては、例えば、逆相クロマトグラフィー等が挙げられる。逆相クロマトグラフィーで使用する充填剤としては、オクタデシルシリル基等のアルキル基が結合したシリカゲル等が挙げられる。 Examples of the separation method in the separation and purification step include reverse phase chromatography. Examples of the packing material used in reversed phase chromatography include silica gel to which an alkyl group such as an octadecylsilyl group is bonded.
［濾過工程］
濾過工程の主たる目的は、試料中の不溶性物質を除去することにある。前処理工程が濾過工程を含むことにより、分析装置及びカラムの汚染を軽減することが可能となる。濾過工程で用いる精密濾過膜の孔径は、分析装置のカラム流路及びイオン化部等に干渉する物質を除去するという観点から、好ましくは０．４５μｍ以下、より好ましくは０．２μｍ以下である。濾過工程で用いる限外濾過膜の分画分子量は、測定対象である２ＳＣ等の修飾アミノ酸を通過させ、それ以外の高分子化合物を除去するという観点から、好ましくは１００００以下、より好ましくは５０００以下、さらに好ましくは３０００以下である。
[Filtration process]
The main purpose of the filtration step is to remove insoluble substances in the sample. By including the filtration step in the pretreatment step, it is possible to reduce contamination of the analytical device and column. The pore diameter of the precision filtration membrane used in the filtration step is preferably 0.45 μm or less, more preferably 0.2 μm or less, from the viewpoint of removing substances that interfere with the column flow path, ionization section, etc. of the analyzer. The molecular weight cutoff of the ultrafiltration membrane used in the filtration step is preferably 10,000 or less, more preferably 5,000 or less, from the viewpoint of allowing modified amino acids such as 2SC to pass through and removing other polymer compounds. , more preferably 3000 or less.
以上のように前処理工程を行って得られた試料に対し、液体クロマトグラフィー－質量分析法を用いて、該試料中の２ＳＣを定量する。液体クロマトグラフィー－質量分析法としては、例えば、ＬＣ－ＭＳ法、ＬＣ－ＭＳ／ＭＳ法、ＬＣ－ＭＳ／ＭＳ／ＭＳ法等が挙げられる。検出感度をより向上させるためには、ＬＣ－ＭＳ／ＭＳ法や、ＬＣ－ＭＳ／ＭＳ／ＭＳ法などの液体クロマトグラフィー－タンデム型質量分析法がより好ましい。 For the sample obtained by performing the pretreatment step as described above, 2SC in the sample is quantified using liquid chromatography-mass spectrometry. Examples of the liquid chromatography-mass spectrometry method include LC-MS method, LC-MS/MS method, and LC-MS/MS/MS method. In order to further improve detection sensitivity, liquid chromatography-tandem mass spectrometry methods such as LC-MS/MS method and LC-MS/MS/MS method are more preferable.
本発明の生体試料の分析方法によれば、生体試料に対し上述のように前処理工程を行っているため、血清を生体試料として用いた場合のみならず、例えば、脳、筋肉、脂肪組織、骨組織、皮膚、肝臓、血液（血漿、血清等）等の脂質が多く含まれる組織を生体試料として用いた場合であっても、該生体試料中の２ＳＣを精度よく定量することができる。つまり本発明の生体試料の分析方法によれば、幅広い種類の生体試料中の２ＳＣを精度よく定量することができる。 According to the biological sample analysis method of the present invention, since the biological sample is subjected to the pretreatment step as described above, it can be used not only when serum is used as a biological sample, but also when using brain, muscle, adipose tissue, etc. Even when a tissue rich in lipids such as bone tissue, skin, liver, blood (plasma, serum, etc.) is used as a biological sample, 2SC in the biological sample can be quantified with high accuracy. In other words, according to the biological sample analysis method of the present invention, 2SC in a wide variety of biological samples can be quantified with high accuracy.
前処理工程を経て得られた試料は、必要に応じて適切な溶媒に溶解させ、分析用試料としてもよい。そのような溶媒としては、液体クロマトグラフィー－質量分析法における液体クロマトグラフィーの移動相の最終条件と同じ溶媒を用いることが好ましい。例えば、メタノールの水溶液やアセトニトリルの水溶液、アセトニトリルとトリフルオロ酢酸の混合水溶液、アセトニトリルとギ酸の混合水溶液等が挙げられるが、特に限定されない。より具体的には、前処理工程を経て得られた試料を乾固処理し、２０体積％アセトニトリル＋０．１質量％ギ酸水溶液に溶解させて、分析用試料としてもよい。あるいは、前処理工程を経て得られた試料を乾固処理し、２０体積％アセトニトリル＋０．１質量％ギ酸水溶液に溶解させた後、孔径０．２μｍのフィルターを用いた精密濾過処理にかけ、回収した濾液に等量の２０体積％アセトニトリル＋０．１質量％ギ酸水溶液を添加して２倍希釈して、分析用試料としてもよい。また、生体試料として血清１００μＬを用いた場合、前処理工程を経て得られた試料に対して、５００～２０００μＬ程度の２０体積％アセトニトリル＋０．１質量％ギ酸水溶液を添加するとよい。 The sample obtained through the pretreatment step may be dissolved in an appropriate solvent as necessary and used as a sample for analysis. As such a solvent, it is preferable to use a solvent that has the same final conditions as the mobile phase of liquid chromatography in liquid chromatography-mass spectrometry. Examples include an aqueous methanol solution, an acetonitrile aqueous solution, a mixed aqueous solution of acetonitrile and trifluoroacetic acid, a mixed aqueous solution of acetonitrile and formic acid, etc., but are not particularly limited. More specifically, the sample obtained through the pretreatment step may be dried and dissolved in a 20% by volume acetonitrile + 0.1% by mass formic acid aqueous solution to prepare a sample for analysis. Alternatively, the sample obtained through the pretreatment step was dried, dissolved in a 20% by volume acetonitrile + 0.1% by mass formic acid aqueous solution, and then subjected to precision filtration using a filter with a pore size of 0.2 μm and collected. An equal amount of 20 volume % acetonitrile + 0.1 mass % formic acid aqueous solution may be added to the filtrate to dilute it twice, and it may be used as a sample for analysis. Further, when 100 μL of serum is used as a biological sample, it is preferable to add about 500 to 2000 μL of a 20 volume % acetonitrile + 0.1 mass % formic acid aqueous solution to the sample obtained through the pretreatment step.
液体クロマトグラフィー－質量分析法により２ＳＣを定量する際の測定条件は、機器の型、試料の状態等に応じて、当業者が通常の知識に基づいて適宜設定すればよい。液体クロマトグラフィーの条件は供される試料によって異なるが、例えば、上述の２０体積％アセトニトリル＋０．１質量％ギ酸水溶液に対しては、移動相にギ酸水溶液とギ酸アセトニトリル溶液でグラジエントを形成させると好ましい。質量分析計としては、二重収束磁場型質量分析計、イオントラップ型質量分析計、四重極型質量分析計などが挙げられるが、これらに限定されない。 Measurement conditions for quantifying 2SC by liquid chromatography-mass spectrometry may be appropriately set by a person skilled in the art based on common knowledge, depending on the type of equipment, the condition of the sample, etc. Liquid chromatography conditions vary depending on the sample provided, but for example, for the above-mentioned 20 volume % acetonitrile + 0.1 mass % formic acid aqueous solution, it is preferable to form a gradient with a formic acid aqueous solution and a formic acid acetonitrile solution in the mobile phase. . Examples of the mass spectrometer include, but are not limited to, a double focusing magnetic field mass spectrometer, an ion trap mass spectrometer, a quadrupole mass spectrometer, and the like.
本発明の生体試料の分析方法により、被験者から採取した生体試料中の２ＳＣを定量し、該生体試料中の２ＳＣの含有量に基づいて、被験者のミトコンドリア機能を評価することもできる。２ＳＣが、ミトコンドリアの機能異常に伴い脂肪細胞に蓄積することは公知であるため（非特許文献２参照）、本発明の生体試料の分析方法により、脂肪細胞を含む組織から得られる生体試料を用いて高精度に定量した２ＳＣの値が、所定の値より高いか低いかによってミトコンドリア機能を評価することができる。より具体的には、標準試料中の２ＳＣの含有量と比較して、被験者から採取した生体試料中の２ＳＣの含有量が有意に高い場合に、被験者のミトコンドリアの機能に障害があると予測又は判断することもできる。標準試料は、健常者から採取した生体試料であってもよいし、２ＳＣを既知の濃度で含有する試料であってもよい。
２ＳＣの定量に使用する生体試料の量に特に制限はないが、本発明の生体試料の分析方法によれば、例えば、組織の場合は０．５ｇ以下、血清の場合は５μｌ以下といった少量の生体試料を用いて高精度な２ＳＣの定量も可能である。
By the biological sample analysis method of the present invention, 2SC in a biological sample collected from a subject can be quantified, and the mitochondrial function of the subject can also be evaluated based on the content of 2SC in the biological sample. Since it is known that 2SC accumulates in adipocytes due to mitochondrial dysfunction (see Non-Patent Document 2), the biological sample analysis method of the present invention uses a biological sample obtained from a tissue containing adipocytes. Mitochondrial function can be evaluated based on whether the value of 2SC, which is quantified with high precision using the method, is higher or lower than a predetermined value. More specifically, if the content of 2SC in the biological sample collected from the subject is significantly higher than the content of 2SC in the standard sample, it is predicted that the mitochondrial function of the subject is impaired. You can also judge. The standard sample may be a biological sample collected from a healthy person, or a sample containing 2SC at a known concentration.
There is no particular limit to the amount of biological sample used for quantification of 2SC, but according to the biological sample analysis method of the present invention, small amounts of biological sample, such as 0.5 g or less for tissue and 5 μl or less for serum, can be used. Highly accurate quantification of 2SC is also possible using a sample.
以上、本発明をその好ましい実施態様に基づき説明したが、本発明は上述した実施態様に制限されない。 Although the present invention has been described above based on its preferred embodiments, the present invention is not limited to the embodiments described above.
以下、実施例を基に本発明を更に詳述するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to the following Examples.
〔実施例１〕
１．生体試料
健常体のマウスを解剖し、約０．４ｇの脳組織を得た。該脳組織に５ｍｌの５％ＴｒｉｔｏｎＸを含む水を添加し、ボルテックスにかけ、懸濁液を得た。
[Example 1]
1. Biological Sample A healthy mouse was dissected and approximately 0.4 g of brain tissue was obtained. 5 ml of water containing 5% TritonX was added to the brain tissue and vortexed to obtain a suspension.
２．前処理工程
（１）脱脂工程
前記の懸濁液に、クロロホルムとメタノールを体積比で２：１の割合で混合した溶液を１ｍｌ添加し、撹拌した。撹拌した後、１２０００ｒｐｍ、４℃で１５分間遠心した。遠心後の上清を除去し、残った沈殿物を風乾させた。遠心後の上清の除去は、有機層及び水層の両方を除去した。風乾後の沈殿物を蒸留水１００μｌに溶解させ、サンプルを得た。
2. Pretreatment Step (1) Degreasing Step To the above suspension, 1 ml of a solution in which chloroform and methanol were mixed at a volume ratio of 2:1 was added and stirred. After stirring, the mixture was centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant after centrifugation was removed, and the remaining precipitate was air-dried. Removal of the supernatant after centrifugation removed both the organic and aqueous layers. The air-dried precipitate was dissolved in 100 μl of distilled water to obtain a sample.
（２）沈殿工程
前記（１）の脱脂工程により得られたサンプルに、１０％トリクロロ酢酸溶液８００μｌを加えて撹拌した。撹拌した後、１２０００ｒｐｍ、４℃で１５分間遠心した。遠心後の上清を除去し、残った沈殿物を風乾させ。サンプルを得た。
（３）内部標準物質の添加
前記（２）の沈殿工程により得られたサンプルに内部標準物質として安定同位体１５Ｎ１３Ｃで標識した２ＳＣを０．０５ｎｍоｌ添加した。
(2) Precipitation step 800 μl of 10% trichloroacetic acid solution was added to the sample obtained in the degreasing step of (1) above and stirred. After stirring, the mixture was centrifuged at 12,000 rpm and 4°C for 15 minutes. After centrifugation, remove the supernatant and air dry the remaining precipitate. Got the sample.
(3) Addition of internal standard substance 0.05 nmole of 2SC labeled with stable isotope 15 N 13 C was added as an internal standard substance to the sample obtained in the precipitation step of (2) above.
（４）加水分解工程
６Ｎ塩酸を１５０μｌ加え、１００℃に加熱し、１８時間の加水分解処理を行った。加水分解後は、遠心濃縮装置にて乾固させた。
（５）除去工程
乾固したサンプルを水７００μｌに溶解させ、２．１ｍｌのメタノール（０．１％ギ酸）の入ったＣａｐｔｉｖａ ＮＤ Ｌｉｐｉｄｓに加えて混合し、４０００ｒｐｍ、室温で１５分遠心した。得られたＣａｐｔｉｖａ ＮＤ Ｌｉｐｉｄｓの通過画分を乾固し、サンプルとした。
（６）分離精製工程
乾固したサンプルを１ｍｌの１％ギ酸を含む水に溶解させ、平衡化したｓｅｐ－ｐａｋ Ｃ１８カラムに加え通過画分を回収した。さらに、１％ギ酸を含む２０％メタノールをカラムに２ｍｌ加え、通過画分を約３ｍｌ回収し、吹付式試験管濃縮装置にて乾固させた。
（７）濾過工程
乾固したサンプルを２０％アセトニトリル（０．１％ギ酸）に溶解させ、分子量３０００カットフィルターにて１２０００ｒｐｍ、室温で６０分遠心した。得られた通過画分をＬＣ－ＭＳ／ＭＳによって分析するための分析用試料とした。
(4) Hydrolysis step 150 μl of 6N hydrochloric acid was added, heated to 100° C., and hydrolyzed for 18 hours. After hydrolysis, it was dried using a centrifugal concentrator.
(5) Removal step The dried sample was dissolved in 700 μl of water, added to Captiva ND Lipids containing 2.1 ml of methanol (0.1% formic acid), mixed, and centrifuged at 4000 rpm for 15 minutes at room temperature. The obtained pass-through fraction of Captiva ND Lipids was dried and used as a sample.
(6) Separation and purification step The dried sample was dissolved in 1 ml of water containing 1% formic acid, added to an equilibrated sep-pak C18 column, and the flow-through fraction was collected. Further, 2 ml of 20% methanol containing 1% formic acid was added to the column, and about 3 ml of the passed-through fraction was collected and dried using a spray test tube concentrator.
(7) Filtration step The dried sample was dissolved in 20% acetonitrile (0.1% formic acid) and centrifuged at 12,000 rpm at room temperature for 60 minutes using a 3,000 molecular weight cut filter. The obtained pass-through fraction was used as an analytical sample for analysis by LC-MS/MS.
〔比較例１〕
脱脂処理を行わなかった以外は、実施例１と同様にして、分析用試料を得た。
[Comparative example 1]
A sample for analysis was obtained in the same manner as in Example 1, except that the degreasing treatment was not performed.
〔実施例２〕
生体試料として、上述の健常体のマウスの脳組織に代えて、ＤＭラット（ストレプトゾトシン誘発糖尿病ラット）の血清を用いた。具体的には、ＤＭラットから採取した血清を１／１０に希釈した。このようにして得た希釈液１０μｌに対し、実施例１と同様にして分析用試料を得た。
[Example 2]
As a biological sample, the serum of a DM rat (streptozotocin-induced diabetic rat) was used instead of the brain tissue of a healthy mouse described above. Specifically, serum collected from DM rats was diluted to 1/10. A sample for analysis was obtained in the same manner as in Example 1 using 10 μl of the diluted solution thus obtained.
〔比較例２〕
脱脂処理を行わなかった以外は、実施例２と同様にして、分析用試料を得た。
[Comparative example 2]
A sample for analysis was obtained in the same manner as in Example 2, except that the degreasing treatment was not performed.
３．液体クロマトグラフィー－質量分析
実施例１及び比較例１で得られた各分析用試料を、ＬＣ－ＭＳ／ＭＳにかけ、２ＳＣの定量分析を行った。その分析結果を図１に示す。図１（ａ）が比較例１を分析した結果であり、図１（ｂ）が実施例１を分析した結果である。ＬＣ－ＭＳ／ＭＳ測定条件は以下のとおりである。
3. Liquid Chromatography-Mass Spectrometry Each analytical sample obtained in Example 1 and Comparative Example 1 was subjected to LC-MS/MS to perform 2SC quantitative analysis. The analysis results are shown in Figure 1. FIG. 1(a) is the result of analyzing Comparative Example 1, and FIG. 1(b) is the result of analyzing Example 1. The LC-MS/MS measurement conditions are as follows.
（ＬＣ－ＭＳ／ＭＳの測定条件）
・クロマトグラフィーカラム：ＺＩＣ（登録商標）－ＨＩＬＩＣ ｃｏｌｕｍｎ （１５０ｘ２．１ｍｍ，５μｍ）
・カラム温度：４０℃
・移動相：アセトニトリル（０．１％ギ酸）、水（０．１％ギ酸）
・グラジエント条件：アセトニトリル９０％から１０％
・流速：２００μＬ／ｍｉｎ
・インジェクション量：１０μＬ
・分析時間：２３分
・溶出時間：１０．２分
・イオン化方法：ＥＳＩ
・キャピラリー温度：２７０℃
・イオン化エネルギー：３５００Ｖ（陽性イオン化時）
・２ＳＣの検出ピーク（ｍ／ｚ）：プレカーサーイオン２３８（ｍ／ｚ）、フラグメントイオン１４９（ｍ／ｚ）
(LC-MS/MS measurement conditions)
・Chromatography column: ZIC (registered trademark)-HILIC column (150x2.1mm, 5μm)
・Column temperature: 40℃
・Mobile phase: Acetonitrile (0.1% formic acid), water (0.1% formic acid)
・Gradient conditions: Acetonitrile 90% to 10%
・Flow rate: 200μL/min
・Injection volume: 10μL
・Analysis time: 23 minutes ・Elution time: 10.2 minutes ・Ionization method: ESI
・Capillary temperature: 270℃
・Ionization energy: 3500V (at the time of positive ionization)
・2SC detection peak (m/z): precursor ion 238 (m/z), fragment ion 149 (m/z)
また、実施例２及び比較例２で得られた各分析用試料を、ＬＣ－ＭＳ／ＭＳにかけ、２ＳＣの定量分析を行った。その分析結果を図２に示す。図２（ａ）が比較例２を分析した結果であり、図２（ｂ）が実施例２を分析した結果である。ＬＣ－ＭＳ／ＭＳ測定条件は、実施例１及び比較例１の分析用試料を分析する際のＬＣ－ＭＳ／ＭＳ測定条件と同じである。 In addition, each analytical sample obtained in Example 2 and Comparative Example 2 was subjected to LC-MS/MS to perform quantitative analysis of 2SC. The analysis results are shown in Figure 2. FIG. 2(a) shows the results of analyzing Comparative Example 2, and FIG. 2(b) shows the results of analyzing Example 2. The LC-MS/MS measurement conditions are the same as those used when analyzing the analytical samples of Example 1 and Comparative Example 1.
４．結果
図１及び２に示すように、実施例１及び２の分析方法により得られた分析結果は、２ＳＣを精度よく定量することができている。一方、比較例１及び２の分析方法により得られた分析結果は、ノイズが多く、２ＳＣを精度よく定量できていない。図１及び２に示す結果から、本発明の生体試料の分析方法によれば、脳等の生体組織を生体試料として用いた場合であっても、該生体試料中の２ＳＣを精度よく定量できることが分かる。したがって、本発明の生体試料の分析方法によれば、幅広い種類の生体試料中の２ＳＣを精度よく定量することができることが分かる。
4. Results As shown in FIGS. 1 and 2, the analysis results obtained by the analysis methods of Examples 1 and 2 were able to accurately quantify 2SC. On the other hand, the analysis results obtained by the analysis methods of Comparative Examples 1 and 2 have a lot of noise, and 2SC cannot be quantified with high accuracy. From the results shown in Figures 1 and 2, it is clear that according to the biological sample analysis method of the present invention, even when biological tissues such as the brain are used as the biological sample, 2SC in the biological sample can be quantified with high accuracy. I understand. Therefore, it can be seen that according to the biological sample analysis method of the present invention, 2SC in a wide variety of biological samples can be quantified with high accuracy.
Claims (7)
分析する前記生体試料に対して、脱脂処理を行う脱脂工程、タンパク質又はその断片を沈殿させ、２ＳＣ化したタンパク質又はその断片を含む沈殿物を回収する沈殿工程、塩酸による加水分解を行う加水分解工程をこの順で含む前処理工程を行うことを特徴とし、
前記脱脂工程は、前記生体試料に対して有機溶媒を加え攪拌した後に遠心分離し、遠心分離後の沈殿物を回収することを含み、
前記沈殿工程は、前記脱脂工程後の試料に塩、アルコール、酸又は水溶性ポリマーを加えることを含み、
前記前処理工程は、前記加水分解工程後に、フィルターを用いて試料を濾過することによりタンパク質及びリン脂質の除去を行う除去工程を更に含む、生体試料の分析方法。 A biological sample analysis method for quantifying S-(2-succinyl)cysteine (2SC) in a biological sample using liquid chromatography-mass spectrometry, the method comprising:
A delipidation step in which the biological sample to be analyzed is subjected to a defatting treatment, a precipitation step in which the protein or its fragments are precipitated and a precipitate containing the 2SC-converted protein or its fragments is recovered, and a hydrolysis step in which hydrolysis is performed with hydrochloric acid. It is characterized by performing a pretreatment step including in this order ,
The delipidation step includes adding an organic solvent to the biological sample, stirring it, centrifuging it, and collecting the precipitate after centrifugation,
The precipitation step includes adding salt, alcohol, acid, or a water-soluble polymer to the sample after the degreasing step,
The pretreatment step further includes, after the hydrolysis step, a removal step of removing proteins and phospholipids by filtering the sample using a filter.
前記生体試料中の２ＳＣの含有量に基づいて前記被験者のミトコンドリアの機能を評価する、ミトコンドリアの機能の評価方法。 Quantifying 2SC in the biological sample collected from a subject by the biological sample analysis method according to any one of claims 1 to 5 ,
A method for evaluating mitochondrial function, comprising evaluating the mitochondrial function of the subject based on the content of 2SC in the biological sample.
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