JP6108805B2 - Laser desorption ionization mass spectrometry - Google Patents
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- 238000001698 laser desorption ionisation Methods 0.000 title claims description 32
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Description
本発明は、レーザー脱離イオン化質量分析法に関し、更に詳しくは、レーザー光を吸収し、その吸収した光エネルギーを測定対象分子に移動させることができる有機シリカ多孔体を用いることによって、均質で再現性のよいスペクトルを取得することができるレーザー脱離イオン化質量分析法に関する。 The present invention relates to laser desorption ionization mass spectrometry, and more specifically, it is homogeneous and reproducible by using a porous organic silica that can absorb laser light and transfer the absorbed light energy to a molecule to be measured. The present invention relates to a laser desorption ionization mass spectrometry method capable of acquiring a good spectrum.
「質量分析（ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｒｙ）（以下、「ＭＳ」と略記することがある）法」とは、測定対象分子を含む試料をイオン化し、測定対象分子由来のイオンを質量電荷比（質量／電荷（ｍ／ｚ））によって分離し検出することによって、その測定対象分子の化学構造に関する情報を得る方法である。 The “mass spectrometry (hereinafter sometimes abbreviated as“ MS ”) method” refers to ionizing a sample containing a molecule to be measured and converting ions derived from the molecule to be measured into a mass-to-charge ratio (mass / charge ( This is a method for obtaining information on the chemical structure of the molecule to be measured by separating and detecting by m / z)).
ＭＳにおいて、試料のイオン化は、分析の可否や得られるスペクトルの質を左右する重要な過程であり、試料を効率よくイオンにするためにこれまで多くのイオン化法が開発されてきた。
特に、生体高分子のイオン化には、レーザー脱離イオン化（ｌａｓｅｒ ｄｅｓｏｒｐｔｉｏｎ／ｉｏｎｉｚａｔｉｏｎ）（以下、「ＬＤＩ」と略記することがある）法のひとつであるマトリックス支援レーザー脱離イオン化（ｍａｔｒｉｘ−ａｓｓｉｓｔｅｄ ｌａｓｅｒ ｄｅｓｏｒｐｔｉｏｎ／ｉｏｎｉｚａｔｉｏｎ）（以下、「ＭＡＬＤＩ」と略記することがある）法が用いられている。このイオン化法を用いた質量分析は、ＮＭＲ等に比べて測定試料の量が少なくても測定が可能であることから、バイオ分野で広く用いられている。
In MS, sample ionization is an important process that determines whether analysis is possible and the quality of the spectrum obtained, and many ionization methods have been developed so far in order to efficiently ionize the sample.
In particular, for the ionization of biopolymers, matrix-assisted laser desorption ionization (laser desorption / ionization) (hereinafter sometimes abbreviated as “LDI”) is a matrix-assisted laser desorption ionization method. / Ionization) (hereinafter may be abbreviated as “MALDI”) method. Mass spectrometry using this ionization method is widely used in the bio field because measurement is possible even when the amount of a measurement sample is small compared to NMR or the like.
ＭＡＬＤＩでは、マトリックスと呼ばれる光吸収を持つ物質の中に、測定対象となる分子（例えば、タンパク質、ペプチド、糖類等がある）を分散させ、そこにパルスレーザーを照射することでマトリックスと共に測定対象となる分子をイオン化する技術である。 In MALDI, molecules to be measured (for example, proteins, peptides, saccharides, etc.) are dispersed in a substance that absorbs light called a matrix, and the target to be measured together with the matrix is irradiated with a pulse laser. It is a technology to ionize the molecule.
使用するレーザーは紫外領域の波長を有する場合が多く、可視領域や赤外領域の波長を使用する場合もあるが、マトリックスの光吸収特性に合わせたレーザーを用いるのが一般的である。現在、最も多用されるレーザーは窒素レーザーであり、波長３３７ｎｍを有する。 The laser to be used often has a wavelength in the ultraviolet region, and a wavelength in the visible region or infrared region may be used, but it is general to use a laser that matches the light absorption characteristics of the matrix. At present, the most frequently used laser is a nitrogen laser, which has a wavelength of 337 nm.
一方、使用するマトリックスの選択が分析の成否を決めるため、これまでに多くのマトリックスが開発され、実際にＭＡＬＤＩに用いられてきた。一般に、マトリックス分子は結晶性の有機分子であり、分析試料中の測定対象分子と共結晶を生成した上で、上記パルスレーザーを照射しイオン化する。近年は様々な液体マトリックスも開発されてきており、測定対象分子やそれを含有する試料に応じ様々な選択肢がある。 On the other hand, since the selection of the matrix to be used determines the success or failure of the analysis, many matrices have been developed and used in MALDI. In general, a matrix molecule is a crystalline organic molecule, and a co-crystal is formed with a molecule to be measured in an analysis sample, and then ionized by irradiation with the pulse laser. In recent years, various liquid matrices have been developed, and there are various options depending on the molecule to be measured and the sample containing it.
一般に、マトリックスと試料は良く混ざり、混合結晶又は混合物となる必要があると考えられている。この試料とマトリックスの混合結晶又は混合物の善し悪しが、感度及び質のよいスペクトルが得られるか否かに影響を与える。
更に、一見同質の混合物に見えても実際には不均一で、特に固体結晶の場合には、結晶が生成した場所すべてから測定対象分子由来のイオンが得られるわけではなく、生成した結晶のごく一部分にレーザー光を照射した場合のみ測定対象分子由来のイオンが得られる。この部分は、「スイートスポット（ｓｗｅｅｔ ｓｐｏｔ）」と呼ばれ、質の高いよいマススペクトルを得るには、試料が消費されるプレスキャンや、経験によって手動でスイートスポットを探し出し、レーザー光照射を行うといった時間のかかる作業が必要となり、特に測定試料全体の自動分析には適さないのが現状であった。
In general, it is considered that the matrix and the sample are well mixed and need to be a mixed crystal or mixture. The quality of the mixed crystal or mixture of the sample and matrix affects whether or not a spectrum with high sensitivity and quality can be obtained.
Furthermore, even though it appears to be a homogeneous mixture, it is actually inhomogeneous. In particular, in the case of a solid crystal, ions derived from the molecule to be measured cannot be obtained from all the places where the crystal is generated. Only when a part of the laser beam is irradiated, ions derived from the molecule to be measured are obtained. This part is called “sweet spot”, and in order to obtain a high-quality mass spectrum, a pre-scan where the sample is consumed or a sweet spot is manually found by experience and laser irradiation is performed. Such a time-consuming work is required, and it is not suitable for automatic analysis of the entire measurement sample.
液体マトリックスにおいても、固体マトリックスほどのばらつきはないが、夾雑塩等の影響でスペクトルの再現性が劣る場合や、イオン源、イオントラップ等を汚染する場合もあり、根本的な解決にはならなかった。 The liquid matrix is not as varied as the solid matrix, but the spectral reproducibility may be inferior due to the influence of contaminated salts, and the ion source and ion trap may be contaminated. It was.
一方、ＭＡＬＤＩにおける試料不均一性やマトリックス由来のクラスターイオンによるバックグランドを改善するために、マトリックスを使用しないソフトＬＤＩ−ＭＳ法が開発されてきた。そこでは、測定プレートとして用いる様々な基材が提案されている。 On the other hand, soft LDI-MS methods that do not use a matrix have been developed in order to improve the sample heterogeneity in MALDI and the background caused by matrix-derived cluster ions. There, various base materials used as measurement plates have been proposed.
その中で、多孔質シリコン基板を用いる方法の一つとして、ＤＩＯＳ−ＭＳ（ｄｅｓｏｒｐｔｉｏｎ／ｉｏｎｉｚａｔｉｏｎ−ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｒｙ ｏｎ ｐｏｒｏｕｓ ｓｉｌｉｃｏｎ）と呼ばれているものがある（特許文献１）。
この方法では、ナノメートルレベルの微細孔を持つ多孔質シリコン基板の表面に試料溶液を塗布し、乾燥させてから、これを質量分析装置のイオン源内に設置し、以降の操作はＭＡＬＤＩ−ＭＳと同様に、試料表面にレーザー光を照射することによって、質量分析が行われる。
ＤＩＯＳ−ＭＳにおけるイオン化の詳細な原理は明らかではないが、ナノシリコン構造体がレーザー光を高効率で吸収し、急速に加熱されることによって、測定対象分子の瞬間的な離脱が起こると共に、該多孔質シリコン基板に結合又は吸着していた成分がイオン化して測定対象分子に電荷を受け渡すことによって、測定対象分子のイオン化が達成されるのではないかと考えられている。
Among them, as one of methods using a porous silicon substrate, there is a method called DIOS-MS (deposition / ionization-mass spectrometry on porous silicon) (Patent Document 1).
In this method, a sample solution is applied to the surface of a porous silicon substrate having nanometer-scale micropores, dried, and then placed in an ion source of a mass spectrometer. The subsequent operations are performed with MALDI-MS. Similarly, mass spectrometry is performed by irradiating the sample surface with laser light.
Although the detailed principle of ionization in DIOS-MS is not clear, the nanosilicon structure absorbs laser light with high efficiency and is heated rapidly, thereby causing instantaneous separation of the molecule to be measured, and It is considered that ionization of the molecule to be measured can be achieved by ionizing the component bonded or adsorbed to the porous silicon substrate and transferring the charge to the molecule to be measured.
ＤＩＯＳ−ＭＳは、試料基板そのものをイオン化媒体として用いるため、試料の均一な塗布が比較的容易であり、ＭＡＬＤＩ−ＭＳで問題となる妨害ピークの発生を回避できるという利点がある。
しかしながら、ＤＩＯＳ−ＭＳでは、多孔質シリコンのイオン化効率は作成条件に大きく左右されること、同一の多孔質構造をもつ試料基板を再現性よく作成することが困難であること、また、測定できる分子量範囲も限られていること等の問題がある。
Since DIOS-MS uses the sample substrate itself as an ionization medium, there is an advantage that uniform application of the sample is relatively easy and generation of a disturbing peak which is a problem in MALDI-MS can be avoided.
However, in DIOS-MS, the ionization efficiency of porous silicon is greatly influenced by the preparation conditions, it is difficult to prepare a sample substrate having the same porous structure with good reproducibility, and the measurable molecular weight. There are problems such as limited range.
表面を２，５−ジヒドロキシ安息香酸（ＤＨＢＡ）やα−シアノ−４−ヒドロキシケイ皮酸（ＣＨＣＡ）で修飾したシリカゲル（非特許文献１）、４，４’−アゾ−ジアニリンで修飾したシリカゲル（非特許文献２）等をマトリックスとして利用する例もある。
しかしながら、これらの方法では、マトリックス由来の妨害ピークは発生しないものの、約１ｎｍｏｌの測定対象分子を検出できるに過ぎず、感度が低いという問題点があった。また、ＤＨＢＡ結晶を用いる通常のＭＡＬＤＩと異なり、固定化されたＤＨＢＡはガス相に気化することができないので、測定対象分子のイオン化効率が低下すると考えられた。
Silica gel modified with 2,5-dihydroxybenzoic acid (DHBA) or α-cyano-4-hydroxycinnamic acid (CHCA) (non-patent document 1), silica gel modified with 4,4′-azo-dianiline ( There is also an example in which Non-Patent Document 2) is used as a matrix.
However, these methods have a problem in that although no interference peak derived from the matrix is generated, only about 1 nmol of the molecule to be measured can be detected, and the sensitivity is low. In addition, unlike normal MALDI using DHBA crystals, immobilized DHBA cannot be vaporized into the gas phase, and it was considered that the ionization efficiency of the molecule to be measured was reduced.
広い範囲の分子量の試料（測定対象分子）に対して、分子を壊すことなく十分な大きさの感度が得られ、ノイズがなく、レーザー照射場所に依存しない均一な感度を有したレーザー脱離イオン化質量分析法は得られていない。
従って、試料溶液を基板上に均一に塗布することができ、レーザー光を照射しても、妨害ピークやフラグメンテーションを発生せずに均一なスペクトルが得られ、高感度な測定が可能な脱離イオン化質量分析法の開発が熱望されていた。
Laser desorption ionization with a sufficient sensitivity for a wide range of molecular weight samples (measuring molecules) without breaking the molecule, no noise, and uniform sensitivity independent of the laser irradiation location Mass spectrometry has not been obtained.
Therefore, the sample solution can be uniformly coated on the substrate, and even when irradiated with laser light, a uniform spectrum can be obtained without generating interference peaks or fragmentation, and desorption ionization that enables highly sensitive measurement. The development of mass spectrometry was eagerly awaited.
本発明は上記背景技術に鑑みてなされたものであり、その課題は、レーザー脱離イオン化質量分析法（以下、「ＬＤＩ質量分析法」と略記することがある）において、測定対象分子の均一で高いイオン生成量を実現させ、短時間で効率良く、質の高いＭＳスペクトルを得ることのできるＬＤＩ質量分析法を提供することにある。 The present invention has been made in view of the above-mentioned background art, and the problem is that in laser desorption ionization mass spectrometry (hereinafter, sometimes abbreviated as “LDI mass spectrometry”), the molecules to be measured are uniform. An object of the present invention is to provide an LDI mass spectrometry method capable of realizing a high-quality MS spectrum in a short time, realizing a high ion production amount.
また、スイートスポットを探すことなく、誰でも容易に解析が可能となり、また、自動分析への応用が可能となり、高スループットのＬＤＩ質量分析法を提供することにある。 Another object of the present invention is to provide a high-throughput LDI mass spectrometry method that allows anyone to easily perform analysis without searching for a sweet spot and enables application to automatic analysis.
本発明者は、上記の課題を解決すべく鋭意検討を重ねた結果、特定の有機シリカ多孔体に、測定対象分子を含む試料を均一に担持させて、レーザー脱離イオン化を行うことによって、上記課題が達成されることを見出して、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the inventor uniformly supports a sample containing a molecule to be measured on a specific organic silica porous material, and performs laser desorption ionization to perform the above-mentioned The inventors have found that the object can be achieved and have completed the present invention.
すなわち、本発明は、
［１］レーザー脱離イオン化質量分析法において、照射レーザー光を吸収可能な有機基を骨格に有する有機シリカ多孔体に、該有機シリカ多孔体の吸収した光エネルギーが移動可能な測定対象分子を含む試料を均一に担持させた後、レーザー光を照射し、該測定対象分子をイオン化させることを特徴とするレーザー脱離イオン化質量分析法を提供するものである。
That is, the present invention
[1] In laser desorption / ionization mass spectrometry, an organic silica porous body having an organic group capable of absorbing irradiated laser light as a skeleton includes a molecule to be measured capable of transferring light energy absorbed by the organic silica porous body. The present invention provides a laser desorption ionization mass spectrometry method characterized in that after a sample is uniformly supported, laser light is irradiated to ionize the molecule to be measured.
［２］上記有機シリカ多孔体が、照射レーザー光を吸収するものであって、該有機シリカ多孔体の発光スペクトルと、上記測定対象分子の吸収スペクトルとが、少なくともある１つの波長において重なる［１］に記載のレーザー脱離イオン化質量分析法を提供するものである。 [2] The organic silica porous body absorbs irradiated laser light, and the emission spectrum of the organic silica porous body and the absorption spectrum of the molecule to be measured overlap at least at one wavelength [1] The laser desorption ionization mass spectrometry method described in the above is provided.
［３］上記有機シリカ多孔体が、照射レーザー光を吸収して発光するものであって、該有機シリカ多孔体の発光スペクトルと、上記測定対象分子の吸収スペクトルとが、少なくともある１つの波長において重なる［１］又は［２］に記載のレーザー脱離イオン化質量分析法を提供するものである。 [3] The organic silica porous body emits light by absorbing irradiated laser light, and the emission spectrum of the organic silica porous body and the absorption spectrum of the molecule to be measured are at least at one wavelength. The laser desorption ionization mass spectrometry method according to [1] or [2], which overlaps, is provided.
［４］上記有機シリカ多孔体の発光スペクトルの短波長端の方が、上記測定対象分子の吸収スペクトルの長波長端より短波長側にあることによって、該有機シリカ多孔体の発光スペクトルと、該測定対象分子の吸収スペクトルとが、少なくともある１つの波長において重なる［２］又は［３］に記載のレーザー脱離イオン化質量分析法を提供するものである。 [4] Since the short wavelength end of the emission spectrum of the organic silica porous material is on the short wavelength side of the long wavelength end of the absorption spectrum of the molecule to be measured, the emission spectrum of the organic silica porous material, The laser desorption ionization mass spectrometry method according to [2] or [3], wherein the absorption spectrum of the molecule to be measured overlaps at least at one wavelength.
［５］上記有機シリカ多孔体が、光捕集アンテナ機能を有するものである［１］ないし［４］の何れかに記載のレーザー脱離イオン化質量分析法を提供するものである。 [5] The laser desorption ionization mass spectrometry method according to any one of [1] to [4], wherein the organic silica porous body has a light collecting antenna function.
［６］上記有機シリカ多孔体が有する孔の平均直径が、１ｎｍ以上１００ｎｍ以下である［１］ないし［５］の何れかに記載のレーザー脱離イオン化質量分析法を提供するものである。 [6] The laser desorption ionization mass spectrometry method according to any one of [1] to [5], wherein the pores of the organic silica porous material have an average diameter of 1 nm to 100 nm.
［７］上記有機シリカ多孔体が有する孔の平均直径が、８ｎｍ以上８０ｎｍ以下である［６］に記載のレーザー脱離イオン化分析法を提供するものである。 [7] The laser desorption ionization analysis method according to [6], wherein the average pore diameter of the porous organic silica material is 8 nm or more and 80 nm or less.
［８］上記測定対象分子が、分子量１６０以上のものである［１］ないし［７］の何れかに記載のレーザー脱離イオン化質量分析法を提供するものである。 [8] The laser desorption / ionization mass spectrometry method according to any one of [1] to [7], wherein the measurement target molecule has a molecular weight of 160 or more.
本発明によれば、前記問題点を解消し、上記課題を解決し、均質で、再現性が良く、ＳＮ比が高いＭＳスペクトルを、簡便で迅速に得ることが可能である。また、自動分析への応用が可能となり、高スループットのＬＤＩ質量分析法を提供できる。 According to the present invention, it is possible to solve the above-mentioned problems, solve the above-described problems, and obtain a MS spectrum that is homogeneous, has good reproducibility, and has a high S / N ratio easily and quickly. In addition, application to automatic analysis becomes possible, and a high-throughput LDI mass spectrometry method can be provided.
すなわち、本発明では、有機シリカ多孔体と測定対象分子との組み合わせによるエネルギー移動を利用しており、有機シリカ多孔体（エネルギー供与体）から測定対象分子（エネルギー受容体）へとエネルギーが効率よく移動できる場合にのみ、測定対象分子がイオン化されるものであり、前記したＤＩＯＳ−ＭＳのように単なる発熱のみによる脱離とは異なる。
このため、基材や夾雑によるシグナルが抑制され、測定対象分子のみを選択的にイオン化させることが可能であり、また、試料のフラグメンテーションが起こらない。
また、測定対象分子が有機シリカ多孔体に均一に担持されることから、マトリックスを用いる通常のＭＡＬＤＩ−ＭＳの測定法と異なりスイートスポットを探す必要がなく、誰でも容易に測定ができ、また、解析を簡便かつ迅速に行うことができる。
That is, in the present invention, energy transfer by the combination of the organic silica porous material and the molecule to be measured is used, and energy is efficiently transferred from the organic silica porous material (energy donor) to the molecule to be measured (energy acceptor). The molecule to be measured is ionized only when it can move, and is different from desorption only by heat generation like the above-mentioned DIOS-MS.
For this reason, the signal by a base material and contamination is suppressed, it is possible to selectively ionize only a measurement object molecule, and the fragmentation of a sample does not occur.
In addition, since the molecules to be measured are uniformly supported on the porous organic silica, unlike the usual MALDI-MS measurement method using a matrix, there is no need to search for a sweet spot, and anyone can easily measure, Analysis can be performed easily and quickly.
更に、有機シリカ多孔体の吸収した光エネルギーが、有機シリカ多孔体内に担持された測定対象分子に効率よくエネルギー移動することにより、イオン化し難い測定対象分子を、通常よりも弱い励起光でイオン化できる。
また、有機シリカは、通常の有機分子に比べ、レーザー光に対して安定であるため、有機シリカ多孔体の骨格が破壊され難く、バックグランドの低いＳＮ比の高いデータを取得できる。
Furthermore, the light energy absorbed by the porous organic silica can be efficiently transferred to the molecules to be measured supported in the porous organic silica, so that the molecules to be measured that are difficult to ionize can be ionized with excitation light weaker than usual. .
Moreover, since organic silica is more stable to laser light than ordinary organic molecules, the skeleton of the organic silica porous body is not easily destroyed, and data with a low background and a high S / N ratio can be obtained.
以下、本発明について説明するが、本発明は以下の実施の具体的態様に限定されるものではなく、本発明の技術思想の範囲内で任意に変形して実施することができる。 Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be implemented with any modifications within the scope of the technical idea of the present invention.
＜有機シリカ多孔体＞
本発明における有機シリカ多孔体は、有機シリカ化学構造を有し、照射レーザー光を吸収可能な有機基をその化学構造の骨格に有する多孔体である。
有機シリカ多孔体は、エネルギー供与体として作用し、有機シリカ多孔体の吸収した光エネルギーは測定対象分子（エネルギー受容体）に移動する。この、有機シリカ多孔体（エネルギー供与体）から測定対象分子（エネルギー受容体）へのエネルギー移動は、発光を経由しない分子間の励起エネルギー移動や電子移動が考えられるし、有機シリカ多孔体から発せられた光を測定対象分子が吸収するエネルギー移動（発光再吸収によるエネルギー移動）、すなわち、発光を経由するエネルギー移動も考えられる。
<Organic silica porous material>
The porous organic silica in the present invention is a porous body having an organic silica chemical structure and having an organic group capable of absorbing irradiation laser light in the skeleton of the chemical structure.
The organic silica porous material acts as an energy donor, and the light energy absorbed by the organic silica porous material is transferred to the molecule to be measured (energy acceptor). The energy transfer from the organosilica porous body (energy donor) to the molecule to be measured (energy acceptor) may be excitation energy transfer or electron transfer between molecules that does not go through light emission. Energy transfer (energy transfer by light emission reabsorption) in which the measurement target molecule absorbs the emitted light, that is, energy transfer via light emission is also conceivable.
発光を経由してエネルギー移動する場合であっても、発光を経由しないでエネルギー移動する場合であっても、上記有機シリカ多孔体が、照射レーザー光を吸収するものであって、該有機シリカ多孔体の発光スペクトルと、上記測定対象分子の吸収スペクトルとが、少なくともある１つの波長において重なっていることが好ましい。このような場合、有機シリカ多孔体が吸収した光エネルギー又は有機シリカ多孔体の励起エネルギーが測定対象分子に移動し易い。 Whether the energy transfer is via light emission or the energy transfer is not via light emission, the organic silica porous body absorbs the irradiated laser beam, and the organic silica porous It is preferable that the emission spectrum of the body and the absorption spectrum of the molecule to be measured overlap at least at one wavelength. In such a case, the light energy absorbed by the organic silica porous body or the excitation energy of the organic silica porous body easily moves to the molecule to be measured.
特に、発光を経由してエネルギー移動する場合、上記有機シリカ多孔体は、照射レーザー光を吸収して発光するものであり、該有機シリカ多孔体の発光スペクトルと、上記測定対象分子の吸収スペクトルとが、少なくともある１つの波長において重なっていることがより好ましい。このような場合、有機シリカ多孔体から出た光エネルギーが測定対象分子に移動し易い。 In particular, in the case of energy transfer via light emission, the organosilica porous body emits light by absorbing irradiated laser light, and the emission spectrum of the organosilica porous body and the absorption spectrum of the measurement target molecule Are more preferably overlapped at least at one wavelength. In such a case, the light energy emitted from the organic silica porous material easily moves to the molecule to be measured.
また、上記何れの場合においても、上記有機シリカ多孔体の発光スペクトルの短波長端の方が、上記測定対象分子の吸収スペクトルの長波長端より短波長側にあることによって、該有機シリカ多孔体の発光スペクトルと、該測定対象分子の吸収スペクトルとが、少なくともある１つの波長において重なっていることがより好ましい。このような場合、有機シリカ多孔体が吸収した光エネルギーが、光エネルギー又は励起エネルギーとして測定対象分子に移動し易い。 Further, in any of the above cases, the organic silica porous material has the short wavelength end of the emission spectrum of the organic silica porous material on the short wavelength side of the long wavelength end of the absorption spectrum of the molecule to be measured. It is more preferable that the emission spectrum of and the absorption spectrum of the molecule to be measured overlap at least at one wavelength. In such a case, the light energy absorbed by the organosilica porous body easily moves to the measurement target molecule as light energy or excitation energy.
本発明において、測定対象分子を含む試料は、有機シリカ多孔体に載置されると、一部は有機シリカ多孔体が有する細孔内に入り込んで、その結果、測定対象分子は有機シリカ多孔体に（特に、有機シリカ多孔体の細孔内壁に）、大きな接触面積をもって接するようになると思われる。
このため、発光を経由してもしなくても、分子間のエネルギー移動が容易になり、上記した効果を発揮する。
In the present invention, when the sample containing the molecule to be measured is placed on the organic silica porous body, a part of the sample enters the pores of the organic silica porous body, and as a result, the molecule to be measured is the organic silica porous body. In particular, it seems to come into contact with a large contact area (especially on the inner wall of the pores of the porous organic silica).
For this reason, energy transfer between molecules is facilitated regardless of whether light is emitted or not, and the above-described effects are exhibited.
本発明に使用できる有機シリカ多孔体の例としては、照射レーザー光を吸収可能な有機基を有する下記の一般式Ａ１、Ａ２、Ａ３、Ａ４、Ａ５、Ａ６、Ｂ、Ｘ１、Ｘ１ａ、Ｘ２、Ｘ３、Ｘ４、Ｘ５、Ｘ６、Ｘ６ａ、Ｃ、Ｄ等で表される有機ケイ素化合物（以下、「有機ケイ素化合物Ｐ」と略記する）の縮重合により得られる有機シリカ多孔体；上記有機基を有する有機ケイ素化合物Ｐと他の有機ケイ素化合物（照射レーザー光を吸収可能な有機基を有さなくてもよい）との共縮合により得られる有機シリカ多孔体；上記有機基を有する有機ケイ素化合物ＰとＳｉ（ＯＲ１１）４［Ｒ１１はメチル基又はエチル基を示す］等で表されるケイ素化合物との共縮合により得られる有機シリカ多孔体；上記有機基を有する有機ケイ素化合物Ｐで表面修飾された有機シリカ多孔体；等が挙げられる。 Examples of the organic silica porous material that can be used in the present invention include the following general formulas A1, A2, A3, A4, A5, A6, B, X1, X1a, X2, and X3 having an organic group capable of absorbing irradiation laser light. , X4, X5, X6, X6a, C, D, etc., an organic silica porous material obtained by polycondensation of an organosilicon compound (hereinafter abbreviated as “organosilicon compound P”); Porous organic silica obtained by co-condensation of silicon compound P with another organic silicon compound (not necessarily having an organic group capable of absorbing irradiated laser light); organic silicon compound P having organic group and Si Table with an organic silicon compound having the organic group P; (oR 11) 4 [ R 11 represents a methyl group or an ethyl group] organic porous silica material obtained by co-condensation of silicon compounds represented by like Modified organic porous silica material; and the like.
これらのうち、レーザー光を効率良く吸収し、かつ、有機シリカ多孔体の細孔内に担持された測定対象分子に効率良く励起エネルギーを移動できる点から、架橋型有機シリカ多孔体が好ましい。
下記で詳述するが、架橋型有機シリカ多孔体では、架橋しているものは、照射レーザー光を吸収可能な有機基を有する架橋有機基であり、「架橋」されているものは、シロキサン構造、すなわち、−（Ｓｉ−Ｏ）ｎ−構造である。
Among these, a crosslinked organic silica porous material is preferable because it absorbs laser light efficiently and can efficiently transfer excitation energy to a molecule to be measured carried in the pores of the organic silica porous material.
As will be described in detail below, in the crosslinked organic silica porous material, what is crosslinked is a crosslinked organic group having an organic group capable of absorbing irradiation laser light, and what is “crosslinked” has a siloxane structure. That is, it is a — (Si—O) n — structure.
＜＜架橋型有機シリカ多孔体＞＞
「有機シリカ多孔体」のうちの「架橋型有機シリカ多孔体」は、好ましくは、鋳型となる界面活性剤の存在下において、前駆体である有機ケイ素化合物を重合させることによって得られる。有機ケイ素化合物は、架橋有機基を有しているので、重合させることによって、架橋型有機シリカ多孔体が得られる。
その後、鋳型となる該界面活性剤を除けば、架橋型有機シリカ多孔体が得られる。
<< Crosslinked organic silica porous material >>
The “crosslinked organic silica porous body” in the “organic silica porous body” is preferably obtained by polymerizing an organosilicon compound as a precursor in the presence of a surfactant as a template. Since the organosilicon compound has a crosslinked organic group, a crosslinked organic silica porous material is obtained by polymerization.
Thereafter, the crosslinked organic silica porous material is obtained by removing the surfactant used as a template.
以下に、本発明に好適に使用することができる架橋型有機シリカ多孔体の例、すなわちその前駆体となる有機ケイ素化合物の例を挙げる。なお、この例の中には重複しているものもある。 Below, the example of the bridge | crosslinking type organic silica porous body which can be used conveniently for this invention, ie, the example of the organosilicon compound used as the precursor, is given. Note that some of these examples overlap.
＜＜＜架橋型有機シリカ多孔体の例（１）＞＞＞
架橋型有機シリカ多孔体の前駆体となる有機ケイ素化合物の例としては、特開２０００−２１９７７０（特許第３８９９７３３号）に記載のものが挙げられる。
<<< Example of crosslinked organic silica porous material (1) >>>
As an example of the organosilicon compound used as the precursor of the crosslinked organic silica porous material, those described in JP-A-2000-219770 (Japanese Patent No. 3899733) can be mentioned.
すなわち、架橋型有機シリカ多孔体は、以下の一般式Ａ１〜Ａ６で示される化合物から選択される何れか１種類以上の有機ケイ素化合物を、好ましくは界面活性剤の存在下で縮重合させることによって得られる多孔体である。 That is, the crosslinked organic silica porous material is obtained by polycondensing any one or more types of organic silicon compounds selected from the compounds represented by the following general formulas A1 to A6, preferably in the presence of a surfactant. It is the porous body obtained.
＜＜＜架橋型有機シリカ多孔体の例（２）＞＞＞
また、架橋型有機シリカ多孔体の前駆体となる有機ケイ素化合物の例として、特開２００８−０８４８３６に記載のものが挙げられる。
<<< Example (2) of crosslinked organic silica porous body >>>
Moreover, as an example of the organosilicon compound used as the precursor of a bridge | crosslinking-type organic silica porous body, the thing of Unexamined-Japanese-Patent No. 2008-084836 is mentioned.
すなわち、架橋型有機シリカ多孔体は、下記一般式Ｂで表される有機ケイ素化合物の重合体からなる多孔体である。 That is, the crosslinked organic silica porous body is a porous body made of a polymer of an organosilicon compound represented by the following general formula B.
上記一般式Ｂ中、Ｘはｍ価の有機基であり、本発明のＬＤＩ質量分析法において、照射レーザー光を吸収可能な有機基であり、シロキサン構造、すなわち、−（Ｓｉ−Ｏ）ｎ−構造を架橋する能力を有する架橋有機基である。Ｘの具体例については後述する。 In the general formula B, X is an m-valent organic group, and in the LDI mass spectrometry of the present invention, is an organic group that can absorb the irradiation laser beam, and has a siloxane structure, that is, — (Si—O) n —. It is a crosslinked organic group having the ability to crosslink the structure. A specific example of X will be described later.
また、上記一般式Ｂ中、Ｒ１は、アルコキシ基（好ましくは炭素数１〜５のアルコキシ基）、ヒドロキシル基（−ＯＨ）、アリル基（ＣＨ２＝ＣＨ−ＣＨ２−）、エステル基（好ましくは炭素数１〜５のエステル基）及びハロゲン原子（塩素原子、フッ素原子、臭素原子、ヨウ素原子）からなる群から選択される少なくとも一つを示し、中でも縮合反応が制御し易いという観点からアルコキシ基及び／又はヒドロキシル基が好ましい。なお、同一分子中に複数のＲ１が存在する場合、Ｒ１は同一でも異なっていてもよい。 In the general formula B, R 1 represents an alkoxy group (preferably an alkoxy group having 1 to 5 carbon atoms), a hydroxyl group (—OH), an allyl group (CH 2 ═CH—CH 2 —), an ester group ( Preferably, it is at least one selected from the group consisting of an ester group having 1 to 5 carbon atoms) and a halogen atom (a chlorine atom, a fluorine atom, a bromine atom, an iodine atom), and from the viewpoint that the condensation reaction is easy to control. Alkoxy groups and / or hydroxyl groups are preferred. In addition, when several R < 1 > exists in the same molecule, R < 1 > may be same or different.
また、上記一般式Ｂ中、Ｒ２は、アルキル基（好ましくは炭素数１〜５のアルキル基）及び水素原子からなる群から選択される少なくとも一つを示す。なお、同一分子中に複数のＲ２が存在する場合、Ｒ２は同一でも異なっていてもよい。 In the general formula B, R 2 represents at least one selected from the group consisting of an alkyl group (preferably an alkyl group having 1 to 5 carbon atoms) and a hydrogen atom. In addition, when two or more R < 2 > exists in the same molecule, R < 2 > may be the same or different.
更に、上記一般式Ｂ中のｎ及び（３−ｎ）はそれぞれケイ素原子（Ｓｉ）に結合しているＲ１及びＲ２の数であり、ｎは１〜３の整数を示すが、縮合した後の構造が安定であるという点から、ｎ＝３であることが特に好ましい。
また、上記一般式Ｂ中のｍは、前記有機基（Ｘ）に結合しているケイ素原子（Ｓｉ）の数であり、ｍは１〜４の整数を示すが、安定なシロキサンネットワークを形成し易いという点から、ｍ＝２であることが特に好ましい。
Furthermore, n and (3-n) in the above general formula B are the numbers of R 1 and R 2 bonded to the silicon atom (Si), respectively, and n represents an integer of 1 to 3, but condensed. It is particularly preferable that n = 3 from the viewpoint that the subsequent structure is stable.
M in the general formula B is the number of silicon atoms (Si) bonded to the organic group (X), and m represents an integer of 1 to 4, but forms a stable siloxane network. It is particularly preferable that m = 2 from the viewpoint of ease.
ｍ＝２の場合の有機基Ｘの具体例を以下に示す。ｍ＝２の場合、以下、一般式Ｂの何れかで表される有機ケイ素化合物をＡ−Ｘ−Ａと表記する。
ここで、Ａは、一般式Ｂにおいて、（ ）ｍで表される括弧内の基を示し、同一であっても異なっていてもよい。Ａに関しては、以下同様である。
Specific examples of the organic group X when m = 2 are shown below. In the case of m = 2, hereinafter, the organosilicon compound represented by any one of the general formula B is represented as AXA.
Here, A represents a group in parentheses represented by () m in the general formula B and may be the same or different. The same applies to A below.
ｍ＝２の場合の有機基Ｘの具体例として、下記一般式Ｘ１で表される「置換基を有していてもよいフルオレン骨格を有する有機基」が挙げられる。 Specific examples of the organic group X in the case of m = 2 include “organic group having a fluorene skeleton which may have a substituent” represented by the following general formula X1.
また、ｍ＝２の場合の有機基Ｘの具体例として、下記一般式Ｘ２で表されるピレン骨格を有する有機基が挙げられる。 Further, specific examples of the organic group X in the case of m = 2 include an organic group having a pyrene skeleton represented by the following general formula X2.
ｍ＝２の場合の有機基Ｘの具体例として、下記一般式Ｘ３で表される置換基を有していてもよいアクリジン骨格を有する有機基が挙げられる。 Specific examples of the organic group X in the case of m = 2 include an organic group having an acridine skeleton that may have a substituent represented by the following general formula X3.
ｍ＝２の場合の有機基Ｘの具体例として、下記一般式Ｘ４で表されるアクリドン骨格を有する有機基が挙げられる。 Specific examples of the organic group X in the case of m = 2 include an organic group having an acridone skeleton represented by the following general formula X4.
ｍ＝２の場合の有機基Ｘの具体例として、下記一般式Ｘ５で表されるクアテルフェニル骨格を有する有機基が挙げられる。 Specific examples of the organic group X in the case of m = 2 include an organic group having a quaterphenyl skeleton represented by the following general formula X5.
ｍ＝２の場合の有機基Ｘの具体例として、下記一般式Ｘ６で表される置換基を有していてもよいアントラセン骨格を有する有機基が挙げられる。 Specific examples of the organic group X in the case of m = 2 include an organic group having an anthracene skeleton which may have a substituent represented by the following general formula X6.
＜＜＜架橋型有機シリカ多孔体の例（３）＞＞＞
また、架橋型有機シリカ多孔体の前駆体となる有機ケイ素化合物の例として、Chem. Mater. 2008; 20: 891-908に記載されている以下の化学式Ｃで表されるものが挙げられる。
<<< Example of crosslinked organic silica porous material (3) >>>
Moreover, as an example of the organosilicon compound used as the precursor of the crosslinked organic silica porous material, there can be mentioned those represented by the following chemical formula C described in Chem. Mater. 2008; 20: 891-908.
＜＜＜架橋型有機シリカ多孔体の例（４）＞＞＞
また、架橋型有機シリカ多孔体の前駆体となる有機ケイ素化合物の例として、ＣＲＥＳＴ ナノ界面技術の基盤構築研究領域 第１回公開シンポジウム「ナノ界面が生み出す次世代機能」の予稿集P19-23の「有機ナノ空間材料の創製と光エネルギー変換系への応用」稲垣伸二（豊田中央研究所）に記載されている以下の化学式Ｄで表されるものが挙げられる。
<<< Example of crosslinked organic silica porous material (4) >>>
In addition, as an example of an organosilicon compound that is a precursor of a crosslinked organic silica porous material, the CREST nano-interface technology foundation construction research area, the first public symposium “Next-generation functions produced by nano-interfaces”, P19-23 Examples include those represented by the following chemical formula D described in “Creation of Organic Nanospace Material and Application to Light Energy Conversion System” Shinji Inagaki (Toyota Central Research Laboratory).
＜＜他の有機シリカ多孔体の前駆体となる有機シリカ化合物＞＞
架橋型有機シリカ多孔体以外で、有機シリカ多孔体の前駆体となる有機シリカ化合物としては、以下の一般式Ａ７、Ａ８で示される化合物が挙げられる。
<< Organic silica compound which is a precursor of other organic silica porous bodies >>
Examples of the organic silica compound that is a precursor of the organic silica porous material other than the crosslinked organic silica porous material include compounds represented by the following general formulas A7 and A8.
＜＜有機シリカ多孔体の物性・態様＞＞
本発明における有機シリカ多孔体は、光捕集アンテナ機能を有するものであることが好ましい。「光捕集アンテナ機能」とは、上記の公開公報又は文献に定義が記載されている通り、光を照射した場合に光エネルギーを吸収して励起したエネルギーを細孔の内部に集約する機能をいう。
光捕集アンテナ機能を有する有機シリカ多孔体であれば、吸収したレーザー光の光エネルギーを細孔の内部に担持された測定対象分子に効率よく移動させることができ、測定対象分子をイオン化し易くする。
<< Physical Properties / Aspects of Porous Organosilica >>
The porous organic silica in the present invention preferably has a light collecting antenna function. The “light collecting antenna function” is a function that aggregates the excited energy in the pores by absorbing light energy when irradiated with light, as defined in the above-mentioned publications or documents. Say.
If the organic silica porous material has a light collecting antenna function, the light energy of the absorbed laser light can be efficiently transferred to the measurement target molecule supported inside the pores, and the measurement target molecule can be easily ionized. To do.
また、本発明の有機シリカ多孔体は、薄膜状、粉末状、鱗片状等の形状に特に限定はない。
また、有機シリカ多孔体が有する細孔の構造、細孔径、細孔深さ等も特に限定はないが、細孔の平均直径については、測定対象分子が細孔内に導入され易いように、下限は、１ｎｍ以上が好ましく、２ｎｍ以上がより好ましく、５ｎｍ以上が特に好ましく、８ｎｍ以上が更に好ましく、２０ｎｍ以上が最も好ましい。また、細孔の平均直径の上限は、１０００ｎｍ以下が好ましく、５００ｎｍ以下がより好ましく、２００ｎｍ以下が特に好ましく、１００ｎｍ以下が更に好ましく、８０ｎｍ以下が最も好ましい。
好ましい「細孔の平均直径」は、測定対象分子の分子量に依存し、測定対象分子の分子量が大きければ、好ましい「細孔の平均直径」は大きくなり、測定対象分子の分子量が小さければ、細孔の平均直径は小さくてもよい。
Moreover, the organic silica porous material of the present invention is not particularly limited to a shape such as a thin film shape, a powder shape, and a scale shape.
The pore structure, pore diameter, pore depth, etc. of the porous organic silica material are not particularly limited, but the average diameter of the pores is such that the molecule to be measured can be easily introduced into the pores. The lower limit is preferably 1 nm or more, more preferably 2 nm or more, particularly preferably 5 nm or more, still more preferably 8 nm or more, and most preferably 20 nm or more. Further, the upper limit of the average diameter of the pores is preferably 1000 nm or less, more preferably 500 nm or less, particularly preferably 200 nm or less, further preferably 100 nm or less, and most preferably 80 nm or less.
The preferred “average diameter of the pore” depends on the molecular weight of the molecule to be measured. If the molecular weight of the molecule to be measured is large, the preferable “average diameter of the pore” is large, and if the molecular weight of the molecule to be measured is small, the fine diameter is small. The average diameter of the holes may be small.
また、細孔の深さは、１０ｎｍ以上１０００ｎｍ以下であることが好ましく、１５ｎｍ以上５００ｎｍ以下であることがより好ましく、２０ｎｍ以上１００ｎｍ以下であることが特に好ましい。 The depth of the pores is preferably 10 nm or more and 1000 nm or less, more preferably 15 nm or more and 500 nm or less, and particularly preferably 20 nm or more and 100 nm or less.
＜測定対象分子＞
本発明のＬＤＩ質量分析法が適用される測定対象分子は特に限定はないが、生体由来の分子又は生体試料中の分子であることが好ましく、具体的には、糖、タンパク質、ペプチド、糖タンパク質、糖ペプチド、核酸、糖脂質等であることが、本発明の効果をより発揮できるので好ましい。「測定対象分子」としては、天然物から調製されるもの、天然物を化学的又は酵素学的に一部改変して調製されるものの他、化学的又は酵素学的に調製されるものも好ましい。また、生体に含まれる分子の部分構造を有するものや生体に含まれる分子を模倣して作製されたものも好ましい。
<Measurement molecule>
The molecule to be measured to which the LDI mass spectrometry method of the present invention is applied is not particularly limited, but is preferably a molecule derived from a living body or a molecule in a biological sample, specifically, sugar, protein, peptide, glycoprotein. Glycopeptides, nucleic acids, glycolipids, and the like are preferable because the effects of the present invention can be further exhibited. As a “molecule to be measured”, those prepared from natural products, those prepared by partially modifying natural products chemically or enzymatically, and those prepared chemically or enzymatically are also preferred. . Moreover, what has the partial structure of the molecule | numerator contained in the biological body, and the thing produced by imitating the molecule | numerator contained in the biological body are also preferable.
また、有機シリカ多孔体に担持する試料、すなわち、測定対象分子を含む試料としては、「測定対象分子」そのものだけでもよいし、「測定対象分子」を含むもの、例えば、生体の組織、細胞、体液や分泌物（例えば、血液、血清、尿、精液、唾液、涙液、汗、糞便等）等でもよい。すなわち、直接生体試料を用いてもよい。また、試料を有機シリカ多孔体に載せ、酵素処理等を行なって、測定対象分子を調製してもよい。 Further, the sample supported on the porous organic silica, that is, the sample containing the molecule to be measured may be only the “molecule to be measured” itself, or one containing the “molecule to be measured”, for example, a tissue, cell, Body fluids and secretions (for example, blood, serum, urine, semen, saliva, tears, sweat, feces, etc.) may be used. That is, a biological sample may be used directly. Alternatively, the molecule to be measured may be prepared by placing a sample on a porous organic silica and performing an enzyme treatment or the like.
また、本発明において「測定対象分子」とは、上記試料に含有されている分子であって、その化学構造を決定したい分子だけではなく、上記試料に含有されている分子であって、その化学構造を決定したい分子を誘導体化した分子、すなわち質量分析に供される分子をも含む。 In the present invention, the “measuring molecule” is a molecule contained in the sample and is not only a molecule whose chemical structure is to be determined, but also a molecule contained in the sample, It also includes molecules derivatized from molecules whose structure is to be determined, that is, molecules subjected to mass spectrometry.
本発明のＬＤＩ質量分析法が適用される測定対象分子の分子量については特に限定はないが、他の測定方法での正確な測定が困難であり本発明の特徴を発揮し易いことから、１６０以上であることが好ましく、５００以上であることがより好ましく、１０００以上であることが特に好ましい。 The molecular weight of the molecule to be measured to which the LDI mass spectrometry method of the present invention is applied is not particularly limited, but it is difficult to accurately measure with other measurement methods and the characteristics of the present invention are easily exhibited. It is preferable that it is 500, it is more preferable that it is 500 or more, and it is especially preferable that it is 1000 or more.
＜誘導体化＞
測定対象分子の誘導体化は、上記有機シリカ多孔体の吸収した光エネルギーを受容可能にする標識分子、好ましくは、上記有機シリカ多孔体の発光スペクトルとスペクトルの重なりを有する吸収帯を有する標識分子と共有結合させることにより行うことが好ましい。
<Derivatization>
Derivatization of the molecule to be measured is a label molecule that can accept the light energy absorbed by the organosilica porous body, and preferably a label molecule having an absorption band that has a spectrum overlap with the emission spectrum of the organosilica porous body. It is preferable to carry out by covalent bonding.
該標識分子は、有機シリカ多孔体から供与されるエネルギーの受容体として効果を有するものであれば特に限定されないが、蛍光標識試薬として市販されている分子を利用してもよい。例えば、ピレン誘導体、ｆｌｕｏｒｅｓｃｅｉｎ誘導体、ｒｈｏｄａｍｉｎｅ誘導体、シアニン色素、Ａｌｅｘａ Ｆｌｕｏｒ（登録商標）等が挙げられる。 The labeling molecule is not particularly limited as long as it has an effect as an acceptor of energy donated from the organic silica porous material, but a molecule commercially available as a fluorescent labeling reagent may be used. Examples thereof include pyrene derivatives, fluorescein derivatives, rhodamine derivatives, cyanine dyes, Alexa Fluor (registered trademark), and the like.
エネルギー供与体である有機シリカ多孔体とエネルギー受容体である標識分子の組合せは、エネルギー移動の効率、有機シリカ多孔体の発光スペクトルと測定対象分子の吸収スペクトルとの重なり、相互作用の強度等の点から適宜決定される。 The combination of the organosilica porous body that is an energy donor and the labeled molecule that is the energy acceptor is the energy transfer efficiency, the overlap between the emission spectrum of the organosilica porous body and the absorption spectrum of the molecule to be measured, the strength of interaction, etc. It is determined appropriately from the point.
例えば、有機シリカ多孔体としてメチルアクリドン基架橋有機シリカ多孔体を選択した場合は、標識分子として、４−Ｆｌｕｏｒｏ−７−ｎｉｔｒｏｂｅｎｚｏｆｕｒａｚａｎ、４−Ｆｌｕｏｒｏ−７−ｓｕｌｆｏｂｅｎｚｏｆｕｒａｚａｎ、３−Ｃｈｌｏｒｏｃａｒｂｏｎｙｌ−６，７−ｄｉｍｅｔｈｏｘｙ−１−ｍｅｔｈｙｌ−２（１Ｈ）−ｑｕｉｎｏｘａｌｉｎｏｎｅ等が好ましい。 For example, when a methylacridone group-crosslinked organic silica porous material is selected as the organic silica porous material, 4-Fluoro-7-nitrobenzofurazane, 4-Fluoro-7-sulfobenzofurazan, 3-Chlorocarbon-6,7- dimethyl-1-methyl-2 (1H) -quinoxaline is preferred.
標識分子は、対象分子と化学結合し易い官能基を有することを特徴とし、誘導体化は別の容器で行ってから使用してもよいし、有機シリカ多孔体上で行ってもよい。 The labeling molecule has a functional group that is easily chemically bonded to the target molecule, and derivatization may be performed after being performed in a separate container or may be performed on a porous organic silica.
＜試料の担持方法＞
（１）有機シリカ多孔体からなる基材と試料を混合して、試料を基材に均一に担持させた後、ＬＤＩ質量分析に供してもよいし、
（２）有機シリカ多孔体からなる基材の分散液を基板に塗布して乾燥後、その上に試料を載置して、試料を基材に均一に担持させた後、ＬＤＩ質量分析に供してもよいし、
（３）有機シリカ多孔体からなる基材を薄膜の状態で調製し、その薄膜の上に試料を載置して、試料を基材に均一に担持させた後、ＬＤＩ質量分析に供してもよい。
<Sample loading method>
(1) A base material composed of a porous organic silica and a sample are mixed, and after the sample is uniformly supported on the base material, it may be subjected to LDI mass spectrometry,
(2) A substrate dispersion composed of a porous organic silica material is applied to a substrate and dried, and then a sample is placed thereon, the sample is uniformly supported on the substrate, and then subjected to LDI mass spectrometry. Or
(3) A substrate made of porous organic silica is prepared in a thin film state, a sample is placed on the thin film, and the sample is uniformly supported on the substrate, and then subjected to LDI mass spectrometry. Good.
上記（１）では、有機シリカ多孔体からなる基材の形状は特に限定はなく、針状、薄片状、球状等の何れでもよい。「基材又は基材の分散液」と「試料又は試料の溶液」を混合して試料を基材に均一に担持させる。分散液の分散媒又は溶液の溶媒は、蒸発させ乾燥後にＬＤＩ質量分析に供する。 In said (1), the shape of the base material which consists of an organic silica porous body does not have limitation in particular, Any of acicular shape, flake shape, spherical shape, etc. may be sufficient. “Substrate or dispersion of substrate” and “sample or sample solution” are mixed so that the sample is uniformly supported on the substrate. The dispersion medium of the dispersion or the solvent of the solution is evaporated and dried, and then subjected to LDI mass spectrometry.
上記（２）では、基材の分散液を基板に塗布して乾燥した後の形態は特に限定はなく、有機シリカ多孔体が、粒状、平滑状、島状等に基板上に存在している形態が挙げられる。その上に、試料又は試料の溶液を載置し、溶媒を乾燥させて試料を基材に均一に担持させる。 In the above (2), the form after the substrate dispersion is applied to the substrate and dried is not particularly limited, and the organic silica porous material is present on the substrate in a granular, smooth, or island shape. A form is mentioned. A sample or a solution of the sample is placed thereon, the solvent is dried, and the sample is uniformly supported on the substrate.
上記（３）では、有機シリカ多孔体を調製段階で薄膜とする。基材を薄膜の状態で調製する方法としては、例えば、以下の実施例で詳述した方法等が挙げられる。 In (3) above, the organic silica porous material is made into a thin film at the preparation stage. Examples of the method for preparing the substrate in the state of a thin film include the methods described in detail in the following examples.
＜質量分析装置＞
イオン化に用いられるレーザーとしては、例えば、窒素レーザー（３３７ｎｍ）、ＹＡＧレーザー３倍波（３５５ｎｍ）、ＮｄＹＡＧレーザー（２５６ｎｍ）、炭酸ガスレーザー（９４００ｎｍ、１０６００ｎｍ）等が挙げられるが、窒素レーザーが好ましい。
イオンの分離検出方法は特に限定はなく、二重収束法、四重極集束法（四重極（Ｑ）フィルター法）、タンデム型四重極（ＱＱ）法、イオントラップ法、飛行時間（ＴＯＦ）法等を用いて、イオン化した分子を質量／電荷比（ｍ／ｚ）に従って分離し検出する。
<Mass spectrometer>
Examples of the laser used for ionization include a nitrogen laser (337 nm), a YAG laser triple wave (355 nm), an NdYAG laser (256 nm), a carbon dioxide laser (9400 nm, 10600 nm) and the like, and a nitrogen laser is preferable.
The ion separation and detection method is not particularly limited. Double focusing method, quadrupole focusing method (quadrupole (Q) filter method), tandem quadrupole (QQ) method, ion trap method, time of flight (TOF) ) Method or the like to separate and detect ionized molecules according to the mass / charge ratio (m / z).
以下に、評価例を挙げて本発明を更に具体的に説明するが、本発明は、その要旨を超えない限りこれらの評価例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to evaluation examples. However, the present invention is not limited to these evaluation examples as long as the gist thereof is not exceeded.
調製例１
＜有機シリカ多孔体の合成＞
（１）メチルアクリドン基架橋有機シリカ多孔体（ＭＡｃｄ−ＰＭＯ）薄膜（評価例１と評価例４で使用）の調製
メチルアクリドン基架橋有機シラン（下記式（１）で表される化合物）１５ｍｇと、鋳型となる界面活性剤であるポリスチレン−ポリエチレンオキシドジブックポリマー（Ｐ４９１１−ＳＥＯ（ｐｏｌｙｍｅｒ ｓｏｕｒｃｅ社製））１５ｍｇを、テトラヒドロフランとエタノールの１：１混合溶液（容積比）１ｍＬに溶解させた後、イオン交換水３μＬ及び２Ｍ塩酸２μＬを滴下し、室温で２４時間撹拌した。
Preparation Example 1
<Synthesis of porous organic silica>
(1) Preparation of a methylacridone group-crosslinked organosilica porous material (MACd-PMO) thin film (used in Evaluation Examples 1 and 4) Methylacridone group-crosslinked organosilane (compound represented by the following formula (1)) 15 mg and 15 mg of a polystyrene-polyethylene oxide dibook polymer (P4911-SEO (manufactured by Polymer Source)) as a template surfactant were dissolved in 1 mL of a 1: 1 mixed solution (volume ratio) of tetrahydrofuran and ethanol. Thereafter, 3 μL of ion-exchanged water and 2 μL of 2M hydrochloric acid were added dropwise and stirred at room temperature for 24 hours.
得られたゾルを、Ｓｉ基板（Ｐ型）上にスピンコート（回転数：２０００ｒｐｍ、回転時間：３０ｓ）した後、すぐに密閉容器の中でトルエン蒸気に室温で一晩暴露した。アンモニア蒸気に６０℃で１２時間暴露した後、８０℃で真空加熱することで、縮合反応を進行させた。トルエンに浸漬し、１０５℃で２４時間加熱する処理を２回行うことで、界面活性剤を除去し、目的のＭＡｃｄ−ＰＭＯ薄膜を得た。 The obtained sol was spin-coated on a Si substrate (P type) (rotation speed: 2000 rpm, rotation time: 30 s) and immediately exposed to toluene vapor in a sealed container overnight at room temperature. After exposure to ammonia vapor at 60 ° C. for 12 hours, the condensation reaction was allowed to proceed by heating in vacuum at 80 ° C. The surfactant was removed by immersing in toluene and heating twice at 105 ° C. for 24 hours to obtain the target MAcd-PMO thin film.
走査型電子顕微鏡写真により、得られた膜が、直径約２０ｎｍの細孔を有していることを確認した（図１）。また、吸収スペクトル及び発光スペクトルより、窒素レーザーの波長である３３７ｎｍに強い吸収帯を有し、かつ４６０ｎｍを中心とした発光を示すことを確認した（図２）。 It was confirmed by scanning electron micrographs that the obtained film had pores having a diameter of about 20 nm (FIG. 1). Further, it was confirmed from the absorption spectrum and the emission spectrum that it has a strong absorption band at 337 nm, which is the wavelength of the nitrogen laser, and emits light centered at 460 nm (FIG. 2).
調製例２
＜有機基を含まないシリカ多孔体の合成＞
（２）シリカ多孔体薄膜（評価例３で使用）の調製
テトラエトキシシラン３０ｍｇと、鋳型となる界面活性剤であるポリスチレン−ポリエチレンオキシドジブックポリマー（Ｐ４９１１−ＳＥＯ（ｐｏｌｙｍｅｒ ｓｏｕｒｃｅ社製））１０ｍｇを、テトラヒドロフランとエタノールの１：１混合溶液（容積比）１ｍＬに溶解させた後、イオン交換水３μＬ、２Ｍ塩酸２μＬを滴下し、室温で２４時間撹拌した。
Preparation Example 2
<Synthesis of porous silica containing no organic group>
(2) Preparation of porous silica thin film (used in evaluation example 3) 30 mg of tetraethoxysilane and 10 mg of polystyrene-polyethylene oxide dibook polymer (P4911-SEO (manufactured by polymer source)) which is a surfactant as a template. After dissolving in 1 mL of a 1: 1 mixed solution (volume ratio) of tetrahydrofuran and ethanol, 3 μL of ion-exchanged water and 2 μL of 2M hydrochloric acid were added dropwise and stirred at room temperature for 24 hours.
得られたゾルを、Ｓｉ基板（Ｐ型）上にスピンコート（回転数：２０００ｒｐｍ、回転時間：３０ｓ）した後、室温で一晩乾燥した。アンモニア蒸気に６０℃で１２時間暴露した後、８０℃で真空加熱することで縮合反応を進行させた。そして、トルエンに浸漬し、１０５℃で２４時間加熱する処理を２回行うことで、界面活性剤を除去し、目的のシリカ多孔体薄膜を得た。 The obtained sol was spin-coated on a Si substrate (P type) (rotation speed: 2000 rpm, rotation time: 30 s) and then dried overnight at room temperature. After exposure to ammonia vapor at 60 ° C. for 12 hours, the condensation reaction was allowed to proceed by vacuum heating at 80 ° C. Then, the surfactant was removed by immersing in toluene and heating at 105 ° C. for 24 hours twice to obtain the target porous silica thin film.
走査型電子顕微鏡写真により、得られた膜が、直径約２０ｎｍの細孔を有していることを確認した（図３）。また、吸収スペクトル及び発光スペクトルより、２００ｎｍ〜８００ｎｍに吸収帯を有しておらず、また、窒素レーザーの波長である３３７ｎｍで励起しても発光を示さないことを確認した（図４）。 It was confirmed by scanning electron micrographs that the obtained film had pores having a diameter of about 20 nm (FIG. 3). Further, from the absorption spectrum and the emission spectrum, it was confirmed that there was no absorption band at 200 nm to 800 nm, and no light was emitted even when excited at 337 nm, which is the wavelength of the nitrogen laser (FIG. 4).
評価例１
＜メチルアクリドン架橋有機シリカ薄膜／ＮＢＤ−ＩＲＮＫＳ＞
１ｍＭのＩＲＮＫＳペプチド水溶液（「ＩＲＮＫＳ」とは、１文字アミノ酸の配列で表わしたペプチドを示す。）５０μＬに０．１Ｍホウ酸緩衝液（ｐＨ ８．０）５０μＬを加え、更に５０ｍＭのＮＢＤ−Ｆ（４−Ｆｌｕｏｒｏ−７−ｎｉｔｒｏｂｅｎｚｏｆｕｒａｚａｎ、下記式（２）で表される化合物）／エタノール溶液１００μＬを加えた後に、遮光条件下、６０℃で１分間反応させた。
Evaluation Example 1
<Methylacridone crosslinked organosilica thin film / NBD-IRNKS>
50 μL of 0.1 M borate buffer (pH 8.0) is added to 50 μL of a 1 mM IRNKS peptide aqueous solution (“IRNKS” indicates a peptide represented by a one-letter amino acid sequence), and 50 mM NBD-F is further added. (4-Fluoro-7-nitrobenzofurazan, compound represented by the following formula (2)) / ethanol solution 100 μL was added, and the mixture was reacted at 60 ° C. for 1 minute under light-shielding conditions.
その後、５０ｍＭのＨＣｌ水溶液４６０μＬを加え、減圧濃縮装置を用いて、反応混合物を乾燥した。Ｃ１８スピンカラム（８ｍｇ）をアセトニトリル、純水で洗浄し、乾燥させた反応物を純水に溶解してカラムに通した。純水でカラムを洗浄した後に８０％アセトニトリル水溶液で溶出することによって、ＮＢＤ標識されたペプチドを得ることができた。この標識ペプチドは、４７０ｎｍに吸収ピークを有した。 Thereafter, 460 μL of 50 mM HCl aqueous solution was added, and the reaction mixture was dried using a vacuum concentrator. A C18 spin column (8 mg) was washed with acetonitrile and pure water, and the dried reaction product was dissolved in pure water and passed through the column. After washing the column with pure water and eluting with 80% acetonitrile aqueous solution, an NBD-labeled peptide could be obtained. This labeled peptide had an absorption peak at 470 nm.
この反応物を６０％アセトニトリル水溶液２０μＬに溶解させた。この溶液を、調製例１で調製したメチルアクリドン架橋有機シリカ薄膜コートした基板の上部に０．３μＬ滴下させ、自然乾燥させた。この基板をＭＡＬＤＩ−ＴＯＦ ＭＳのスライド装着式カートリッジプレートに装着し、ＭＡＬＤＩ−ＱＩＴ−ＴＯＦ ＭＳ、Ａｘｉｍａ−ＱＩＴ（Ｓｈｉｍａｄｚｕ／Ｋｒａｔｏｓ）を用いて、基板上の異なる３点にレーザー光を照射し、測定を行った。 This reaction product was dissolved in 20 μL of a 60% acetonitrile aqueous solution. 0.3 μL of this solution was dropped onto the upper part of the substrate coated with the methylacridone-crosslinked organic silica thin film prepared in Preparation Example 1, and allowed to air dry. This substrate is mounted on a MALDI-TOF MS slide-mounted cartridge plate, and MALDI-QIT-TOF MS and Axima-QIT (Shimadzu / Kratos) are used to irradiate laser light to three different points on the substrate. Went.
その結果、負イオンモードで図５に示すように、基板上の何れの点においても、ＩＲＮＫＳペプチドにＮＢＤが２分子結合したイオン（ｍ／ｚ９４１）のみが検出され、更に、正イオンモードにおいても、ＩＲＮＫＳペプチドにＮＢＤが２分子結合したイオン（ｍ／ｚ９４３）が検出された。 As a result, as shown in FIG. 5 in the negative ion mode, only an ion (m / z 941) in which two molecules of NBD are bonded to the IRNKS peptide is detected at any point on the substrate, and also in the positive ion mode. , An ion (m / z 943) in which two molecules of NBD were bound to the IRNKS peptide was detected.
本発明によれば、何れのモードにおいても、ＩＲＮＫＳペプチドにＮＢＤが２分子結合したイオンのみが検出され、本発明のレーザー脱離イオン化質量分析法は、よりフラグメンテーションが起こり難いソフトなイオン化であり、均質かつ再現性のよいスペクトルを得ることができることが示された。 According to the present invention, in any mode, only ions in which two molecules of NBD are bound to the IRNKS peptide are detected, and the laser desorption ionization mass spectrometry method of the present invention is a soft ionization that is less prone to fragmentation. It was shown that a homogeneous and reproducible spectrum can be obtained.
評価例２
＜メチルアクリドン架橋有機シリカ薄膜／ＮＢＤ−ＩＲＮＫＳ＞
８ｎｍよりも小さい平均直径を有するメチルアクリドン架橋有機シリカよりなる有機シリカ多孔体を用いて、評価例１と同一試料を同様に測定したところ、何れのモードにおいても、シグナルがほとんど検出されなかった。
分子量約１０００のペプチドは、例えば、αヘリックス構造をとった場合、１ｎｍ×１．５ｎｍの大きさになるとされる。このことから、少なくとも、糖鎖、ペプチド、糖ペプチドについては、有機シリカ多孔体が有する孔の平均直径が８ｎｍ以上あるメチルアクリドン架橋有機シリカの方が、試料を担持する能力が大きいと思われる。
Evaluation example 2
<Methylacridone crosslinked organosilica thin film / NBD-IRNKS>
When the same sample as in Evaluation Example 1 was measured using an organosilica porous material made of methylacridone-crosslinked organosilica having an average diameter of less than 8 nm, almost no signal was detected in any mode. .
For example, a peptide having a molecular weight of about 1000 has a size of 1 nm × 1.5 nm when it has an α-helix structure. Therefore, at least for sugar chains, peptides, and glycopeptides, methylacridone-crosslinked organosilica having an average pore diameter of 8 nm or more in the organosilica porous body seems to have a larger ability to carry a sample. .
従って、８ｎｍよりも小さい平均直径を有する有機シリカ多孔体の細孔の内部には、上記試料が入り難かったからシグナルがほとんど検出されなかったと考えられる。
すなわち、有機シリカ多孔体の細孔内に測定対象分子が存在することによって本発明の効果が奏されることが確認された。
Therefore, it is considered that almost no signal was detected because it was difficult for the sample to enter the pores of the porous organic silica having an average diameter smaller than 8 nm.
That is, it was confirmed that the effect of the present invention is exhibited by the presence of the molecule to be measured in the pores of the organic silica porous body.
評価例３
＜シリカ多孔体薄膜／ＮＢＤ−ＩＲＮＫＳ＞
調製例１で調製したメチルアクリドン架橋有機シリカ薄膜コートした基板の代わりに、調製例２で調製した有機基を含まないシリカ多孔体薄膜コート基板を用いた以外は、評価例１と同様に、ＮＢＤ標識されたペプチドのｎｅｇａｔｉｖｅ ｉｏｎを測定した。
Evaluation Example 3
<Silica porous film / NBD-IRNKS>
Instead of the methylacridone-crosslinked organic silica thin film coated substrate prepared in Preparation Example 1, the same porous porous thin film coated substrate containing no organic group prepared in Preparation Example 2 was used as in Evaluation Example 1, The negative ion of NBD-labeled peptide was measured.
その結果、図７に示すように、ノイズが見られたのみでシグナルとなるイオンは検出されなかった。Ｐｏｓｉｔｉｖｅ ｉｏｎは、シリカ多孔体薄膜コート基板では、有機シリカ薄膜コート基板に比べてシグナル強度が低かった。これらのことは、多数のメチルアクリドン基が導入されたことによってイオン化効率が向上したことを示している。 As a result, as shown in FIG. 7, no noise was detected and no ions serving as signals were detected. In the positive ion, the signal intensity of the porous silica thin film coated substrate was lower than that of the organic silica thin film coated substrate. These facts show that ionization efficiency is improved by introducing a large number of methylacridone groups.
評価例４
＜メチルアクリドン架橋有機シリカ薄膜／Ｆｍｏｃ−ＩＲＮＫＳ＞
１ｍＭのＩＲＮＫＳペプチド水溶液（「ＩＲＮＫＳ」とは、１文字アミノ酸の配列で表わしたペプチドを示す。）５０μＬに、０．１Ｍ炭酸緩衝液（ｐＨ１１．０）５０μＬを加え、更に、５０ｍＭの「Ｆｍｏｃ−ＯＳｕ（Ｎ−（９−Ｆｌｕｏｒｅｎｙｌｍｅｔｈｏｘｙｃａｒｂｏｎｙｌｏｘｙ）ｓｕｃｃｉｎｉｍｉｄｅ、下記式（３）で表される化合物）」のアセトン溶液１００μＬを加えた後に、遮光条件下、室温で１時間反応させた。
Evaluation Example 4
<Methylacridone crosslinked organic silica thin film / Fmoc-IRNKS>
To 50 μL of a 1 mM IRNKS peptide aqueous solution (“IRNKS” indicates a peptide represented by a one-letter amino acid sequence), 50 μL of 0.1 M carbonate buffer (pH 11.0) is added, and 50 mM “Fmoc− After adding 100 μL of an acetone solution of OSu (N- (9-Fluorenylmethoxycarbonyl) succinimide, a compound represented by the following formula (3)), the mixture was reacted at room temperature for 1 hour under light-shielding conditions.
その後、５０ｍＭのＨＣｌ水溶液４６０μＬを加え、減圧濃縮装置を用いて、反応混合物を乾燥した。Ｃ１８スピンカラム（８ｍｇ）をアセトニトリル、純水で洗浄し、乾燥させた反応物を純水に溶解してカラムに通した。純水でカラムを洗浄した後に８０％アセトニトリル水溶液で溶出することによって、Ｆｍｏｃで標識されたペプチド（分子量１０６１）を得ることができた。この標識ペプチドは、４００ｎｍ以上に吸収極大を持たなかった。この反応物を６０％アセトニトリル水溶液２０μＬに溶解させた。この溶液を、調製例１で調製したメチルアクリドン架橋有機シリカ薄膜コートした基板の上部に０．３μＬ滴下させ、自然乾燥させた。評価例１と同様に質量分析装置で測定した。 Thereafter, 460 μL of 50 mM HCl aqueous solution was added, and the reaction mixture was dried using a vacuum concentrator. A C18 spin column (8 mg) was washed with acetonitrile and pure water, and the dried reaction product was dissolved in pure water and passed through the column. After washing the column with pure water and eluting with 80% acetonitrile aqueous solution, a peptide (molecular weight 1061) labeled with Fmoc could be obtained. This labeled peptide did not have an absorption maximum at 400 nm or more. This reaction product was dissolved in 20 μL of a 60% acetonitrile aqueous solution. 0.3 μL of this solution was dropped onto the upper part of the substrate coated with the methylacridone-crosslinked organic silica thin film prepared in Preparation Example 1, and allowed to air dry. It measured with the mass spectrometer similarly to the evaluation example 1.
その結果、測定対象分子は正イオンモードにおいても（図８）、負イオンモードにおいても検出できなかった。すなわち、メチルアクリドン架橋有機シリカ薄膜がレーザー光を吸収して生じる発光波長を吸収しないＦｍｏｃ標識ペプチドはイオン化されないことが示された。 As a result, the molecule to be measured could not be detected in the positive ion mode (FIG. 8) or in the negative ion mode. That is, it was shown that the Fmoc-labeled peptide that does not absorb the emission wavelength generated when the methylacridone-crosslinked organic silica thin film absorbs laser light is not ionized.
評価例５
＜ＤＨＢＡ／ＮＢＤ−ＩＲＮＫＳ＞
メチルアクリドン架橋有機シリカ薄膜を用いず、マトリックスとして、２，５−ジヒドロキシ安息香酸（ＤＨＢＡ）を用いて、評価例１と同一試料を同様の質量分析装置を用いて、ＭＡＬＤＩ−ＭＳ測定をした。
Evaluation Example 5
<DHBA / NBD-IRNKS>
MALDI-MS measurement was performed on the same sample as in Evaluation Example 1 using the same mass spectrometer, using 2,5-dihydroxybenzoic acid (DHBA) as a matrix without using a methylacridone-crosslinked organic silica thin film. .
その結果、フラグメンテーションが起こり、ＩＲＮＫＳペプチドにＮＢＤが２分子結合したイオンと共に、ＩＲＮＫＳペプチドにＮＢＤが１分子結合したイオンも観測された（図６）。
評価例１を評価例５と比較することによって、本発明の優位性が示された。
As a result, fragmentation occurred, and an ion in which one molecule of NBD was bonded to the IRNKS peptide was observed together with an ion in which two molecules of NBD were bonded to the IRNKS peptide (FIG. 6).
By comparing Evaluation Example 1 with Evaluation Example 5, the superiority of the present invention was shown.
本発明のレーザー脱離イオン化質量分析法は、測定対象分子のみを選択的にイオン化させることが可能であり、試料のフラグメンテーションが起こらず、マトリックスを用いる通常の測定法と異なりスイートスポットを探す必要がなく、イオン化し難い対象分子を通常よりも弱い励起光でイオン化できるため、ＭＳスペクトルを使用する全ての分析分野に、特に、微量試料しか入手できない場合がある生体分析の分野等に広く利用されるものである。 The laser desorption ionization mass spectrometry method of the present invention can selectively ionize only the molecule to be measured, does not cause fragmentation of the sample, and it is necessary to search for a sweet spot unlike a normal measurement method using a matrix. The target molecules that are difficult to ionize can be ionized with weaker excitation light than usual, so it is widely used in all analysis fields that use MS spectra, especially in the field of biological analysis where only a small amount of sample may be available. Is.
Claims (5)
該有機シリカ多孔体に、該測定対象分子を含む試料を均一に担持させた後、レーザー光を照射することにより該測定対象分子をイオン化させることを特徴とするレーザー脱離イオン化質量分析法。 In laser desorption ionization mass spectrometry, “a porous organic silica having an organic group capable of absorbing irradiated laser light in its skeleton ” and “ a target molecule to which the light energy absorbed by the porous organic silica can be transferred”. A measurement target molecule configured such that an emission spectrum of the organic silica porous material and an absorption spectrum thereof overlap at least at one wavelength,
A laser desorption ionization mass spectrometry method, wherein after a sample containing the molecule to be measured is uniformly supported on the porous organic silica, the molecule to be measured is ionized by irradiation with laser light.
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