JP2008157905A - Quantity determination method for protein and peptide in biological sample, by addition method - Google Patents
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本発明は、血清、血しょう、血液、尿などの試料中のアルブミン、ヘモグロビン、グロブリン、Ｃ−反応性タンパク、ホルモン等の特定のタンパク質（修飾されたものも含む）やペプチドを正確に求める方法に関する。The present invention is a method for accurately determining specific proteins (including modified ones) and peptides such as albumin, hemoglobin, globulin, C-reactive protein, and hormone in samples such as serum, plasma, blood and urine. About.
総蛋白を定量する方法としては、ビュレット法が広く使われている。これはたんぱく質を総て求める方法であるが、特定の蛋白質、例えば、アルブミン、グロブリン、マイクロアルブミン等を求める方法としては、電気泳動法、イオン交換クロマトグラフィー、ゲルろ過クロマトグラフィー、アフイニテイークロマトグラフィーなどが広く用いられている。アルブミンに関して言えば、ＢＣＤ法、ＢＣＰ法などの発色させて比色法により求められる方法が簡使法として用いられる。また、質量分析法により微量の蛋白質を測定する方法が広く用いられているが、蛋白質の安定同位体の作製が困難であることと、目的蛋白質を損失なく精製することが困難であるため正確さ及び精密さにおいて十分ではない。さらに、マルチイオン化した蛋白質を質量分析法で測定する場合、感度が低下することと、干渉が飛躍的に増大するという問題がある。このように、特定の蛋白質を正確に且つ高精度に求める方法は知られていない。The Burette method is widely used as a method for quantifying total protein. This is a method for obtaining all proteins, but methods for obtaining specific proteins such as albumin, globulin, microalbumin, etc. include electrophoresis, ion exchange chromatography, gel filtration chromatography, affinity chromatography. Are widely used. As for albumin, a method such as a BCD method, a BCP method, or the like that is obtained by a colorimetric method by color development is used as a simple method. In addition, a method for measuring a minute amount of protein by mass spectrometry is widely used. However, it is difficult to produce a stable isotope of the protein and it is difficult to purify the target protein without loss. And not enough in precision. Furthermore, when measuring a multiionized protein by mass spectrometry, there are problems that sensitivity is lowered and interference is greatly increased. Thus, there is no known method for obtaining a specific protein accurately and with high accuracy.
発明者は、構成アミノ酸による蛋白質・ペプチドの定量方法［特願２００５−３５５３９３］を出願し、特定の蛋白質やペプチドを同位体希釈質量分析法により、高感度で、正確かつ高精度に定量する方法について開示した。しかし、この方法では、複雑なマトリックスを有する生体試料中の特定の蛋白質やペプチドを定量する場合、他成分の干渉が除去できない場合が存在する。さらに、目的の蛋白質やペプチドを操作の過程で損失なく回収することは極めて困難な場合が多く存在する。そのような場合でも適用可能な方法を開発することを目的とした。The inventor has filed a method for quantifying proteins / peptides with constituent amino acids [Japanese Patent Application No. 2005-355393], and quantifies specific proteins and peptides with high sensitivity, accuracy and high accuracy by isotope dilution mass spectrometry. Disclosed. However, in this method, when a specific protein or peptide in a biological sample having a complicated matrix is quantified, there are cases where interference from other components cannot be removed. Furthermore, there are many cases where it is extremely difficult to recover the target protein or peptide without any loss in the process of operation. The purpose was to develop a method that could be applied even in such a case.
本発明のポイントはあらかじめ生体試料に、求めようとする蛋白質／分子中の特定の修飾アミノ基又はその他の修飾基の比率を特願２００５−３５５３９３の方法で確定しておき、それを一定添加後、試料を十分に精製し、目的の蛋白質のみを得る。精製した試料について、再び特願２００５−３５５３９３の方法により特定の修飾アミノ基又はその他修飾基の比率を測定し、計算によりもとの試料中に含まれる目的蛋白質の量を求める。The point of the present invention is that a ratio of a specific modified amino group or other modified group in a protein / molecule to be obtained is determined in advance in a biological sample by the method of Japanese Patent Application No. 2005-355393, and after adding a certain amount thereof The sample is sufficiently purified to obtain only the target protein. For the purified sample, the ratio of the specific modified amino group or other modified group is measured again by the method of Japanese Patent Application No. 2005-355393, and the amount of the target protein contained in the original sample is determined by calculation.
ここで、生体試料とは、血液、血しょう、血清、組織、尿、髄液などを言う。Here, the biological sample refers to blood, plasma, serum, tissue, urine, cerebrospinal fluid, and the like.
特定の蛋白質では、生体試料中の総ての蛋白質を対象とするが、生体試料から各種クロマトグラフィー、溶媒抽出、分取電気泳動法、超遠心法、限外ろ過法、抗原抗体法などの方法により、分離して、精製することが可能なものに限られる。For specific proteins, all proteins in biological samples are targeted, but various methods such as chromatography, solvent extraction, preparative electrophoresis, ultracentrifugation, ultrafiltration, and antigen-antibody methods are used from biological samples. Therefore, it is limited to those that can be separated and purified.
ペプチドも同じように精製できなければならない。ペプチドについては、もともと生体試料中に存在していたものの他に、蛋白からある一定の反応式により分解して生じたものも本発明の対象に含まれる。The peptide must be able to be purified in the same way. As for peptides, in addition to those originally present in biological samples, peptides generated by decomposing from proteins according to a certain reaction formula are also included in the object of the present invention.
本発明で言う精製においては、試料中のアルブミンを例に取ると精製においてその総てを回収する必要はなく、他の蛋白が混入していない画分のみを分取すればよい。この点は本発明の本質をなす部分といえる。In the purification referred to in the present invention, when albumin in a sample is taken as an example, it is not necessary to collect all of the albumin during purification, and only a fraction not contaminated with other proteins may be collected. This point can be said to be an essential part of the present invention.
一方特願２００５−３５５３９３や、その他の質量分析法では総てを回収しなければならない。総てを回収するとなれば、極めて困難な精製を行わねばならない場合が多く、種々の蛋白質定量に適応する事は難しい。On the other hand, in Japanese Patent Application No. 2005-355393 and other mass spectrometry, all must be collected. If all are recovered, it is often necessary to carry out extremely difficult purification, and it is difficult to adapt to various protein quantifications.
血清試料を例にとり修飾基をグルコースとすると、血清中のアルブミン濃度をＡｘ、その糖化割合をＧｘ，サンプリング量をＷｘとする。糖化割合を求めたアルブミン標準液の濃度をＡｓ、その糖化割合でＧｓ，サンプリング量をＷｓとすると、両者を混合して均一とした試料の糖化割合Ｇｍは
Ｇｍ ＝Ａｘ・Ｇｘ・Ｗｘ＋Ａｓ・Ｇｓ・Ｗｓ
Ａｘ・Ｗｘ＋Ａｓ・Ｗｓ
となる。 よって
Ａｘ ＝ Ａｓ・Ｗｓ（Ｇｍ−Ｇｓ）
Ｗｘ（Ｇｘ−Ｇｍ）
となり、この式により、血清中のアルブミン濃度Ａｘが求まる。Taking a serum sample as an example, if the modifying group is glucose, the serum albumin concentration is Ax, the saccharification ratio is Gx, and the sampling amount is Wx. The concentration of the albumin standard solutions was determined glycation ratio As, Gs at the glycation rate and the sampling amount to Ws, glycation ratio Gm of a uniform mixture of both samples Gm = Ax · Gx · Wx + As · Gs · Ws
Ax / Wx + As / Ws
It becomes. Therefore, Ax = As · Ws (Gm−Gs)
Wx (Gx-Gm)
From this equation, the albumin concentration Ax in serum is obtained.
本発明では、直接定量を行うのは修飾したアミノ酸であり、質量分析においては一価の分子イオンが測定対象となるので、極めて高感度であり、且つ干渉は小さい。他のエレクトロンスプレーイオン化法による質量分析では、蛋白のマルチイオンが測定対象となるので、感度が低く干渉は飛躍的に増大する。また、あらかじめ修飾蛋白を添加するので、これが内標準となり精製による損失が補正されるので、十分な精製が可能である。このように、本法により高感度に、正確且つ高精度に蛋白質を測定できるようになった。In the present invention, it is a modified amino acid that is directly quantified, and since monovalent molecular ions are to be measured in mass spectrometry, the sensitivity is extremely high and interference is small. In mass spectrometry based on other electron spray ionization methods, protein multi-ions become the measurement target, so the sensitivity is low and interference increases dramatically. In addition, since the modified protein is added in advance, this becomes an internal standard and the loss due to purification is corrected, so that sufficient purification is possible. As described above, the present method has made it possible to measure proteins with high sensitivity, accuracy and accuracy.
（１）アルブミン標準液の調製とそのアルブミン濃度と糖化割合の測定
溶離液（２２０ｍＭ 塩化マグネシウムを含む５０ｍＭグリシン緩衝液 ｐＨ８．５）で３倍に希釈した血清５００μＬをイオン交換カラム（Ｓｈｏｄｅｘ ＡｓａｈｉｐａｃｋＥＳ−５０２Ｎ ２０Ｃ）注入し，流量５ｍＬ／ｍｉｎ、波長２８０ｎｍでモニターしながら保持時間５〜７分のアルブミン分画１０ｍＬを分取した。次に遠心限外ろ過フィルター（Ａｍｉｃｏｎ Ｕｌｔｒａ−１５ ３０，０００ＣＵＴ）により分子量３万以下の成分を水で洗浄除去して、濃度約１０ｍｇ／ｍＬ（ＢＣＧ比色法で測定）アルブミン溶液５００μＬを得、これをアルブミン標準液とした。Ｎａｔｉｖｅ ＰＡＧＥ電気泳動法ではアルブミンのシングルバンドのみが検出され、また分析専用イオン交換カラムを用いたＨＰＬＣ測定の結果から標準液のアルブミン純度は９８％以上であると確認できた。このアルブミン標準液１００ｍｇ（アルブミン約１５ｎｍｏＬ）と５０ｐｍｏＬ／ｍｇ１３Ｃ６−グルコース糖化リジン１００ｍｇ、１０μｍｏＬ／ｍｇＤ４−リジン１００ｍｇの２種類のスパイク用の安定同位体化合物溶液、それぞれを１本の封菅用のガラス管に精秤して十分に混合した。ついで５０ｍＭ水素化ホウ素ナトリウム３０ｕＬを加え室温で３時間かけて糖化リジン基を水素化して化学的に安定なヘキシトールリジン基としたものに、１０ｍｏＬ／Ｌ塩酸４００ｕＬと水を加え溶液中の塩酸濃度を５ｍｏＬ／Ｌに調整した。ガラス管内を窒素ガスで十分にパージしてから減圧状態でガスバーナーにより封管後、１１０℃に設定した加温器内で２０時間保持してアルブミンのペプチド結合を完全に加水分解した。加水分解が終了して開封したガラス管を真空遠心濃縮機に入れ、５０℃に加温して１５００ＲＰＭで約３時間かけて塩酸と水を揮発除去した得られた乾固物を、０．０６％ＨＦＢＡ（ヘプタフルオロ酪酸）５００ｕＬに溶解したものをＬＣ／ＭＳ測定溶液とした。アルブミンに含まれる自然界の同位体組成の糖化リジンとスパイクした安定同位体化合物の糖化リジンのヘキシトールリジン基にプロトンが付加した各々Ｍ／Ｚ＝３１１．２，Ｍ／Ｚ＝３１７．２のＳＩＭピークの面積比をＬＣ／ＭＳで測定した。また同時に、リジンについてはリジン基にプロトンが付加した各々Ｍ／Ｚ＝１４７．２，Ｍ／Ｚ＝１５１．２のＳＩＭピークの面積比をＬＣ／ＭＳで測定した。高純度の糖化リジン（純度９８％以上）と高純度のリジン（シグマ社、純度９８％以上）を水に溶解して調製した混合標準液（３５ｐｍｏＬ／ｍｇ糖化リジン＋１０μｍｏＬ／ｍｇリジン）に、５０ｐｍｏＬ／ｍｇ１３Ｃ６−グルコース糖化リジンと１０μｍｏＬ／ｍｇＤ４−リジンの２種類のスパイク用の安定同位体化合物溶液それぞれを精秤して加え、各々５種の同位体比組成（糖化リジンの同位体比０．５，１．０，１．５，２．０、２．５、リジンの同位体比０．８，０．９，１．０，１．１，１．２）の検量溶液について、前記と同様にＳＩＭピークの面積比を測定した。得られた検量線から、アルブミン標準液のリジン及び糖化リジンのモル濃度を求めた。またアルブミンのモル濃度は得られたリジンのモル濃度を５９（アルブミン１分子当たりのリジン基の数）で除して求めた。さらに、糖化割合は糖化リジンモル濃度をアルブミンのモル濃度で除して算出した。
（２）血清試料中の糖化割合の測定
前記（１）と同様に、血清試料からアルブミンを精製してその糖化リジン及びアルブミンを測定し糖化割合を求めた。
（３）血清試料とアルブミン標準液の混合液の糖化割合の測定
前記（１）で調製したアルブミン標準液を用いて、本発明で血清試料中のアルブミンを定量するためには、両者の糖化割合（糖化リジン／アルブミン）の値ができるだけ大きく異なる必要がある。つまり、糖化割合が正常域（２０〜４０モル％）の血清試料を定量するには、アルブミン標準液の方を本実施例のように８０モル％程度に大きくしておくと本発明が適用できる。一方、糖化割合が大きい糖尿病患者の血清試料などを定量するには、糖化割合が正常域程度の低いアルブミン標準液を用いる。血清試料１００ｍｇとアルブミン標準液２００ｍｇをそれぞれ精秤して十分に混和したものを、前記（１）と同様に処理して濃度約１０ｍｇ／ｍＬの精製アルブミン試料０．４ｍＬを得た。この精製アルブミン試料１００ｍｇ（アルブミン約１５ｎｍｏＬ）を用いて、前記（１）と同様に糖化リジン及びアルブミンを測定し糖化割合を求めた。
（４）血清試料中のアルブミン定量結果
５種類の血清試料（いずれも糖化割合は正常域）を本法で測定して（式２）を用いて定量した結果と日常検査法であるＢＣＧ法の測定値は５％以内で一致した。(1) Preparation of albumin standard solution and measurement of albumin concentration and saccharification ratio 500 μL of serum diluted 3-fold with an eluent (50 mM glycine buffer solution containing 8.5 mM magnesium chloride, pH 8.5) was ion-exchanged column (Shodex Asahipack ES-502N) 20C) The albumin fraction was collected at a flow rate of 5 mL / min and a wavelength of 280 nm, and 10 mL of the albumin fraction was collected for 5 to 7 minutes. Next, a component having a molecular weight of 30,000 or less was washed and removed with water using a centrifugal ultrafiltration filter (Amicon Ultra-15 30,000 CUT) to obtain 500 μL of an albumin solution having a concentration of about 10 mg / mL (measured by BCG colorimetric method). This was used as an albumin standard solution. In Native PAGE electrophoresis, only a single band of albumin was detected, and from the results of HPLC measurement using an ion exchange column for analysis, it was confirmed that the albumin purity of the standard solution was 98% or more. This albumin standard solution 100 mg (albumin about 15 nmoL) and 50 pmoL / mg 13 C 6- glucosylated lysine 100 mg, 10 μmoL / mgD 4- lysine 100 mg of stable isotope compound solutions for spikes, each containing one sealant Weighed thoroughly in a glass tube and mixed well. Next, 30 uL of 50 mM sodium borohydride was added, and the saccharified lysine group was hydrogenated at room temperature for 3 hours to form a chemically stable hexitol lysine group. 400 mol of 10 mol / L hydrochloric acid and water were added to the hydrochloric acid in the solution. The concentration was adjusted to 5 moL / L. The inside of the glass tube was sufficiently purged with nitrogen gas, sealed with a gas burner under reduced pressure, and kept in a warmer set at 110 ° C. for 20 hours to completely hydrolyze the peptide bond of albumin. The glass tube opened after hydrolysis was placed in a vacuum centrifugal concentrator, heated to 50 ° C. and volatilized and removed from hydrochloric acid and water at 1500 RPM for about 3 hours. A solution dissolved in 500 uL of% HFBA (heptafluorobutyric acid) was used as an LC / MS measurement solution. Proton added to the hexitol lysine group of the glycated lysine of the glycated lysine of the spiked stable isotope compound of natural isotope composition contained in albumin, M / Z = 311.2, M / Z = 317.2 respectively The area ratio of the SIM peak was measured by LC / MS. At the same time, for lysine, the area ratio of SIM peaks of M / Z = 147.2 and M / Z = 151.2 in which protons were added to the lysine group was measured by LC / MS. To a mixed standard solution (35 pmoL / mg glycated lysine + 10 μmol / mg lysine) prepared by dissolving high purity glycated lysine (purity 98% or more) and high purity lysine (Sigma, purity 98% or more) in water, 50 pmoL Each of the two stable isotope compound solutions for spikes of 10 mg / L 13 mg 6- glucose saccharified lysine and 10 μmol / mgD 4- lysine was precisely weighed and added, and each of the five isotopic composition ratios Calibration solutions with ratios of 0.5, 1.0, 1.5, 2.0, 2.5 and lysine isotope ratios of 0.8, 0.9, 1.0, 1.1, 1.2) The area ratio of the SIM peak was measured in the same manner as described above. From the obtained calibration curve, the molar concentrations of lysine and glycated lysine in the albumin standard solution were determined. The molar concentration of albumin was determined by dividing the molar concentration of lysine obtained by 59 (number of lysine groups per albumin molecule). Furthermore, the saccharification ratio was calculated by dividing the glycated lysine molar concentration by the molar concentration of albumin.
(2) Measurement of saccharification ratio in serum sample In the same manner as in (1) above, albumin was purified from a serum sample and its glycated lysine and albumin were measured to determine the saccharification ratio.
(3) Measurement of saccharification ratio of a mixture of a serum sample and an albumin standard solution In order to quantify albumin in a serum sample using the albumin standard solution prepared in (1) above, The value of (glycated lysine / albumin) should be as different as possible. That is, in order to quantify a serum sample having a normal saccharification ratio (20 to 40 mol%), the present invention can be applied by increasing the albumin standard solution to about 80 mol% as in this embodiment. . On the other hand, in order to quantify a serum sample or the like of a diabetic patient having a large glycation ratio, an albumin standard solution having a low glycation ratio in the normal range is used. 100 mg of serum sample and 200 mg of albumin standard solution were precisely weighed and mixed thoroughly, and treated in the same manner as in (1) to obtain 0.4 mL of purified albumin sample having a concentration of about 10 mg / mL. Using 100 mg of this purified albumin sample (albumin about 15 nmoL), glycated lysine and albumin were measured in the same manner as in (1) above to determine the saccharification ratio.
(4) Results of quantification of albumin in serum samples Results of quantification using 5 types of serum samples (all glycation ratios are in normal range) using this method and formula (2) and BCG method which is a daily test method The measured values agreed within 5%.
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| US10386371B2 (en) | 2011-09-08 | 2019-08-20 | The Regents Of The University Of California | Metabolic flux measurement, imaging and microscopy |
| JP2018138914A (en) * | 2011-12-07 | 2018-09-06 | グラクソスミスクライン エルエルシー | Methods for determining total body skeletal muscle mass |
| JP2015513370A (en) * | 2012-03-13 | 2015-05-11 | ザ・ジョンズ・ホプキンス・ユニバーシティ | Citrullinated brain and neuroproteins as biomarkers of brain injury or neurodegeneration |
| US9709573B2 (en) | 2012-03-13 | 2017-07-18 | The Johns Hopkins University | Citrullinated brain and neurological proteins as biomarkers of brain injury or neurodegeneration |
| US10365288B2 (en) | 2012-03-13 | 2019-07-30 | The Johns Hopkins University | Citrullinated brain and neurological proteins as biomarkers of brain injury or neurodegeneration |
| US12007395B2 (en) | 2012-03-13 | 2024-06-11 | The Johns Hopkins University | Citrullinated brain and neurological proteins as biomarkers of brain injury or neurodegeneration |
| CN103837592A (en) * | 2014-03-18 | 2014-06-04 | 中国计量科学研究院 | Method for in-situ measuring iron content in biological tissue sample by adopting isotopic dilution LA-ICP-MS (laser ablation inductively coupled plasma mass spectrometry) |
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