JP4447458B2 - Mass spectrometry plate, preparation method thereof and use thereof - Google Patents
Mass spectrometry plate, preparation method thereof and use thereof Download PDFInfo
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- JP4447458B2 JP4447458B2 JP2004541284A JP2004541284A JP4447458B2 JP 4447458 B2 JP4447458 B2 JP 4447458B2 JP 2004541284 A JP2004541284 A JP 2004541284A JP 2004541284 A JP2004541284 A JP 2004541284A JP 4447458 B2 JP4447458 B2 JP 4447458B2
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Description
本発明は、プロテオーム解析を多量、迅速、かつ高感度に実施できる、新規な質量分析用プレート、その調製方法およびその用途に関する。 The present invention relates to a novel plate for mass spectrometry that can perform proteome analysis in a large amount, quickly, and with high sensitivity, a preparation method thereof, and an application thereof.
分子生物学やゲノミクス（ゲノム科学）をはじめとする創薬の基盤研究の進歩により、ここ数年間で創薬の様相が急速に変化し、ゲノム創薬に代表される新しい創薬方法が開発されつつある。
しかしながら、ゲノミクスが明らかにした塩基配列からはまだ蛋白質の機能（生理作用）は予測できず、遺伝情報を新薬に結び付ける手法の確立がポストゲノムに求められている。その一つとして、上述の塩基配列から翻訳される蛋白質（１０万種以上とも言われる）を全て単離・同定し、その機能を帰属することを目的とするプロテオミクス（蛋白質解析科学）が注目を浴びている。
プロテオームとは、狭義には一個の生物体を構成する全ての蛋白質を意味するが、広義にはある細胞の細胞液中の全蛋白質、血清中に含まれる全ての蛋白質、ある組織に含まれる全ての蛋白質等のように、生物体の組織学的、解剖学的に特定される部位に含まれる全ての蛋白質を意味する場合もある。本明細書では、広義で使用しているが、広義のプロテオームの完全な集合体が狭義のプロテオームであることは明らかである。
プロテオーム解析では、従来より二次元電気泳動法と質量分析法を組合わせる方法が汎用されてきたが、以下の問題点が未だ解決されずにいる。すなわち、従来法では、泳動後の試料を質量分析法にかけるまでに、泳動ゲルを小片に分断し、特殊な溶液でそれぞれの小片から蛋白質を抽出しなければならない。このように長時間を消費し、多段階の煩雑な操作が要求されるために、測定時間の短縮化、装置の小型化、大量検体の処理、全装置のロボット化が極めて困難であった。
さらに、いわゆる「多数の微量蛋白質」（ｌｏｗ−ａｂｕｎｄａｎｃｅ ｐｒｏｔｅｉｎｓ）の問題がある。例えば、酵母では、たった１００個の遺伝子が酵母の全蛋白質重量の５０％を製造している。これは、残り５０％の蛋白質が何千もの遺伝子の産物であることを意味する。大量の微量蛋白質には、調節蛋白質、受容体を含めた情報伝達蛋白質等、生体にとって最も重要な蛋白質が大量に含まれる。しかしながら、従来法では電気泳動により分離した多数かつ微量の蛋白質試料を回収することが不可能な状況にある。
このような電気泳動法と質量分析法の組合せ技術の限界を解決し、かつ蛋白質−蛋白質相互作用を解明すべく、現在様々な取り組みが行われている。例えば、同位体標識化アフィニティタグ法（ＩＣＡＴ：ｉｓｏｔｏｐｅ−ｃｏｄｅｄ ａｆｆｉｎｉｔｙ ｔａｇ；Ｎａｔ．Ｂｉｏｔｅｃｈ．，１７：９９４−９９９（１９９９））、酵母でのｔｗｏ−ｈｙｂｒｉｄ ｓｙｓｔｅｍ、ＢＩＡ−ＭＳ−ＭＳ、蛋白質アレイ法（溶液、チップ；Ｔｒｅｎｄｓ Ｂｉｏｔｅｃｈｎｏｌ．，１９：Ｓ３４−３９（２００１））、ＬＣ−ＭＳ−ＭＳによるペプチドミクス等である。このうち、蛋白質アレイ法としては、固相蛋白質アレイ法（チップ法；Ｃｕｒｒ．Ｏｐｉｎ．Ｂｉｏｔｅｃｈｎｏｌ．，１２：６５−６９（２００１））、ナノ粒子に情報を組み込んだ液相アレイ法（ｆｌｕｏｒｅｓｃｅｎｃｅ−ｅｎｃｏｄｅｄｂｅａｄｓ；Ｃｌｉｎ，Ｃｈｅｍ．，４３：１７４９−１７５６（１９９７）；Ｎａｔ，Ｂｉｏｔｅｃｈ．，１９：６３１−６３５（２００１）；ｂａｒｃｏｄｅｄ ｎａｎｏｐａｒｔｉｃｌｅｓ；Ｔｒｅｎｄｓ Ｂｉｏｔｅｃｈｎｏｌ．（２００１）前記）などが挙げられる。
一方、本発明者らは、膜蛋白質と当該蛋白質と相互作用可能な化合物とを各々グループ化し、両者を同時分析することが可能なプロテオーム解析法を提案している（ＷＯ ０２／５６０２６）。
このような測定方法（システム）の改良とは別に、プロテオーム解析を目的としないか、あるいは技術的にプロテオーム解析への使用は困難であるが、蛋白質の質量分析分野での改善を目指した幾つかの報告がある。電気泳動により分離した蛋白質をポリビニリデンジフロリド（ｐｏｌｙｖｉｎｙｌｉｄｅｎｅ ｄｉｆｌｕｏｒｉｄｅ；ＰＶＤＦ）膜に転写して、ＭＡＬＤＩ型質量分析用のステンレス製プレートに両面テープ（Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ，１７：９５４−９６１（１９９６））、フレーム（Ａｎａｌ．Ｃｈｅｍ．，６９：２８８８−２８９２（１９９７））あるいはグリース（Ａｎａｌ．Ｃｈｅｍ．，７１：４８００−４８０７（１９９９）；Ａｎａｌ．Ｃｈｅｍ．，７１：４９８１−４９８８（１９９９）；ＷＯ ００／４５１６８）で固定化して測定する方法が報告されている。これらの方法は、一旦ＰＶＤＦ膜を測定途中で質量分析用プレートに固定化する点で操作が煩雑となるだけでなく、バックグラウンドが高く、相対ピーク強度が極端に低いという欠点があり、高い検出感度が求められるプロテオーム解析には不向きである。
また、予めマトリックスを塗布した質量分析用プレートに電気泳動後のゲルを直接転写させて質量分析する方法や、一旦ブロット用のＰＶＤＦ膜に蛋白質を電気転写した後に、予めマトリックスを塗布した質量分析用プレートに拡散転写する方法も報告されている（ＵＳ ５，５９５，６３６）。しかしながら、これらの方法においては、電気転写を用いた場合、マトリックスが転写中に転写緩衝液中に溶出して測定自体が不可能になる恐れがある。一方、拡散転写を用いた場合は転写効率が低いため、特に微量蛋白質を質量分析用プレートに転写するには検出感度の面から不十分である。
さらに上記の技術を改良して、マトリックスにニトロセルロース（ブロット用の膜成分である）を混合して質量分析用プレートに塗布する方法が報告されている（ＧＢ ２３１２７８２ Ａ）。しかし、この方法でも電気転写、拡散転写のいずれの場合における問題点も完全に解決されたとはいえない。
ところで、質量分析用プレートとしては、通常用いられるアルミニウムあるいはステンレス・スチール製プレートの他に、これらを改良し、シリカでコートした、あるいは、疎水性基を付加した質量分析用プレート等も市販されている（Ｃｉｐｈｅｒｇｅｎ社製、ＷＯ ９４／２８４１８）。しかし、これらの技術・製品も、大量、迅速かつ高感度にプロテオーム解析を行うという研究開発のニーズを必ずしも満たすものではない。
本発明の目的は、従来の電気泳動法と質量分析法の組合せ技術によるプロテオーム解析法に新しい技術を導入することにある。詳しくは、プロテオーム解析を多量、迅速かつ高感度に実施できる技術を提供することにある。Advances in basic drug discovery research, including molecular biology and genomics, have led to rapid changes in drug discovery over the past few years, and new drug discovery methods typified by genome drug discovery have been developed. It's getting on.
However, the function (physiological action) of the protein cannot be predicted from the nucleotide sequence revealed by genomics, and the establishment of a method for linking genetic information to a new drug is required for the post-genome. As one of them, proteomics (protein analysis science) aimed at isolating and identifying all proteins (also said to be more than 100,000 species) translated from the above-mentioned base sequences and assigning their functions attracts attention. I'm bathing.
Proteome means, in a narrow sense, all the proteins that constitute an organism, but in a broad sense, all proteins in the cell fluid of a cell, all proteins contained in serum, and all contained in a certain tissue. In some cases, it means all proteins contained in histologically and anatomically specified parts of an organism, such as Although used herein in a broad sense, it is clear that a complete collection of broad proteomes is a narrow proteome.
In proteomic analysis, a method combining two-dimensional electrophoresis and mass spectrometry has been widely used, but the following problems have not been solved yet. In other words, in the conventional method, before the sample after electrophoresis is subjected to mass spectrometry, the electrophoresis gel must be divided into small pieces, and the protein must be extracted from each small piece with a special solution. As described above, since a long time is consumed and a multi-step complicated operation is required, it is extremely difficult to shorten the measurement time, to reduce the size of the apparatus, to process a large amount of samples, and to form a robot for all apparatuses.
Furthermore, there is a problem of so-called “multiple trace proteins” (low-abundance proteins). For example, in yeast, only 100 genes produce 50% of the total protein weight of yeast. This means that the remaining 50% protein is the product of thousands of genes. A large amount of a small amount of protein contains a large amount of proteins that are most important for the living body, such as regulatory proteins and information transfer proteins including receptors. However, in the conventional method, it is impossible to collect a large number of minute protein samples separated by electrophoresis.
Currently, various efforts are being made to solve the limitations of the combination technique of electrophoresis and mass spectrometry and to elucidate the protein-protein interaction. For example, isotope-labeled affinity tag method (ICAT: isotopic-coded affinity tag; Nat. Biotech., 17: 994-999 (1999)), two-hybrid system in yeast, BIA-MS-MS, protein array method (Solution, chip; Trends Biotechnol., 19: S34-39 (2001)), peptide mix by LC-MS-MS, and the like. Among these, as the protein array method, a solid phase protein array method (chip method; Curr. Opin. Biotechnol., 12: 65-69 (2001)), a liquid phase array method in which information is incorporated into nanoparticles (fluorescence-encoded beads) Clin, Chem., 43: 1749-1756 (1997); Nat, Biotech., 19: 631-635 (2001); barcoded nanoparticulates; Trends Biotechnol. (2001) supra).
On the other hand, the present inventors have proposed a proteome analysis method capable of grouping membrane proteins and compounds capable of interacting with the proteins, and simultaneously analyzing them (WO 02/56026).
Apart from the improvement of such measurement methods (systems), it is not intended for proteome analysis or technically difficult to use for proteome analysis, but there are some improvements aimed at improving protein mass spectrometry. There is a report. Proteins separated by electrophoresis are transferred to a polyvinylidene difluoride (PVDF) membrane, and a double-sided tape (Electrophoresis, 17: 954-961 (1996)), frame to a stainless steel plate for MALDI-type mass spectrometry. (Anal. Chem., 69: 2888-2892 (1997)) or grease (Anal. Chem., 71: 4800-4807 (1999); Anal.Chem., 71: 4981-4988 (1999); WO 00/45168 ) Has been reported for immobilization and measurement. These methods not only make the operation complicated in that the PVDF membrane is once immobilized on the plate for mass spectrometry during measurement, but also have the disadvantages of high background and extremely low relative peak intensity, and high detection. It is not suitable for proteome analysis that requires sensitivity.
In addition, the gel after electrophoresis is directly transferred to a mass spectrometry plate coated with a matrix in advance, and mass spectrometry is performed, or the protein is electrotransferred once to a PVDF membrane for blotting, and then the matrix is coated in advance. A method of diffusion transfer to a plate has also been reported (US 5,595,636). However, in these methods, when electric transfer is used, the matrix may elute in the transfer buffer during transfer, making measurement impossible. On the other hand, since the transfer efficiency is low when diffusion transfer is used, it is particularly insufficient from the viewpoint of detection sensitivity to transfer a trace amount of protein to a plate for mass spectrometry.
Furthermore, a method of improving the above technique and mixing nitrocellulose (which is a membrane component for blotting) in a matrix and applying it to a plate for mass spectrometry has been reported (GB 2312782 A). However, this method has not completely solved the problems in both the electric transfer and the diffusion transfer.
By the way, as a plate for mass spectrometry, in addition to a commonly used aluminum or stainless steel plate, a mass spectrometry plate or the like obtained by improving these and coating with silica or adding a hydrophobic group is also commercially available. (Ciphergen, WO 94/28418). However, these technologies and products do not necessarily meet the research and development needs for proteome analysis in large quantities, quickly and with high sensitivity.
An object of the present invention is to introduce a new technique into a proteome analysis method based on a conventional combination technique of electrophoresis and mass spectrometry. Specifically, it is to provide a technique capable of performing a large amount of proteome analysis quickly and with high sensitivity.
本発明は、ＰＶＤＦで基板をコーティングして該基板上にＰＶＤＦの薄層を形成させた質量分析用プレートは、ＰＶＤＦを膜の形態で基板上に固定化したものに比べて、質量分析の検出感度および精度が飛躍的に向上することの発見に基づいている。また、このような質量分析用プレートは、転写時にマトリックスを含まないので、電気転写によっても溶出することがないというさらなる利点を有する。
すなわち、本発明は、
１）支持体上にＰＶＤＦを含有するコーティングを有してなる質量分析用プレート；
２）ＰＶＤＦで支持体表面をコーティングすることを特徴とする質量分析用プレートの調製方法；
３）分析対象物含有試料をゲル電気泳動して分析対象物を分離し、分離した分析対象物を前記１）の、もしくは前記２）の方法により得られる質量分析用プレートに転写し、転写した分析対象物を質量分析することを特徴とする分析対象物の同定方法；
に関するものである。
本発明のさらなる目的および特徴、並びに本発明の効果は、以下の発明の詳細な説明においてより明確となるであろう。In the present invention, a mass spectrometry plate in which a substrate is coated with PVDF and a thin layer of PVDF is formed on the substrate is detected by mass spectrometry compared to a plate in which PVDF is immobilized on the substrate in the form of a film. It is based on the discovery that sensitivity and accuracy are dramatically improved. Such a plate for mass spectrometry does not contain a matrix at the time of transfer, and therefore has an additional advantage that it is not eluted by electrotransfer.
That is, the present invention
1) a plate for mass spectrometry comprising a coating containing PVDF on a support;
2) A method for preparing a plate for mass spectrometry, characterized by coating the surface of a support with PVDF;
3) Analyte-containing sample is subjected to gel electrophoresis to separate the analyte, and the separated analyte is transferred to the mass spectrometric plate obtained by the method of 1) or 2) and transferred. A method for identifying an analyte, comprising mass-analyzing the analyte;
It is about.
Further objects and features of the present invention, as well as the advantages of the present invention, will become more apparent in the detailed description of the invention that follows.
図１は、泳動ゲルから質量分析用プレートへの電気転写の度合いを示す。Ａは泳動ゲル（転写前）、Ｂは泳動ゲル（転写後）、Ｃは本発明の質量分析用プレート（転写後）を示す。左端の数字はバンドに対応する蛋白質の分子量を表す。電気転写によって蛋白質の種類によってはゲル中のほぼ全量が質量分析用プレートへ移行することを示している。
図２は、泳動ゲルと質量分析用プレートの位置関係（図２Ａ）およびプレート上の移行した蛋白質が受けるレーザー光量の差異（図２Ｂ）を示す。図中、１は質量分析用プレート、２は泳動ゲル、３は蛋白質を各々示す。また、Ａは光量最大、Ｂは光量小、Ｃは光量ゼロ、の場合を各々示す。
図３は、本発明の質量分析用プレート（図３Ａ）、アルミプレート（図３Ｂ）、ＰｒｏｔｅｉｎＣｈｉｐ ａｒｒａｙ（Ｈ４、図３Ｃ）を用いたときの質量分析ピークの相対強度を示す。
図４は、本発明の質量分析用プレートまたはアルミプレートを用いて得られるＳＣＵＰＡとＨＳＡの質量分析スペクトル（ピーク像）を示す。横軸は分子量、縦軸は相対強度を示す。図４ＡおよびＢはＳＣＵＰＡを、図４ＣおよびＤはＨＳＡを、各々示す。また、図４ＡおよびＣは本発明の質量分析用プレートを用いた場合を、図４ＢおよびＤはアルミプレートを用いた場合を、各々示す。
図５は、本発明の質量分析用プレートを用いて電気転写した蛋白質（ＳＣＵＰＡ）とそれに特異的に反応する抗体（抗ＳＣＵＰＡ抗体）との蛋白質−蛋白質相互作用を質量分析法により解析した結果を示す。横軸は分子量、縦軸は相対強度を示す。図中の数字はピークの分子量を示す。図５Ａは分離・転写されたＳＣＵＰＡと抗ＳＣＵＰＡ抗体の相互作用を示す。また、図５Ｂは抗ＳＣＵＰＡ抗体を単独で用いた場合の結果を示す。
図６は、電気泳動により分離した蛋白質をＰＶＤＦ膜に転写した場合の質量分析スペクトルを示す。横軸は分子量、縦軸は相対強度を示す。図中の数字はピークの分子量を示す。図６ＡはＳＣＵＰＡを、図６ＢはＨＳＡを、各々示す。FIG. 1 shows the degree of electrical transfer from the electrophoresis gel to the mass spectrometric plate. A shows an electrophoresis gel (before transfer), B shows an electrophoresis gel (after transfer), and C shows a plate for mass spectrometry of the present invention (after transfer). The leftmost number represents the molecular weight of the protein corresponding to the band. Electrotransfer indicates that almost all of the gel is transferred to the plate for mass spectrometry depending on the type of protein.
FIG. 2 shows the positional relationship between the electrophoresis gel and the plate for mass spectrometry (FIG. 2A) and the difference in the amount of laser light received by the transferred protein on the plate (FIG. 2B). In the figure, 1 is a plate for mass spectrometry, 2 is an electrophoresis gel, and 3 is a protein. A shows the case where the light quantity is maximum, B the light quantity is small, and C the light quantity is zero.
FIG. 3 shows the relative intensities of mass spectrometry peaks when using the plate for mass spectrometry (FIG. 3A), the aluminum plate (FIG. 3B), and the protein chip array (H4, FIG. 3C) of the present invention.
FIG. 4 shows mass spectrometry spectra (peak images) of SCUPA and HSA obtained using the mass spectrometry plate or aluminum plate of the present invention. The horizontal axis represents molecular weight, and the vertical axis represents relative intensity. 4A and B show SCUPA and FIGS. 4C and D show HSA, respectively. 4A and 4C show the case where the plate for mass spectrometry of the present invention is used, and FIGS. 4B and 4D show the case where an aluminum plate is used.
FIG. 5 shows the results of analyzing the protein-protein interaction between the protein (SCUPA) electrotransferred using the plate for mass spectrometry of the present invention and the antibody (anti-SCUPA antibody) specifically reacting to the protein by mass spectrometry. Show. The horizontal axis represents molecular weight, and the vertical axis represents relative intensity. The numbers in the figure indicate the molecular weight of the peak. FIG. 5A shows the interaction between the separated and transcribed SCUPA and the anti-SCUPA antibody. FIG. 5B shows the results when the anti-SCUPA antibody was used alone.
FIG. 6 shows a mass spectrometry spectrum when the protein separated by electrophoresis is transferred to a PVDF membrane. The horizontal axis represents molecular weight, and the vertical axis represents relative intensity. The numbers in the figure indicate the molecular weight of the peak. 6A shows SCUPA, and FIG. 6B shows HSA.
質量分析用プレート
本発明の質量分析用プレートは、支持体（基本構造）上にポリビニリデンジフロリド（ｐｏｌｙｖｉｎｙｌｉｄｅｎｅ ｄｉｆｌｏｒｉｄｅ；ＰＶＤＦ）を含有するコーティング（薄層）を有するものである。支持体の素材は、通常質量分析用プレートにおいて使用されているものであれば特に限定されない。例えば、絶縁体（ガラス、セラミクス、プラスチック・樹脂等）、金属（アルミニウム、ステンレス・スチール等）、導電性ポリマー、それらの複合体などが挙げられる。好ましくはアルミニウムプレートを用いる。
本発明の質量分析用プレートの形状は、使用する質量分析装置の、特に試料導入口に適合するように考案される。例えば、電気泳動後のゲルのサイズに合わせた集合型の質量分析用プレートに蛋白質を転写した後に、質量分析装置の試料導入口にフィットするように一本一本が簡単に分離できるようにあらかじめ切れ目を入れたクラスター型質量分析用プレート等が挙げられるが、これに限定されるものではない。
本発明において「ＰＶＤＦを含有するコーティング」とは、従来公知のＰＶＤＦ膜のように予め成型された構造体を支持体上に重層するのではなく、ＰＶＤＦ分子が分散した状態で支持体上に堆積されて形成される薄層をいう。ＰＶＤＦが堆積される態様は特に制限されないが、後述の質量分析用プレートの調製方法において例示される手段が好ましく用いられる。
薄層の厚さは、蛋白質の転写効率および質量分析の測定感度等に好ましくない影響を与えない範囲で適宜選択することができるが、例えば、約０．１〜約１０００μｍ、好ましくは約１〜約３００μｍである。
質量分析用プレートの調製方法
本発明の質量分析用プレートは、ＰＶＤＦで支持体表面をコーティングすることにより調製される。コーティングの手段としては、塗布、噴霧、蒸着、浸漬、印刷（プリント）、スパッタリングなどが好ましく例示される。
「塗布」する場合、ＰＶＤＦを、適当な溶媒、例えば、ジメチルホルムアミド（ｄｉｍｅｔｈｙｌ ｆｏｒｍａｍｉｄｅ；ＤＭＦ）などの有機溶媒に適当な濃度（例えば、約１〜約１００ｍｇ／ｍＬ程度）で溶解したもの（以下、「ＰＶＤＦ含有溶液」という）を、刷毛などの適当な道具を用いて支持体（基本構造，基板）に塗布することができる。
「噴霧」する場合、上記と同様にして調製したＰＶＤＦ含有溶液を噴霧器に入れ、支持体上に均一にＰＶＤＦが堆積されるように噴霧すればよい。
「蒸着」する場合、通常の有機薄膜作製用真空蒸着装置を用い、支持体を入れた真空槽中でＰＶＤＦ（固体でも溶液でもよい）を加熱・気化させることにより、支持体表面上にＰＶＤＦの薄層を形成させることができる。
「浸漬」させる場合、上記と同様にして調製したＰＶＤＦ含有溶液中に支持体を浸漬させればよい。
「印刷（プリント）」する場合は、支持体の材質に応じて通常使用され得る各種印刷技術を適宜選択して利用することができ、例えば、スクリーン印刷などが好ましく用いられる。
「スパッタリング」する場合は、例えば、真空中に不活性ガス（例、Ａｒガス等）を導入しながら支持体とＰＶＤＦ間に直流高電圧を印加し、イオン化したガスをＰＶＤＦに衝突させて、はじき飛ばされたＰＶＤＦ分子を支持体上に堆積させて薄層を形成させることができる。
コーティングは支持体全面に施してもよいし、質量分析に供される一面のみに施してもよい。
ＰＶＤＦはコーティング手段に応じて適宜好ましい形態で使用することができ、例えば、ＰＶＤＦ含有溶液、ＰＶＤＦ含有蒸気、ＰＶＤＦ固体分子などの形態で支持体にアプライされ得るが、ＰＶＤＦ含有溶液の形態でアプライすることが好ましい。「アプライする」とは、接触後にＰＶＤＦが支持体上に残留・堆積されるように支持体に接触させることをいう。アプライ量は特に制限はないが、ＰＶＤＦ量として、例えば、約１〜約１００μｇ／ｃｍ２が挙げられる。アプライ後に溶媒は自然乾燥、真空乾燥などにより除去する。
本発明の質量分析用プレートにおける支持体（基本構造，基板）は、ＰＶＤＦでコーティングする前に予め適当な物理的、化学的手法により、その表面を修飾（加工）しておいてもよい。具体的には、プレート表面を磨く、傷を付ける、酸処理、アルカリ処理、ガラス処理（テトラメトキシシランなど）等の手法が例示される。
本発明の質量分析用プレートは安定性に優れている。すなわち、ｐＨ２〜１０あるいは各種塩を含む水溶液、メタノール、アセトニトリルなどの溶媒、これらの混合溶媒への浸漬、電気的な負荷、湿潤と乾燥の繰り返し、高度な真空状態、などの条件下であっても接着面は剥がれないという特性を有する。
当該プレートの用途
Ａ．蛋白質等の同定
本発明の質量分析用プレートを用いて蛋白質をはじめとする種々の化合物を同定することができる。すなわち、分析対象物（例：蛋白質、核酸、オリゴヌクレオチド、糖、オリゴ糖、細胞膜レセプターに対するアゴニストもしくはアンタゴニスト、毒素、ウイルスエピトープ、ホルモン、ペプチド、酵素、酵素の基質もしくはインヒビター、コファクター、薬物、レクチン、抗体等）含有試料を電気泳動（例：ポリアクリルアミドゲル電気泳動）に付して分析対象物を分離し、分離された分析対象物を本発明の質量分析用プレートに転写し、転写された分析対象物を質量分析することにより、（その分子量に関する情報から）分析対象物を同定するものである。
分析対象物含有試料
分析対象物含有試料は、公知のいかなる手法によって（生体）材料より調製されてもよい。ここで（生体）材料としては、例えば、動植物や微生物等の、任意の生物の細胞または組織、あるいは細胞外液（例えば、血液、血漿、尿、骨髄液、腹水等）、細胞内液、細胞小顆粒内液などが挙げられる。また、細胞培養液、遺伝子組換えにより得られる培養液なども含まれる。
電気泳動に供される分析対象物含有試料を調製するには公知の方法が用いられる。例えば、蛋白質含有試料の場合、標的細胞を入手後、適切な緩衝液中で種々の蛋白質分解酵素阻害剤の存在下にホモゲナイズするか、ポリトロン等の細胞破壊装置で懸濁化するか、低浸透圧ショックにより細胞を破壊するか、超音波処理により細胞膜を破壊した後、遠心分離して上清を採取することにより可溶性画分を得ることができる。また得られた沈殿（不溶性）画分を、界面活性剤や蛋白質変性剤により可溶化して用いることもできる。
電気泳動
試料中の分析対象物を電気泳動により分離（ゲル上に展開）する工程である。電気泳動装置は公知のものを用いることができる。また市販品を用いてもよい。目的に応じて一次元ゲル電気泳動、二次元ゲル電気泳動のどちらも使用することができる。二次元ゲル電気泳動では、一次元の泳動は分析対象物の等電点による分離、二次元の泳動は分析対象物の分子量による分離が基本である。電気泳動に使用されるゲルのサイズは特に制限されない。１０ｃｍ×１０ｃｍが一般的であるが、必要ならば２０ｃｍ×２０ｃｍあるいは他のサイズも使用できる。また、ゲルの素材もポリアクリルアミドが基本であるが、目的によってはアガロースゲル、セルロースアセテート膜等、他の媒体の利用も可能である。ゲルの濃度も均一な濃度あるいはグラジエントな濃度の両方が使用できる。
転写
ゲル電気泳動により分離した分析対象物を、ゲルから本発明の質量分析用プレートに移行させる工程である。転写装置としては公知のものを用いることができる。また市販品を用いてもよい。転写の方法自体は公知である。泳動後ゲルに展開された分析対象物は、種々の方法（拡散、電気力その他）によって質量分析用プレートに移行される。この工程は一般に転写（ｂｌｏｔ）と呼ばれる。拡散転写、電気転写などが挙げられる。特に好ましくは電気転写である。
電気転写時に使用する緩衝液としては、ｐＨ７〜９、低塩濃度のものを用いることが好ましい。具体的には、トリス緩衝液、リン酸緩衝液、ホウ酸緩衝液、酢酸緩衝液などが例示される。トリス緩衝液としては、トリス／グリシン／メタノール緩衝液、ＳＤＳ−トリス−トリシン緩衝液など、リン酸緩衝液としては、ＡＣＮ／ＮａＣｌ／等張リン酸緩衝液、リン酸ナトリウム／ＡＣＮなど、ホウ酸緩衝液としては、ホウ酸ナトリウム−塩酸緩衝液、トリス−ホウ酸塩／ＥＤＴＡ、ホウ酸塩／ＡＣＮなど、酢酸緩衝液としては、トリス−酢酸塩／ＥＤＴＡなどが挙げられる。好ましくは、トリス／グリシン／メタノール緩衝液、ホウ酸ナトリウム−塩酸緩衝液である。トリス／グリシン／メタノール緩衝液の組成としては、トリス１０〜１５ｍＭ、グリシン７０〜１２０ｍＭ、メタノール７〜１３％程度が例示される。ホウ酸ナトリウム−塩酸緩衝液の組成としては、ホウ酸ナトリウム５〜２０ｍＭ程度が例示される。
また、当該転写後に、後の質量分析（ＭＡＬＤＩ法による）に有利なように、レーザー光を吸収し、エネルギー移動を通じて分析対象物分子のイオン化を促進するためにマトリックスと呼ばれる試薬を添加することもできる。当該マトリックスとしては、質量分析において公知のものを用いることができる。例えば、シナピン酸（ｓｉｎａｐｉｎｉｃ ａｃｉｄ；ＳＰＡ（＝３，５−ｄｉｍｅｔｈｏｘｙ−４−ｈｙｄｏｒｏｘｙｃｉｎａｍｍｉｃ ａｃｉｄ））、インドールアクリル酸（Ｉｎｄｏｌｅａｃｒｙｌｉｃ ａｃｉｄ；ＩＡＡ）、２，５−ジヒドロキシ安息香酸（２，５−ｄｉｈｙｄｒｏｘｙｂｅｎｚｏｉｃ ａｃｉｄ；ＤＨＢ）、α−シアノ−４−ヒドロキシ桂皮酸（α−ｃｙａｎｏ−４−ｈｙｄｒｏｘｙｃｉｎａｍｍｉｃ ａｃｉｄ；ＣＨＣＡ）等が挙げられるが、これらに限定されない。好ましくは、ＤＨＢまたはＣＨＣＡである。ＣＨＣＡの場合、少なくとも２１％飽和濃度のものを添加することが好ましい。具体的には２１〜１００％飽和濃度のもの、好ましくは４０〜１００％飽和濃度のもの、特に好ましくは５０〜１００％飽和濃度のものが挙げられる。
質量分析
本発明の質量分析用プレート上に転写された分析対象物を質量分析することにより、（分子量に関する情報から）分析対象物を同定する工程である。質量分析装置は、ガス状の試料をイオン化した後、その分子や分子断片を電磁場に投入し、その移動状況から質量数／電荷数によって分離、物質のスペクトルを求めることにより、物質の分子量を測定・検出する装置である。試料とレーザー光を吸収するマトリックスを混合、乾燥させて結晶化し、マトリックスからのエネルギー移動によるイオン化とレーザー照射による瞬間加熱により、イオン化した分析対象物を真空中に導くマトリックス支援レーザー脱イオン化（ＭＡＬＤＩ）と、初期加速による試料分子イオンの飛行時間差で質量数を分析する飛行時間型質量分析（ＴＯＦＭＳ）とをあわせて用いるＭＡＬＤＩ−ＴＯＦＭＳ法、１分析対象物を１液滴にのせて液体から直接電気的にイオン化する方法、試料溶液を電気的に大気中にスプレーして、個々の分析対象物多価イオンをｕｎｆｏｌｄの状態で気相に導くナノエレクトロスプレー質量分析（ｎａｎｏ−ＥＳＭＳ）法等の原理に基づく質量分析装置を使用することができる。
本発明の質量分析用プレート上に存在する分析対象物を質量分析する方法自体は公知である。
また、本発明によるプロテオーム解析の完全自動化のためには、レーザー射出口か、あるいは逆に質量分析用プレートを乗せた架台を一次元または二次元方向へ移動させ、連続全面スキャンすることにより、電気泳動により一次元または二次元に展開された分析対象物を全て分析にかけることが可能となる（断続的なスキャン法では、レーザー光の照射されない、すなわち分析されない分析対象物が残存する）。この方式に断続スキャン法を取り入れれば、９６穴プレートに分注された９６種のサンプルを一括して質量分析用プレートにマウントし（矩形チップに９６種類のサンプルが等間隔で配列する）、そのまま質量分析に供することも可能である。
Ｂ．分析対象物群の同定
本発明によれば、複数種の分析対象物の集合体（分析対象物群）を一度に解析することができる。すなわち、上述の種々の生体材料から調製される分析対象物含有試料は、通常、多種多様な分析対象物を含有している。かかる分析対象物群を一次元もしくは二次元のゲル電気泳動によって分離し、ゲル上に分離・展開された分析対象物群を、例えば、泳動ゲルと適合する大きさの本発明の質量分析用プレート（好ましくは、転写後、質量分析装置の試料導入口にフィットするように一本一本が簡単に分離できるようにあらかじめ切れ目を入れたクラスター型質量分析用プレート）に転写することにより、原則的に分析対象物含有試料中に含まれるすべての分析対象物（分析対象物が蛋白質であれば、プロテオーム）を、個別の位置に分画して質量分析用プレート上に転写することができる。これらの分析対象物群を上記の連続スキャン型質量分析装置を用いて測定することにより、転写された分析対象物群を一括して同定することが可能となる。
Ｃ．分析対象物複合体の同定
本発明によれば、分析対象物と当該分析対象物と親和性を有するもの（相互作用を示すもの）の関連性を一度に解析することができる。すなわち、電気泳動により分析対象物を分離し、分離した分析対象物を本発明の質量分析用プレートに転写し、その後に当該分析対象物と親和性を有する（相互作用を示す）化合物を含有する試料（例えば、ペプチド、蛋白質、核酸、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液、細胞膜画分、オルガネラ膜画分など）を添加して当該プレート上で複合体を形成させ、形成された複合体を質量分析することにより、（分子量に関する情報から）転写された分析対象物、当該分析対象物と親和性を有する（相互作用を示す）化合物、および／またはそれらの複合体を同定するものである。
本発明をより詳細に説明するために以下に実施例を挙げるが、本発明はこれらにより何ら限定されるものではない。
実施例１：質量分析用プレートの調製
ＰｒｏｔｅｉｎＣｈｉｐ Ｓｙｓｔｅｍ（Ｃｉｐｈｅｒｇｅｎ社製）の質量分析用プレート導入部に適合するように調製したアルミニウム製の基本構造（アルミプレート）を１％テトラメトキシシラン／１％酢酸溶液に浸漬し、風乾した後に焼成（１３０℃、３時間）した。このプレートに、ポリビニリデンジフロリド（ＰＶＤＦ）をジメチルホルムアミド（Ｄｆ）に１０ｍｇ／ｍＬとなるように溶解したものを塗布した。調製された質量分析用プレートは大きさが７８ｍｍ×８ｍｍ×２ｍｍ、表面が白色の板状であった。
本質量分析用プレートは、その後の有機溶媒への浸漬による前工程、電気泳動ゲルとの接触、種々の緩衝液中での電気転写、その後のマトリックスの塗布・乾燥及び質量分析時の高度真空下、などの各工程で、亀裂、剥離、損傷、変色等の化学的、電気的、機械的、物理的変性が一切生じなかった。
本質量分析用プレートを使用して以下の検討を行った。
実施例２：質量分析用プレートへの蛋白質の電気転写
プレステインド分子量マーカー（Ｂｉｏ−Ｒａｄ社製）を１２％ＳＤＳ−ポリアクリルアミドゲル電気泳動した後に、ゲル上の蛋白質を１２．５ｍＭ Ｔｒｉｓ／９６ｍＭ グリシン／１０％メタノール緩衝液中で、実施例１で調製した質量分析用プレートに対して、９０ｍＡで３時間電気転写した。その結果、転写後のポリアクリルアミドゲルには分子量９万と１１万のプレステインド蛋白質が若干残存するものの（図１Ｂ）、いずれの蛋白質とも本発明の質量分析用プレートに効率よく転写された（図１Ｃ）。
実施例３：質量分析用プレートに転写された蛋白質に対する質量分析
サンプル（蛋白質混合物）を１２％ＳＤＳ−ポリアクリルアミドゲル電気泳動した後にゲルを直接、実施例１の質量分析用プレートに接触させ、電気泳動で分離された蛋白質を実施例１で調製した質量分析用プレートに電気転写させ、本質量分析用プレートを直ちに質量分析装置に導入して測定した。マトリックスとして２，５−ジヒドロキシ安息香酸（ＤＨＢ；７５ｍｇ／ｍＬのエタノール溶液）を用い、測定装置はＰｒｏｔｅｉｎＣｈｉｐ Ｓｙｓｔｅｍ（前記）を使用して、Ｄｅｔｅｃｔｏｒ ｖｏｌｔａｇｅ：１８００ Ｖ；Ｄｅｔｅｃｔｏｒ Ｓｅｎｓｉｔｉｖｉｔｙ：８；Ｌａｓｅｒ Ｉｎｔｅｎｓｉｔｙ：２８０の条件で測定を行った。質量校正はミオグロビン（ウマ筋肉由来）、ＧＡＰＤＨ（ウサギ由来）、アルブミン（ウシ血清由来）を用いて外部校正を行った。本実験では蛋白質混合物として、一本鎖尿性プラスミノーゲンアクチベータ（ｓｉｎｇｌｅ ｃｈａｉｎ ｕｒｉｎａｒｙ ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｔｏｒ；ＳＣＵＰＡ）とヒト血清アルブミン（ｈｕｍａｎ ｓｅｒｕｍ ａｌｂｕｍｉｎ；ＨＳＡ）の二種類の蛋白質を使用し、１２％ＳＤＳ−ポリアクリルアミドゲルに各４μｇの蛋白質混合物を還元下でタンデムに２回電気泳動した。すなわち、初回サンプルを添加し、３０ｍＡで４０分間電気泳動した後、電流を一旦止め、２回目に同じレーンに同じサンプルを添加し、さらに３０ｍＡで２３分間電気泳動した。泳動後、ゲルを泳動レーンごとにカットし、さらにゲル上面の添加位置から３９ｍｍの位置でカットし、二枚の泳動ゲルの上面同士が接触するように並べた後に、質量分析用プレートを接触させた（図２Ａ）。この時、泳動サンプルはＳＣＵＰＡ、ＨＳＡ、ＳＣＵＰＡ、ＨＳＡ、ＨＳＡ、ＳＣＵＰＡ、ＨＳＡ、ＳＣＵＰＡ、の順序に配列し、質量分析用プレートには各蛋白質が４バンド、合計８バンド転写された。
使用した質量分析装置は、一本の質量分析用プレートに対してレーザー光が連続的に照射されず、２４個所にわたって等間隔で照射されるために、転写された蛋白質の質量分析用プレート上の位置によっては、レーザー光が全く当たらない場合、一部分当たる場合、完全に当たる場合が想定される（図２Ｂ）。このような分析装置の制約の下でレーザー光が最も多最に当たリピーク強度が最も高くなる確率を向上させるために、先に述べた手法で各モデル蛋白質を質量分析用プレートの異なる位置にそれぞれ４箇所転写させるように工夫した。その結果、理論的には同じ質量の蛋白質が転写されても、質量分析の結果得られたピークの高さは様々であった（図３）。したがって、本実験では、得られた複数のピークの中で相対強度の最も高いピークをレーザー光が完全に当たった真の値に最も近い値とみなして考察を行った。
質量分析用プレートは事前にメタノールに浸漬し、転写用緩衝液（１０ｍＭホウ酸ナトリウム緩衝液、ｐＨ８．０）で平衡化した後に、９０ｍＡで２時間電気転写を行った。さらに質量分析して相対ピーク強度を測定した。比較対照として、ＰＶＤＦをコートしない通常のアルミプレート、ヘキサデシル基を導入したアルミプレート（ＰｒｏｔｅｉｎＣｈｉｐ ａｒｒａｙ、Ｃｉｐｈｅｒｇｅｎ社製、Ｈ４）も用いて同様の実験を行った。結果を図３、図４に示す。
ＳＣＵＰＡの場合、本発明の質量分析用プレート（図３Ａ）での相対ピーク強度は１２．３１で、通常のアルミプレート（図３Ｂ）の値（０．８７）の１４倍高いものであった。ＨＳＡについて同様に比較すると、本質量分析用プレートでの相対ピーク強度はアルミプレートに比較して４９倍（６．２６：０．１２９）高いものであった。また、質量分析用プレートはＨ４（図３Ｃ）と比較しても２倍から４倍相対ピーク強度が高いものであった。さらに、本発明の質量分析用プレート（図４ＡおよびＣ）はアルミプレート（図４ＢおよびＤ）に比較してスペクトルが鮮明であった。これらの結果から、本発明の質量分析用プレートを用いることにより、複数種の蛋白質を電気泳動で分離した後、迅速かつ高感度に一括して質量分析できることが判明した。
実施例４：電気泳動分離後の電気転写蛋白質と結合蛋白質の相互作用の解析
本発明の質量分析用プレートを用いた応用例の一つとして、質量分析用プレートに転写された蛋白質と相互作用しうる蛋白質との結合およびその後の質量分析によるそれら蛋白質複合体の同定を検討した。材料（マテリアル）としてＳＣＵＰＡとその抗体（抗ＳＣＵＰＡ抗体）を用いた。
ＳＣＵＰＡ（４μｇ）を１２％ＳＤＳ−ポリアクリルアミドゲルに添加後、１時間電気泳動した。泳動後にゲルをカットし、ゲル上のＳＣＵＰＡを実施例１で調製した質量分析用プレートに実施例３と同様に電気転写した。ＳＣＵＰＡが転写された質量分析用プレートをブロッキング後、抗血清（硫安により粗精製したもの、抗ＳＣＵＰＡ抗体を含んでいる）を添加し、一晩反応させた。反応終了後、プレートをＰＢＳ緩衝液で洗浄し、マトリックスを添加して、質量分析を行った。その結果を図５に示す。
電気泳動後の質量分析用プレートに転写されたＳＣＵＰＡは４８，６１６にピークが現れ、同プレート上に固定されたＳＣＵＰＡと相互作用した抗ＳＣＵＰＡ抗体のピークは１４５，３００（７３，１１５は同抗体の２価イオン）に認められた（図５Ａ）。また、コントロールとしてＳＣＵＰＡを含まないゲルでは、電気転写、ブロッキング、抗ＳＣＵＰＡ抗体添加、ＰＢＳ洗浄等、同様の操作を行ったにもかかわらず、本質量分析用プレートには一切ピークが観察されなかった（図５Ｂ）。
以上の結果から、電気泳動分離後、本質量分析用プレートに転写されたＳＣＵＰＡは、自身と相互作用しうる蛋白質（抗ＳＣＵＰＡ抗体）との結合活性を保持したこと、及び転写された蛋白質とそれと相互作用する蛋白質の双方の分子量が実際に一括して測定され、その結果、両分子が同定されたことから、本質量分析用プレート上で蛋白質−蛋白質複合体の検出と、本質量分析用プレート上で生成された複合体の同定が可能であることが証明された。
比較例１：ＰＶＤＦ膜への転写と質量分析
１２％ＳＤＳ−ポリアクリルアミドゲルにＳＣＵＰＡとＨＳＡの二種類の蛋白質各４μｇの混合物を還元下でタンデムに２回電気泳動した。初回サンプルを添加し、３０ｍＡで４０分間電気泳動した後、電流を一旦止め、２回目に同じレーンに同じサンプルを添加し、さらに３０ｍＡで２３分間電気泳動した。泳動後ゲルを泳動レーンごとにカットし、さらにゲル上面の添加位置から３９ｍｍの位置でカットし、図２Ａに示すように二枚の泳動ゲルの上面同士が接触するように並べたゲル上に、７８ｍｍ×８ｍｍにカットしたＰＶＤＦ膜を載せ、１０ｍＭホウ酸ナトリウム緩衝液（ｐＨ８．０）中で９０ｍＡ、２時間電気転写した。転写終了後、ＰＶＤＦ膜をＰＢＳで洗浄し、蒸留水でリンス、乾燥させ、さらに透明両面テープ（住友スリーエム社製）でアルミプレートに貼り付けた。これにマトリックス（ＤＨＢ）を添加してＰｒｏｔｅｉｎＣｈｉｐ Ｓｙｓｔｅｍ（前記）で質量分析を行った。
その結果、ＰＶＤＦ膜に転写されたＳＣＵＰＡは５０，０９６．９に、ＨＳＡは６７，３９４．５に、各々ピークが現れたが、相対ピーク強度が極めて低く、さらにＳ／Ｎ比も低いためピークの同定が困難であった（図６）。また、通常の質量分析用プレート（本発明の質量分析用プレートも同様であるが）では高度真空状態に到達する時間が２〜３分で、その後すぐに測定可能となるのに対して、ＰＶＤＦ膜を使用した場合は、真空状態に到達する時間が４５分もかかり、使用した質量分析装置に過大な負荷をかけ、頻繁な使用に耐えられないことが判明した。
実施例５：本発明の質量分析用プレートを用いた種々のプロテオーム解析
種々の細胞もしくは組織抽出サンプルを用いて、比較的低分子量域（分子量１，０００〜２０，０００）での本発明の質量分析用プレートの性能を確認した。以下の実験では、検体を１６％ＳＤＳ−ポリアクリルアミドゲル（トリス−トリシン緩衝液中）にアプライし、９０分間電気泳動した後、１０ｍＭホウ酸ナトリウム緩衝液（塩酸でｐＨ８．０に調整したもの）中、ゲルから本発明の質量分析用プレートに１〜２時間電気転写した。転写終了後、プレートをリンスし、マトリックスをスポットして質量分析を行った。
（１）ブタ小脳を、４℃、１Ｎ酢酸でホモジナイズし、４℃、３，０００ｒｐｍで３０分間遠心して上清を回収した。アセトニトリルを終濃度１０％となるように添加し、逆相クロマト用カラムにアプライした。１０％アセトニトリルを含む０．１％トリフルオロ酢酸（ＴＦＡ）で洗浄し、６０％アセトニトリルを含む０．１％ＴＦＡで溶出した。溶出物を凍結乾燥し、ブタ小脳抽出物とした。この抽出物を、ＳＤＳ−ＰＡＧＥで分離後、本発明の質量分析用プレートに電気転写した。マトリックスとして、０．５％ＴＦＡ／５０％ＡＣＮ中に飽和α−シアノ−４−ヒドロキシ桂皮酸（ＣＨＣＡ）を含むものを用いて質量分析を行った。その結果、分子量３，０００〜２０，０００の範囲でピークを検出可能であった。
これを、マトリックスとして飽和ＣＨＣＡの代わりに、ＤＨＢ（１５０ｍｇ／ｍＬエタノール）、飽和ＳＰＡ（０．５％ＴＦＡ／５０％ＡＣＮ中）を用いた場合と比較してみた。ＤＨＢの場合は、分子量３，０００〜２０，０００の範囲では明確なピークを認めなかった。飽和ＳＰＡの場合は、同じ分子量の範囲で数個のピークを認めただけであった。これに対して、飽和ＣＨＣＡでは、分子量２，０００〜１０，０００の範囲および５，０００〜２０，０００の範囲で多数のピークを認めた。これらの結果から、比較的低分子量域の蛋白質の同定にはＣＨＣＡがより好ましいことが示された。
（２）マトリックスとして５０％飽和ＣＨＣＡを用いた以外は上記（１）と同様にして、ブタ小脳抽出物の質量分析を行った。その結果、分子量１，０００以上の範囲でピークを検出可能であった。特に、分子量３，０００〜２０，０００の範囲でピークを検出するのに優れていた。
これを、５０％飽和濃度のＣＨＣＡの代わりに、１０または２０％飽和濃度のものを用いた場合と、分子量１，０００〜１０，０００の範囲での質量分析におけるピークの出現の程度を比較してみた。１０％飽和濃度のＣＨＣＡの場合は数個のピークを認めた。２０％飽和濃度のＣＨＣＡの場合はピークの数が１０％飽和濃度のものに比べて若干多く認められた。これに対し、５０％飽和濃度のＣＨＣＡの場合は多数のピークを認めた。
（３）Ｕ９３７細胞（１×１０５〜２×１０６個／ｍｌ）を、１０％ＦＣＳを含むＲＰＭ１１６４０培地中、１００ｎｇ／ｍｌホルボール１２−ミリスチン酸エステル１３−酢酸塩（ＰＭＡ）の共存下で４８時間培養した。ＰＭＡ非共存下で同様に実験を行ったものを対照とした。培養終了後にリン酸緩衝生理食塩水（ＰＢＳ）で細胞を洗浄し、セルペレットを調製した。さらに、プロテイン抽出試薬（ノバジュン）およびプロテアーゼインヒビターを添加して４℃で１５分間放置した後、遠心して上清を回収し、セルライセートとした。これをＳＤＳ−ＰＡＧＥにて分離後、本発明の質量分析用プレートに電気転写した。さらに質量分析を行い、分子量３，０００〜２０，０００の範囲で検出されたピークの強度について、ＰＭＡで刺激したＵ９３７細胞での結果を対照のＵ９３７細胞での結果と比較した。その結果、ＰＭＡ刺激による蛋白質発現（ピーク強度）の変動は以下の通りであった（表１）。
実施例６：電気転写時の緩衝液の効果の検討
電気転写時の緩衝液として、トリス緩衝液（２５ｍＭトリス／１９２ｍＭグリシン／２０％メタノール、ｐＨ８．３）、リン酸緩衝液（５％ＡＣＮ／１２５ｍＭ ＮａＣｌ／ＰＢＳ、ｐＨ７．２）、酢酸緩衝液（４０ｍＭトリス−酢酸塩／１ｍＭ ＥＤＴＡ、ｐＨ８．０）、ホウ酸緩衝液（１０ｍＭホウ酸ナトリウム−塩酸、ｐＨ８．０）を用いた。質量分析用の蛋白質としては、ＨＳＡ、ＳＣＵＰＡを用いた。その他は実施例５の方法に準じて実験を行った。その結果、各緩衝液の中では、ホウ酸緩衝液を用いた場合に質量分析のピーク強度が最大となった。例えば、ピーク強度の最大値は、ホウ酸緩衝液対リン酸緩衝液ではＳＣＵＰＡで約２１倍、ＨＳＡで約４倍であった。 Mass spectrometry plate
The plate for mass spectrometry of the present invention has a coating (thin layer) containing polyvinylidene difluoride (PVDF) on a support (basic structure). The material for the support is not particularly limited as long as it is usually used in a plate for mass spectrometry. Examples thereof include insulators (glass, ceramics, plastics / resins, etc.), metals (aluminum, stainless steel, etc.), conductive polymers, and composites thereof. An aluminum plate is preferably used.
The shape of the plate for mass spectrometry of the present invention is devised so as to be suitable for the sample inlet of the mass spectrometer to be used. For example, after transferring the protein to a mass spectrometric plate that matches the size of the gel after electrophoresis, it can be separated in advance so that it can be easily separated to fit the sample inlet of the mass spectrometer. Examples include, but are not limited to, a cluster-type mass spectrometry plate with a cut.
In the present invention, the “coating containing PVDF” means that a previously formed structure is not layered on the support as in a conventionally known PVDF film, but is deposited on the support in a state where PVDF molecules are dispersed. This is a thin layer formed. Although the aspect in which PVDF is deposited is not particularly limited, means exemplified in a method for preparing a plate for mass spectrometry described later is preferably used.
The thickness of the thin layer can be appropriately selected within a range that does not unfavorably affect the protein transfer efficiency, the measurement sensitivity of mass spectrometry, etc., for example, about 0.1 to about 1000 μm, preferably about 1 to About 300 μm.
Method for preparing a plate for mass spectrometry
The plate for mass spectrometry of the present invention is prepared by coating the support surface with PVDF. Preferred examples of the coating means include application, spraying, vapor deposition, dipping, printing (printing), and sputtering.
When “coating”, PVDF is dissolved in an appropriate solvent (for example, about 1 to about 100 mg / mL) in an organic solvent such as dimethylformamide (DMF) (hereinafter, about 1 to about 100 mg / mL). (Referred to as “PVDF-containing solution”) can be applied to a support (basic structure, substrate) using a suitable tool such as a brush.
In the case of “spraying”, a PVDF-containing solution prepared in the same manner as described above may be put in a sprayer and sprayed so that PVDF is uniformly deposited on the support.
In the case of “evaporation”, PVDF (which may be solid or solution) is heated and vaporized in a vacuum tank containing a support using a normal vacuum deposition apparatus for producing an organic thin film. A thin layer can be formed.
When “immersing”, the support may be immersed in a PVDF-containing solution prepared in the same manner as described above.
In the case of “printing”, various printing techniques that can be normally used depending on the material of the support can be appropriately selected and used. For example, screen printing is preferably used.
In the case of “sputtering”, for example, a DC high voltage is applied between the support and the PVDF while introducing an inert gas (eg, Ar gas) in a vacuum, and the ionized gas is collided with the PVDF to be repelled. The PVDF molecules deposited can be deposited on a support to form a thin layer.
The coating may be applied to the entire surface of the support or may be applied only to one surface subjected to mass spectrometry.
PVDF can be used in a preferred form as appropriate depending on the coating means. For example, PVDF can be applied to the support in the form of a PVDF-containing solution, PVDF-containing vapor, PVDF solid molecule, etc., but is applied in the form of a PVDF-containing solution. It is preferable. “Apply” means that the PVDF is brought into contact with the support so that it remains and is deposited on the support after the contact. The amount of Apply is not particularly limited, but the amount of PVDF is, for example, about 1 to about 100 μg / cm.2Is mentioned. After the application, the solvent is removed by natural drying, vacuum drying or the like.
The support (basic structure, substrate) in the mass spectrometric plate of the present invention may be modified (processed) in advance by an appropriate physical or chemical technique before coating with PVDF. Specifically, techniques such as polishing the plate surface, scratching, acid treatment, alkali treatment, glass treatment (tetramethoxysilane, etc.) are exemplified.
The plate for mass spectrometry of the present invention is excellent in stability. That is, under conditions such as aqueous
Uses of the plate
A. Identification of proteins, etc.
Various compounds including proteins can be identified using the plate for mass spectrometry of the present invention. Analytes (eg, proteins, nucleic acids, oligonucleotides, sugars, oligosaccharides, agonists or antagonists to cell membrane receptors, toxins, viral epitopes, hormones, peptides, enzymes, enzyme substrates or inhibitors, cofactors, drugs, lectins The sample containing the sample was subjected to electrophoresis (eg, polyacrylamide gel electrophoresis) to separate the analyte, and the separated analyte was transferred to the mass spectrometric plate of the present invention and transferred. The analysis object is identified by mass analysis of the analysis object (from information on its molecular weight).
Sample containing analyte
The analyte-containing sample may be prepared from the (biological) material by any known technique. Here, as the (biological) material, for example, cells or tissues of any living organisms such as animals and plants and microorganisms, or extracellular fluids (for example, blood, plasma, urine, bone marrow fluid, ascites fluid), intracellular fluids, cells Examples include small intragranular fluid. Also included are cell culture fluids, culture fluids obtained by genetic recombination, and the like.
A known method is used to prepare an analyte-containing sample to be subjected to electrophoresis. For example, in the case of a protein-containing sample, after obtaining the target cells, homogenize in the presence of various protease inhibitors in an appropriate buffer, suspend with a cell disruption device such as polytron, or low penetration A soluble fraction can be obtained by disrupting cells by pressure shock or disrupting a cell membrane by sonication and then collecting the supernatant by centrifugation. The obtained precipitated (insoluble) fraction can be used after being solubilized with a surfactant or a protein denaturant.
Electrophoresis
This is a step of separating the analyte in the sample by electrophoresis (developing on a gel). A well-known thing can be used for an electrophoresis apparatus. Commercial products may also be used. Either one-dimensional gel electrophoresis or two-dimensional gel electrophoresis can be used depending on the purpose. In two-dimensional gel electrophoresis, one-dimensional electrophoresis is basically based on separation of the analyte by the isoelectric point, and two-dimensional electrophoresis is based on separation based on the molecular weight of the analyte. The size of the gel used for electrophoresis is not particularly limited. 10 cm x 10 cm is common, but 20 cm x 20 cm or other sizes can be used if desired. The gel material is basically polyacrylamide, but other media such as agarose gel and cellulose acetate film can be used depending on the purpose. The gel concentration can be either a uniform concentration or a gradient concentration.
Transcription
This is a step of transferring the analyte separated by gel electrophoresis from the gel to the mass spectrometric plate of the present invention. A known transfer device can be used. Commercial products may also be used. The transfer method itself is known. The analyte developed on the gel after electrophoresis is transferred to the plate for mass spectrometry by various methods (diffusion, electric force, etc.). This process is generally called blotting. Examples include diffusion transfer and electric transfer. Particularly preferred is electrotransfer.
As the buffer used at the time of electrotransfer, it is preferable to use a buffer having a pH of 7-9 and a low salt concentration. Specific examples include Tris buffer, phosphate buffer, borate buffer, and acetate buffer. Tris buffer as tris / glycine / methanol buffer, SDS-tris-tricine buffer, etc., phosphate buffer as ACN / NaCl / isotonic phosphate buffer, sodium phosphate / ACN, borate, etc. Examples of the buffer solution include sodium borate-hydrochloric acid buffer solution, Tris-borate / EDTA, and borate / ACN. Examples of the acetate buffer include Tris-acetate / EDTA. Preferred are tris / glycine / methanol buffer and sodium borate-hydrochloric acid buffer. Examples of the composition of the tris / glycine / methanol buffer include Tris 10-15 mM, glycine 70-120 mM, and methanol 7-13%. Examples of the composition of the sodium borate-hydrochloric acid buffer include about 5 to 20 mM sodium borate.
In addition, after the transfer, a reagent called a matrix may be added so as to absorb laser light and promote ionization of analyte molecules through energy transfer, so as to be advantageous for subsequent mass spectrometry (by MALDI method). it can. As the matrix, those known in mass spectrometry can be used. For example, sinapinic acid (SPA = 3,5-dimethyl-4-hydroxycinaminic acid), indoleacrylic acid (IAA), 2,5-dihydroxybenzoic acid (2,5-dihydroxybenzoic acid; 2,5-dihydroxybenzoic acid; ), Α-cyano-4-hydroxycinnamic acid (CHCA) and the like, but not limited thereto. Preferably, it is DHB or CHCA. In the case of CHCA, it is preferable to add at least 21% saturated concentration. Specifically, those having a saturation concentration of 21 to 100%, preferably those having a saturation concentration of 40 to 100%, particularly preferably those having a saturation concentration of 50 to 100%.
Mass spectrometry
This is a step of identifying an analysis object (from information on molecular weight) by mass-analyzing the analysis object transferred onto the plate for mass spectrometry of the present invention. The mass spectrometer measures the molecular weight of a substance by ionizing a gaseous sample and then putting the molecule or molecular fragment into an electromagnetic field, separating it by mass number / charge number from its movement, and obtaining the spectrum of the substance. -It is a device to detect. Matrix-assisted laser deionization (MALDI), in which a sample and a matrix that absorbs laser light are mixed, dried, crystallized, and ionized analyte is brought into vacuum by ionization by energy transfer from the matrix and instantaneous heating by laser irradiation. And the MALDI-TOFMS method using time-of-flight mass spectrometry (TOFMS), which analyzes the mass number based on the time-of-flight difference of sample molecular ions by initial acceleration. Principles of ionization, nanoelectrospray mass spectrometry (nano-ESMS), etc., in which sample solutions are electrically sprayed into the atmosphere and individual analyte multivalent ions are led to the gas phase in an unfolded state A mass spectrometer based on can be used.
A method for mass spectrometry of an analysis object present on the mass spectrometry plate of the present invention is known per se.
In order to fully automate the proteome analysis according to the present invention, the laser injection port, or conversely, the platform on which the mass analysis plate is placed is moved in the one-dimensional or two-dimensional direction, and the entire surface is scanned, so that It is possible to analyze all the analysis objects developed one-dimensionally or two-dimensionally by electrophoresis (in the intermittent scanning method, an analysis object that is not irradiated with laser light, that is, is not analyzed remains). If the intermittent scan method is adopted in this method, 96 types of samples dispensed in a 96-well plate are collectively mounted on a plate for mass spectrometry (96 types of samples are arranged at equal intervals on a rectangular chip), It can also be used for mass spectrometry as it is.
B. Identification of analyte group
ADVANTAGE OF THE INVENTION According to this invention, the aggregate | assembly (analysis object group) of a multiple types of analysis target object can be analyzed at once. That is, the analyte-containing sample prepared from the various biomaterials described above usually contains a wide variety of analytes. The analysis object group is separated by one-dimensional or two-dimensional gel electrophoresis, and the analysis object group separated and developed on the gel is, for example, a mass spectrometry plate of the present invention having a size compatible with the electrophoresis gel. (Preferably, after transfer, cluster-type mass spectrometry plate that has been cut in advance so that it can be easily separated one by one so that it fits the sample inlet of the mass spectrometer.) All the analytes contained in the analyte-containing sample (if the analyte is a protein, the proteome) can be fractionated at individual positions and transferred onto the mass spectrometry plate. By measuring these analysis object groups using the above-described continuous scanning mass spectrometer, the transferred analysis object groups can be collectively identified.
C. Identification of analyte complexes
According to the present invention, it is possible to analyze the relationship between an analysis object and an object having an affinity for the analysis object (indicating an interaction) at a time. That is, the analyte is separated by electrophoresis, the separated analyte is transferred to the mass spectrometric plate of the present invention, and then contains a compound having affinity (showing interaction) with the analyte. Add a sample (eg, peptide, protein, nucleic acid, non-peptidic compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract, cell membrane fraction, organelle membrane fraction, etc.) Compound formed on plate and mass-analyzed to form complex, transferred analyte (from molecular weight information), compound having affinity (showing interaction) with the analyte , And / or their complexes.
In order to describe the present invention in more detail, examples are given below, but the present invention is not limited to these examples.
Example 1: Preparation of a plate for mass spectrometry
A basic structure made of aluminum (aluminum plate) prepared so as to be compatible with a plate portion for mass spectrometry of ProteinChip System (manufactured by Ciphergen) is immersed in a 1% tetramethoxysilane / 1% acetic acid solution, air-dried and then fired ( (130 ° C., 3 hours). A solution of polyvinylidene difluoride (PVDF) dissolved in dimethylformamide (Df) at 10 mg / mL was applied to this plate. The prepared plate for mass spectrometry was a plate having a size of 78 mm × 8 mm × 2 mm and a white surface.
The plate for mass spectrometry is pre-processed by subsequent immersion in an organic solvent, contact with an electrophoresis gel, electrotransfer in various buffers, subsequent application and drying of the matrix, and high vacuum during mass spectrometry. No chemical, electrical, mechanical, or physical modification such as cracking, peeling, damage, or discoloration occurred in each process such as.
The following examination was performed using this plate for mass spectrometry.
Example 2: Electrotransfer of protein to a plate for mass spectrometry
After prestained molecular weight marker (Bio-Rad) was subjected to 12% SDS-polyacrylamide gel electrophoresis, the protein on the gel was prepared in Example 1 in 12.5 mM Tris / 96 mM glycine / 10% methanol buffer. The resulting mass spectrometry plate was electrotransferred at 90 mA for 3 hours. As a result, although some pre-tained proteins with molecular weights of 90,000 and 110,000 remained in the polyacrylamide gel after transfer (FIG. 1B), both proteins were efficiently transferred to the plate for mass spectrometry of the present invention (FIG. 1C).
Example 3: Mass spectrometry for proteins transferred to a plate for mass spectrometry
After the sample (protein mixture) was subjected to 12% SDS-polyacrylamide gel electrophoresis, the gel was directly brought into contact with the mass spectrometry plate of Example 1, and the protein separated by electrophoresis was prepared in Example 1. The plate was electrotransferred, and the plate for mass spectrometry was immediately introduced into the mass spectrometer and measured. 2,5-Dihydroxybenzoic acid (DHB; 75 mg / mL ethanol solution) was used as the matrix, and the measuring apparatus was Protein Chip System (described above), Detector voltage: 1800 V; Detector Sensitivity: 8; Laser Intensity: 280 The measurement was performed under the following conditions. Mass calibration performed external calibration using myoglobin (derived from horse muscle), GAPDH (derived from rabbit), and albumin (derived from bovine serum). In this experiment, two types of proteins, single chain urinary plasminogen activator (SCUPA) and human serum albumin (HSA), were used as a protein mixture, and 12% SDS-poly An acrylamide gel was electrophoresed twice in tandem with 4 μg of each protein mixture under reduction. That is, after adding the first sample and performing electrophoresis at 30 mA for 40 minutes, the current was temporarily stopped, the same sample was added to the same lane for the second time, and electrophoresis was further performed at 30 mA for 23 minutes. After the electrophoresis, the gel is cut into each electrophoresis lane, further cut at a position 39 mm from the addition position on the upper surface of the gel, and arranged so that the upper surfaces of the two electrophoresis gels are in contact with each other. (FIG. 2A). At this time, the electrophoresis sample was arranged in the order of SCUPA, HSA, SCUPA, HSA, HSA, SCUPA, HSA, SCUPA, and 4 bands of each protein were transferred to the mass spectrometric plate.
Since the used mass spectrometer is not continuously irradiated with a laser beam to one mass analysis plate but is irradiated at equal intervals over 24 locations, the transferred protein is on the mass analysis plate. Depending on the position, it is assumed that the laser beam does not hit at all, the laser beam hits a part, or the laser beam hits completely (FIG. 2B). In order to improve the probability that the re-peak intensity is the highest when the laser beam hits the most under such analyzer restrictions, each model protein is placed at a different position on the plate for mass spectrometry using the method described above. It was devised so that each of the four points was transferred. As a result, even if proteins with the same mass were theoretically transcribed, the heights of the peaks obtained as a result of mass spectrometry varied (FIG. 3). Therefore, in this experiment, the peak having the highest relative intensity among the plurality of obtained peaks was considered as the value closest to the true value when the laser beam was completely irradiated.
The plate for mass spectrometry was immersed in methanol in advance, equilibrated with a transfer buffer (10 mM sodium borate buffer, pH 8.0), and then subjected to electrotransfer at 90 mA for 2 hours. Further, the relative peak intensity was measured by mass spectrometry. As a comparative control, the same experiment was performed using a normal aluminum plate not coated with PVDF and an aluminum plate into which a hexadecyl group was introduced (ProteinChip array, manufactured by Ciphergen, H4). The results are shown in FIGS.
In the case of SCUPA, the relative peak intensity in the mass spectrometry plate of the present invention (FIG. 3A) was 12.31, which was 14 times higher than the value (0.87) of the normal aluminum plate (FIG. 3B). When HSA was compared in the same manner, the relative peak intensity in the plate for mass spectrometry was 49 times (6.26: 0.129) higher than that in the aluminum plate. Moreover, the plate for mass spectrometry had a
Example 4: Analysis of interaction between electrotranscribed protein and binding protein after electrophoretic separation
As an application example using the plate for mass spectrometry of the present invention, the binding of the protein transcribed to the plate for mass spectrometry and the protein capable of interacting with each other and the identification of those protein complexes by mass spectrometry were examined. . SCUPA and its antibody (anti-SCUPA antibody) were used as materials.
SCUPA (4 μg) was added to a 12% SDS-polyacrylamide gel, followed by electrophoresis for 1 hour. After electrophoresis, the gel was cut, and SCUPA on the gel was electrotransferred to the mass spectrometry plate prepared in Example 1 in the same manner as in Example 3. After blocking the SCUPA-transferred mass spectrometry plate, antiserum (crudely purified with ammonium sulfate, containing anti-SCUPA antibody) was added and allowed to react overnight. After completion of the reaction, the plate was washed with PBS buffer, a matrix was added, and mass spectrometry was performed. The result is shown in FIG.
The peak of SCUPA transferred to the plate for mass spectrometry after electrophoresis appears at 48,616, and the peak of the anti-SCUPA antibody interacting with SCUPA immobilized on the plate is 145,300 (73,115 are the same antibody). (Fig. 5A). In addition, in the gel without SCUPA as a control, no peaks were observed on the plate for mass spectrometry even though the same operations such as electrotransfer, blocking, addition of anti-SCUPA antibody, and PBS washing were performed. (FIG. 5B).
From the above results, after electrophoresis separation, the SCUPA transferred to the plate for mass spectrometry retained the binding activity with the protein (anti-SCUPA antibody) capable of interacting with itself, and the transferred protein and the Since the molecular weights of both interacting proteins were actually measured together and as a result, both molecules were identified, the detection of the protein-protein complex on the plate for mass spectrometry and the plate for mass spectrometry It proved possible to identify the complex produced above.
Comparative Example 1: Transfer to PVDF membrane and mass spectrometry
A 12% SDS-polyacrylamide gel was electrophoresed twice in tandem with a mixture of 4 μg each of the two types of proteins SCUPA and HSA. After the first sample was added and electrophoresis was performed at 30 mA for 40 minutes, the current was temporarily stopped, the same sample was added to the same lane for the second time, and electrophoresis was further performed at 30 mA for 23 minutes. After the electrophoresis, the gel is cut for each electrophoresis lane, further cut at a position 39 mm from the addition position on the upper surface of the gel, and on the gel arranged so that the upper surfaces of the two electrophoresis gels are in contact with each other as shown in FIG. A PVDF membrane cut to 78 mm × 8 mm was mounted, and electrotransferred at 90 mA for 2 hours in 10 mM sodium borate buffer (pH 8.0). After completion of the transfer, the PVDF membrane was washed with PBS, rinsed with distilled water, dried, and further adhered to an aluminum plate with a transparent double-sided tape (Sumitomo 3M). Matrix (DHB) was added thereto, and mass spectrometry was performed using ProteinChip System (described above).
As a result, SCUPA transferred to the PVDF film showed peaks at 50,096.9 and HSA at 67,394.5, respectively. However, the relative peak intensity is extremely low, and the S / N ratio is also low. Was difficult to identify (FIG. 6). In addition, the normal plate for mass spectrometry (the same applies to the plate for mass spectrometry of the present invention) takes 2 to 3 minutes to reach a high vacuum state, and can be measured immediately thereafter. When a membrane was used, it took 45 minutes to reach a vacuum state, and it was found that an excessive load was applied to the used mass spectrometer and it could not withstand frequent use.
Example 5: Various proteome analyzes using the plate for mass spectrometry of the present invention
Using various cell or tissue extraction samples, the performance of the plate for mass spectrometry of the present invention in a relatively low molecular weight range (molecular weight 1,000 to 20,000) was confirmed. In the following experiment, the sample was applied to 16% SDS-polyacrylamide gel (in Tris-Tricine buffer), electrophoresed for 90 minutes, and then 10 mM sodium borate buffer (pH adjusted to 8.0 with hydrochloric acid). The gel was electrotransferred from the gel to the plate for mass spectrometry of the present invention for 1 to 2 hours. After the transfer, the plate was rinsed and the matrix was spotted for mass spectrometry.
(1) Porcine cerebellum was homogenized with 1N acetic acid at 4 ° C., and centrifuged at 4 ° C. and 3,000 rpm for 30 minutes to collect the supernatant. Acetonitrile was added to a final concentration of 10% and applied to the column for reverse phase chromatography. Washed with 0.1% trifluoroacetic acid (TFA) containing 10% acetonitrile and eluted with 0.1% TFA containing 60% acetonitrile. The eluate was freeze-dried to obtain a pig cerebellum extract. The extract was separated by SDS-PAGE and electrotransferred to the plate for mass spectrometry of the present invention. Mass spectrometry was performed using a matrix containing saturated α-cyano-4-hydroxycinnamic acid (CHCA) in 0.5% TFA / 50% ACN. As a result, a peak was detectable in the molecular weight range of 3,000 to 20,000.
This was compared with the case of using DHB (150 mg / mL ethanol) and saturated SPA (in 0.5% TFA / 50% ACN) instead of saturated CHCA as a matrix. In the case of DHB, no clear peak was observed in the molecular weight range of 3,000 to 20,000. In the case of saturated SPA, only a few peaks were observed in the same molecular weight range. In contrast, in saturated CHCA, a number of peaks were observed in the molecular weight range of 2,000 to 10,000 and in the range of 5,000 to 20,000. From these results, it was shown that CHCA is more preferable for identification of proteins having a relatively low molecular weight range.
(2) Mass analysis of porcine cerebellum extract was performed in the same manner as (1) except that 50% saturated CHCA was used as a matrix. As a result, peaks could be detected in the molecular weight range of 1,000 or more. In particular, it was excellent in detecting a peak in the molecular weight range of 3,000 to 20,000.
Compared with the case of using 10 or 20% saturated concentration instead of 50% saturated CHCA, the degree of appearance of peaks in the mass analysis in the molecular weight range of 1,000 to 10,000 is compared. I tried. In the case of 10% saturated CHCA, several peaks were observed. In the case of 20% saturated CHCA, the number of peaks was slightly larger than that of the 10% saturated concentration. On the other hand, many peaks were observed in the case of 50% saturated CHCH.
(3) U937 cells (1 × 105~ 2x106/ Ml) was cultured in RPM11640 medium containing 10% FCS for 48 hours in the presence of 100 ng / ml phorbol 12-myristate 13-acetate (PMA). The same experiment conducted in the absence of PMA was used as a control. After completion of the culture, the cells were washed with phosphate buffered saline (PBS) to prepare a cell pellet. Further, a protein extraction reagent (Novajung) and a protease inhibitor were added and allowed to stand at 4 ° C. for 15 minutes, and then centrifuged to collect the supernatant to obtain cell lysate. This was separated by SDS-PAGE and electrotransferred to the plate for mass spectrometry of the present invention. Further, mass spectrometry was performed, and the results of the U937 cells stimulated with PMA were compared with the results of the control U937 cells for the intensity of the peak detected in the molecular weight range of 3,000 to 20,000. As a result, changes in protein expression (peak intensity) by PMA stimulation were as follows (Table 1).
Example 6: Examination of the effect of buffer solution during electrical transfer
As a buffer at the time of electrotransfer, Tris buffer (25 mM Tris / 192 mM glycine / 20% methanol, pH 8.3), phosphate buffer (5% ACN / 125 mM NaCl / PBS, pH 7.2), acetate buffer ( 40 mM Tris-acetate / 1 mM EDTA, pH 8.0) and borate buffer (10 mM sodium borate-hydrochloric acid, pH 8.0) were used. HSA and SCUPA were used as proteins for mass spectrometry. Other than that, the experiment was conducted according to the method of Example 5. As a result, in each buffer solution, the peak intensity of mass spectrometry was maximized when the borate buffer solution was used. For example, the maximum peak intensity was about 21 times for SCUPA and about 4 times for HSA for borate buffer versus phosphate buffer.
本発明の質量分析用プレートは、リガンド吸着素材としてＰＶＤＦを用いたことにより、各種の蛋白質を均等に吸着でき、転写効率に優れ、蛋白質の三次元構造・機能を正常に保持することが可能となる。また、非特異吸着が起こらない、転写後瞬時に質量分析を行うことができるなどの特徴を有する。
本発明の質量分析用プレートのこのような特徴を活かして、多量、迅速かつ高感度に蛋白質を分析・同定することができる。蛋白質は１種でもよく、複数種の集合体（蛋白質群）でもよく、また、当該蛋白質と親和性を有する（相互作用を示す）化合物との複合体であっても同様に（つまり、多量、迅速かつ高感度に）分析・同定することができる。
また、操作時間の大幅な短縮化と工程の簡略化、装置の小型化、低廉化、自動化、大量の検体処理が可能となり、大規模なプロテオーム解析が容易になる。さらに、生理的に重要な微量蛋白質のプロテオーム解析が可能となり、診断用のバイオマーカーの発見、新規な医薬品開発のシードの発見に貢献できる。
従って本発明は、電気泳動法と質量分析法を組合せた従来のプロテオーム解析に新たな手法を導入することを可能とするものである。
本出願は、米国で出願されたＵＳ １０／２６４，５０５および日本で出願された特願２００２−３４４７１０を基礎としており、それらの内容は本明細書に全て包含されるものである。
ここで述べられた刊行物および特許を含めたすべての引用文献は、ここに言及したことで、引用により組み込まれるべく、個々に、詳細に示され、ここにそのすべてが明示されたと同程度に、明細書中に組み込まれるものである。
本発明は、好適な具体例を強調して記載したものであるが、好適な具体例を変更してもよいことは当業者には明白である。ここで詳細に記載された以外の方法で本発明を実行してもよい。したがって、本発明は添付の請求の範囲の精神および範囲内に包含されるすべての変更を含むものである。Since the plate for mass spectrometry of the present invention uses PVDF as a ligand adsorbing material, it can adsorb various proteins equally, has excellent transfer efficiency, and can normally maintain the three-dimensional structure and function of the protein. Become. In addition, non-specific adsorption does not occur and mass spectrometry can be performed immediately after transfer.
By utilizing such characteristics of the plate for mass spectrometry of the present invention, proteins can be analyzed and identified in large quantities, quickly and with high sensitivity. The protein may be one kind, a plurality of kinds of aggregates (protein group), or a complex with a compound having affinity (indicating interaction) with the protein (that is, a large amount, Analysis and identification can be performed quickly and with high sensitivity.
In addition, the operation time can be greatly shortened, the process can be simplified, the apparatus can be miniaturized, the cost can be reduced, the automation can be performed, and a large amount of samples can be processed. In addition, proteome analysis of physiologically important trace proteins is possible, contributing to the discovery of diagnostic biomarkers and the discovery of new drug development seeds.
Therefore, the present invention makes it possible to introduce a new method to the conventional proteome analysis that combines electrophoresis and mass spectrometry.
This application is based on
All cited references, including the publications and patents mentioned herein, are hereby incorporated by reference and are shown individually and in detail to the extent that they are expressly incorporated herein by reference. Are incorporated into the specification.
While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments may be varied. The present invention may be implemented in ways other than those described in detail herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.
Claims (12)
(a) 支持体およびそれに接着するコーティングを含んでなる質量分析用プレートであって、該コーティングがポリビニリデンジフロリドを含有し、かつ少なくとも該支持体の質量分析に供される面全体に施されている質量分析用プレートを提供し、
(b) 分析対象物含有試料をゲル電気泳動に供し、
(c) 泳動後のゲルを該質量分析用プレートに転写して分析対象物を該質量分析用プレートに移行させ、
(d) 該質量分析用プレートを質量分析に供して移行した分析対象物を分析する。Analyte identification method comprising the following steps (a) to (d):
(a) a mass spectrometric plate comprising a support and a coating adhering thereto, the coating containing polyvinylidene difluoride and applied to at least the entire surface of the support subjected to mass analysis; A plate for mass spectrometry that is
(b) subjecting the analyte-containing sample to gel electrophoresis;
(c) The gel after electrophoresis is transferred to the plate for mass spectrometry to transfer the analyte to the plate for mass spectrometry,
(d) The mass analysis plate is subjected to mass spectrometry to analyze the transferred analyte.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
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| US10/264,505 US20030129666A1 (en) | 2001-01-09 | 2002-10-04 | Novel proteome analysis method and devices therefor |
| US10/264,505 | 2002-10-04 | ||
| JP2002344710 | 2002-11-27 | ||
| JP2002344710 | 2002-11-27 | ||
| PCT/JP2003/012711 WO2004031759A1 (en) | 2002-10-04 | 2003-10-03 | Plate for mass spectrometry, process for preparing the same and use thereof |
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| JPWO2004031759A1 JPWO2004031759A1 (en) | 2006-02-02 |
| JP4447458B2 true JP4447458B2 (en) | 2010-04-07 |
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| CN114778851A (en) * | 2022-04-25 | 2022-07-22 | 武汉翌圣生物科技有限公司 | Method for transferring biological macromolecular protein into membrane |
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| CN111896345A (en) * | 2020-07-30 | 2020-11-06 | 上海化工研究院有限公司 | Device for uniformly coating medicine film on inner wall of container in toxicity contact experiment |
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