出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2014/11/22 06:38:05」(JST)
マトリックス支援レーザー脱離イオン化法（マトリックスしえんレーザーだつりイオンかほう、英: Matrix Assisted Laser Desorption / Ionization, MALDI）は質量分析におけるサンプルのイオン化法の一つである。日本では「MALDI」をマルディーと呼ばれることが多いが、英語での発音はモールディーに近い。
MALDIの開発と実用化は島津製作所の田中耕一の研究成果に拠るところが大きく、かかる功績により、田中には2002年にノーベル化学賞が授与されている。
MALDI法はESI法と並ぶ代表的なソフトイオン化法で、従来のイオン化法では壊れやすかった大型の生体分子（タンパク質、ペプチド、多糖など）のイオン化に向く。これにより分子量の大きな高分子化合物の質量分析が可能となり、医学や生物学、特に生化学分野を中心に非常に大きな発展をもたらした。
また、分析に必要なサンプル量がごく微量で良いという利点があり、ESI法を凌ぐフェムトモル（fmol）オーダーから測定可能である。加えてサンプルの純度に対する要求性も比較的低い。これらの特徴が、大量の高純度試料を用意することが難しい生体由来の試料の分析を、一層容易なものにしている。
物質に紫外レーザー光を照射すると、物質が光を吸収して光電子移動が進行し、イオン化される。この直接的なレーザー照射によるイオン化法をレーザー脱離イオン化法（Laser Desorption / Ionization、LDI）という。しかし、LDIでは物質の種類によっては効率的な電子移動が行われず、試料がレーザーでダメージを受けてしまうという欠点があった。そこで、レーザー光によってイオン化されやすい物質をマトリックスとしてサンプルと予め混合しておき、これにレーザーを照射する事でイオン化する手法、すなわちMALDIが開発された。
サンプルとマトリックスの混合物（混晶）に窒素レーザー（波長337 nm）のパルスを当てると、マトリックスは瞬時に励起され、受け取ったレーザーの余剰エネルギーを熱エネルギーとして放出する。その結果、マトリックスとサンプルは気化され、同時にマトリックス-サンプル間でプロトンの授受が起こってサンプルがイオン化される。このとき生じるイオンは主に［M+H］+、 ［M+Na］+、［M-H］- 等である。サンプルの種類によっては［M+］や［M-H］- も観測される。また、MALDIで生じるイオンは多くの場合一価であるが、二価イオン（［M+2H］2+）が生成される場合もある
MALDIには多くの場合TOF型（Time of Flight、飛行時間質量分析計）の分析部が組み合わされる。生成したイオンは加速電圧（20～25kV前後）を印加されて運動エネルギーを生じ、イオン検出器まで飛行していく。イオンが受け取るエネルギーは電荷量のみに依存する為、電荷に対する質量（質量電荷比）が大きい分子は低速で、逆に小さい分子は高速で飛行する。この差異により、検出器に到達するまでの時間差からサンプルの質量を割り出す事が可能となる。TOFの場合、原理的には検出時間を延長すれば質量に検出上限は無く、実際に分子量数百～数十万の幅広い質量に対応した測定が可能である。
最近ではTOFの実装はイオン反射装置であるリフレクトロンを伴うものが多く、飛行距離を伸ばすと共にイオンの運動エネルギー誤差を相殺し、より高精度の分析が可能となっている。また、混晶にレーザーを当てた直後の数百～数十nsは加速電圧を印加せず、その後一斉に加速すること（delayed extraction、遅延引き出し）で初期状態の違いによる検出時間のバラつきを抑える事が可能である。
マトリックスの役目は、前述の通りレーザーエネルギー伝達の仲介にある。質量分析のスペクトルはサンプルとマトリックスの混晶の状態に大きく左右され、従ってサンプルに応じた適切なマトリックスを選択しなければならない。
いわゆるプロテオミクスの現場では、MALDIは通常のSDS-PAGEや二次元電気泳動による分離操作と組み合わせて用いられる。ペプチドマスフィンガープリンティング（PMF）は利用の代表例である。
通常のMALDIは高真空条件下でイオン化を行うので、クロマトグラフィーと連結した自動解析には向かない。前処理としてクロマトグラフィーを用いる場合は、分離した試料を手作業で分取してMALDIにかけるという操作が必要になる。
AP（atmospheric pressure、大気圧）の名の通り、大気圧下でイオン化が可能なMALDIである。イオン化部が高真空を要求しない事で、接続可能な前処理用の分離装置や質量分析部の種類が増え、多彩な分析系の構築が可能となる。
通常のMALDIには前述の通り窒素レーザーが用いられるが、IRレーザーによるイオン化も実用化が進んでいる。IRによるイオン化は窒素レーザーのようなUVと比較して多価イオンやクラスターイオンが生成されやすく、分解能が落ちる傾向がある。しかしながらIR-MALDIでは選択可能なマトリックスの種類が多く、通常使用されるものの他にコハク酸、グリセリン、尿素、あるいは水などを用いる事ができる。また、多価イオンが生じやすい一方でフラグメントイオンの生成が抑えられるので、UV-MALDIでは分解してしまうサンプルもIR-MALDIで分析できる可能性がある。
Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to electrospray ionization (ESI) in that both techniques are relatively soft ways of obtaining large ions in the gas phase, though MALDI produces far fewer multiply charged ions.
MALDI is thought to be a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them. However, considerable evidence suggests analyte ions are produced from charged particles produced in the ablation process.
The term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by Franz Hillenkamp, Michael Karas and their colleagues.[1] These researchers found that the amino acid alanine could be ionized more easily if it was mixed with the amino acid tryptophan and irradiated with a pulsed 266 nm laser. The tryptophan was absorbing the laser energy and helping to ionize the non-absorbing alanine. Peptides up to the 2843 Da peptide melittin could be ionized when mixed with this kind of “matrix”.[2] The breakthrough for large molecule laser desorption ionization came in 1987 when Koichi Tanaka of Shimadzu Corporation and his co-workers used what they called the “ultra fine metal plus liquid matrix method” that combined 30 nm cobalt particles in glycerol with a 337 nm nitrogen laser for ionization.[3] Using this laser and matrix combination, Tanaka was able to ionize biomolecules as large as the 34,472 Da protein carboxypeptidase-A. Tanaka received one-quarter of the 2002 Nobel Prize in Chemistry for demonstrating that, with the proper combination of laser wavelength and matrix, a protein can be ionized.[4] Karas and Hillenkamp were subsequently able to ionize the 67 kDa protein albumin using a nicotinic acid matrix and a 266 nm laser.[5] Further improvements were realized through the use of a 355 nm laser and the cinnamic acid derivatives ferulic acid, caffeic acid and sinapinic acid as the matrix.[6] The availability of small and relatively inexpensive nitrogen lasers operating at 337 nm wavelength and the first commercial instruments introduced in the early 1990s brought MALDI to an increasing number of researchers.[7] Today, mostly organic matrices are used for MALDI mass spectrometry.
| Compound | Other Names | Solvent | Wavelength (nm) | Applications |
|---|---|---|---|---|
| 2,5-dihydroxy benzoic acid[8] | DHB, Gentisic acid | acetonitrile, water, methanol, acetone, chloroform | 337, 355, 266 | peptides, nucleotides, oligonucleotides, oligosaccharides |
| 3,5-dimethoxy-4-hydroxycinnamic acid[6][9] | sinapic acid; sinapinic acid; SA | acetonitrile, water, acetone, chloroform | 337, 355, 266 | peptides, proteins, lipids |
| 4-hydroxy-3-methoxycinnamic acid[6][9] | ferulic acid | acetonitrile, water, propanol | 337, 355, 266 | proteins |
| α-Cyano-4-hydroxycinnamic acid[10] | CHCA | acetonitrile, water, ethanol, acetone | 337, 355 | peptides, lipids, nucleotides |
| Picolinic acid[11] | PA | Ethanol | 266 | oligonucleotides |
| 3-hydroxy picolinic acid[12] | HPA | Ethanol | 337, 355 | oligonucleotides |
The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (CHCA, alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB).[13] A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0.1).
The identification of suitable matrix compounds is determined to some extent by trial and error, but they are based on some specific molecular design considerations:
The matrix solution is mixed with the analyte (e.g. protein-sample). A mixture of water and organic solvent allows both hydrophobic and water-soluble (hydrophilic) molecules to dissolve into the solution. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte molecules embedded into MALDI crystals. The matrix and the analyte are said to be co-crystallized. Co-crystallization is a key issue in selecting a proper matrix to obtain a good quality mass spectrum of the analyte of interest.
The matrix can be used to tune the instrument to ionize the sample in different ways. As mentioned above, acid-base like reactions are often utilized to ionize the sample, however, molecules with conjugated pi systems, such as naphthalene like compounds, can also serve as an electron acceptor and thus a matrix for MALDI/TOF.[16] This is particularly useful in studying molecules that also possess conjugated pi systems.[17] The most widely used application for these matrices is studying porphyrin like compounds such as chlorophyll. These matrices have been shown to have better ionization patterns that do not result in odd fragmentation patterns or complete loss of side chains.[18] It has also been suggested that conjugated porphryin like molecules can serve as a matrix and cleave themselves eliminating the need for a separate matrix compound.[19]
MALDI techniques typically employ the use of UV lasers such as nitrogen lasers (337 nm) and frequency-tripled and quadrupled Nd:YAG lasers (355 nm and 266 nm respectively). Although not as common, infrared lasers are used due to their softer mode of ionization. IR-MALDI also has the advantage of greater material removal (useful for biological samples), less low-mass interferences, and compatibility with other matrix-free laser desorption mass spectrometry methods.[20]
The laser is fired at the matrix crystals in the dried-droplet spot. The matrix absorbs the laser energy and it is thought that primarily the matrix is desorbed and ionized (by addition of a proton) by this event. The hot plume produced during ablation contains many species: neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters and nanodroplets. Ablated species may participate in the ionization of analyte, though the mechanism of MALDI is still debated. The matrix is then thought to transfer protons to the analyte molecules (e.g., protein molecules), thus charging the analyte.[21] An ion observed after this process will consist of the initial neutral molecule [M] with ions added or removed. This is called a quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na]+ in the case of an added sodium ion, or [M-H]- in the case of a removed proton. MALDI is capable of creating singly charged ions or multiply charged ions ([M+nH]n+) depending on the nature of the matrix, the laser intensity, and/or the voltage used. Note that these are all even-electron species. Ion signals of radical cations (photoionized molecules) can be observed, e.g., in the case of matrix molecules and other organic molecules.
A recent method termed matrix-assisted ionization [MAI] uses matrix preparation identical to MALDI but does not require laser ablation to produce analyte ions of volatile or nonvolatile compounds. Simply exposing the matrix [e.g. 3-nitrobenzonitrile] with analyte to the vacuum of the mass spectrometer creates ions with nearly identical charge states to electrospray ionization.[22] It is suggested that there are likely mechanistic commonality between this process and MALDI.[23]
Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ion source) that in contrast to vacuum MALDI operates at normal atmospheric environment.[24] The main difference between vacuum MALDI and AP-MALDI is the pressure in which the ions are created. In vacuum MALDI, ions are typically produced at 10 mTorr or less while in AP-MALDI ions are formed in atmospheric pressure. In the past the main disadvantage of AP MALDI technique compared to the conventional vacuum MALDI has been its limited sensitivity; however, ions can be transferred into the mass spectrometer with high efficiency and attomole detection limits have been reported.[25]
AP-MALDI is used in mass spectrometry (MS) in a variety of applications ranging from proteomics to drug discovery. Popular topics that are addressed by AP-MALDI mass spectrometry include: proteomics; mass analysis of DNA, RNA, PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers, similar applications as available also for vacuum MALDI instruments.
The AP-MALDI ion source is easily coupled to an ion trap mass spectrometer[26] or any other MS system equipped with ESI (electrospray ionization) or nanoESI source.
The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range. The TOF measurement procedure is also ideally suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation. MALDI-TOF instrument or reflectron is equipped with an "ion mirror" that reflects ions using an electric field, thereby doubling the ion flight path and increasing the resolution. Today, commercial reflectron TOF instruments reach a resolving power m/Δm of well above 20,000 FWHM (full-width half-maximum, Δm defined as the peak width at 50% of peak height).
MALDI has been coupled with IMS-TOF MS to identify phosphorylated and non-phosphorylated peptides.[27][28]
MALDI-FT-ICR MS has been demonstrated to be a useful technique where high resolution MALDI-MS measurements are desired.[29]
In proteomics, MALDI is used for the rapid identification of proteins isolated by using gel electrophoresis: SDS-PAGE, size exclusion chromatography, affinity chromatography, strong/weak ion exchange, isotope coded protein labelling (ICPL),and two-dimensional gel electrophoresis. Peptide mass fingerprinting is the most popular analytical application of MALDI-TOF mass spectrometers. MALDI TOF/TOF mass spectrometers are used to reveal amino acid sequence of peptides using post-source decay or high energy collision-induced dissociation (further use see mass spectrometry).
Loss of sialic acid has been identified in papers when DHB has been used as a matrix for MALDI MS analysis of glycosylated peptides. Using sinapinic acid, 4-HCCA and DHB as matrices, S. Martin studied loss of sialic acid in glycosylated peptides by metastable decay in MALDI/TOF in linear mode and reflector mode.[30] A group at Shimadzu Corporation derivatized the sialic acid by an amidation reaction as a way to improve detection sensitivity[31] and also demonstrated that ionic liquid matrix reduces a loss of sialic acid during MALDI/TOF MS analysis of sialylated oligosaccharides.[32] THAP,[33] DHAP,[34] and a mixture of 2-aza-2-thiothymine and phenylhydrazine [35] have been identified as matrices that could be used to minimize loss of sialic acid during MALDI MS analysis of glycosylated peptides.
It has been reported that a reduction in loss of some post-translational modifications can be accomplished if IR MALDI is used instead of UV MALDI [36]
In molecular biology, a mixture of 5-methoxysalicylic acid and spermine can be used as a matrix for oligonucleotides analysis in MALDI mass spectrometry,[37] for instance after oligonucleotide synthesis.
Some synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and hyperbranched polymers, and other assemblies, have molecular weights extending into the thousands or tens of thousands, where most ionization techniques have difficulty producing molecular ions. MALDI is a simple and fast analytical method that can allow chemists to rapidly analyze the results of such syntheses and verify their results.
In polymer chemistry MALDI can be used to determine the molar mass distribution.[38] Polymers with polydispersity greater than 1.2 are difficult to characterize with MALDI due to the signal intensity discrimination against higher mass oligomers.[39][40][41] A good matrix for polymers is dithranol and AgTFA. The sample must first be mixed with dithranol and the AgTFA added afterwards; otherwise the sample would precipitate out of solution.
MALDI/TOF spectra are used for the identification of microorganisms such as bacteria or fungi. A colony of the microbe in question is smeared directly on the sample target and overlaid with matrix. The mass spectra generated are analyzed by dedicated software and compared with stored profiles. Species diagnosis by this procedure is much faster, more accurate and cheaper than other procedures based on immunological or biochemical tests. MALDI/TOF may become the standard method for species identification in medical microbiological laboratories over the next few years.[42]
MALDI/TOF spectra are often utilized in tandem with other analysis and spectroscopy techniques in the diagnosis of diseases. MALDI/TOF is a diagnostic tool with much potential because it allows for the rapid identification of proteins and changes to proteins without the cost or computing power of sequencing nor the skill or time needed to solve a crystal structure in X-ray crystallography.
One of example of this is necrotizing enterocolitis (NEC), which is a devastating disease that affects the bowels of premature infants. The symptoms of NEC are very similar to those of sepsis, and many infants die awaiting diagnosis and treatment. MALDI/TOF was used to quickly analyze fecal samples and find differences between the mutant and the functional protein responsible for NEC. There is hope that a similar technique could be used as a quick, diagnostic tool that would not require sequencing.[43]
Another example of the diagnostic power of MALDI/TOF is in the area of cancer. Pancreatic cancer remains one of the most deadly and difficult to diagnose cancers.[44] Impaired cellular signaling due to mutations in membrane proteins has been long suspected to contribute to pancreatic cancer.[45] MALDI/TOF has been used to identify a membrane protein associated with pancreatic cancer and at one point may even serve as an early detection technique.[46]
MALDI/TOF can also potentially be used to dictate treatment as well as diagnosis. Since the use of antibacterial hand-creams and soaps has becoming popular, the question of “super bugs” and increased bacterial drug resistance has been raised. MALDI/TOF serves as a method for determining the drug resistance of bacteria, especially to β-lactamases (Penicillin family). The MALDI/TOF detects the presence of carbapenemases, which indicates drug resistance to standard antibiotics. It is predicted that this could serve as a method for identifying a bacteria as drug resistant in as little as three hours. This technique could help physicians decide whether to prescribe more aggressive antibiotics initially.[47]
The sample preparation for MALDI is important for both sensitivity, reproducibility, and quantification of mass analysis. Inorganic salts which are also part of protein extracts interfere with the ionization process. The salts can be removed by solid phase extraction or by washing the dried-droplet MALDI spots with cold water. Both methods can also remove other substances from the sample. The matrix-protein mixture is not homogenous because the polarity difference leads to a separation of the two substances during co-crystallization. The spot diameter of the target is much larger than that of the laser, which makes it necessary to make many laser shots at different places of the target, to get the statistical average of the substance concentration within the target spot. The matrix chemical composition, the addition of trifluoroacetic acid, formic acid, fructose, delay time between the end of laser pulse and start of ion acceleration in the ion source (in vacuum MALDI sources), laser wavelength, UV energy (as well as its density and homogeneity)in a focused light spot produced by pulsed laser, and the impact angle of the laser on the target are among critical parameters for the quality and reproducibility of the MALDI-TOF MS method.
Additionally, the thickness of the MALDI plate can affect the TOF measurements.[48] The smaller plates (less thick) will have longer TOF measurements. This can potentially shift peaks in the spectra and make it difficult to compare to other published results.
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| リンク元 | 「マトリックス支援レーザー脱離イオン化法」「matrix-assisted laser desorption-ionization mass spectrometry」「matrix-assisted laser desorption ionization」 |
| 拡張検索 | 「MALDI-MS」 |
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