Name: whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)
Uploaded: 2016-03-24T15:00:00
Description: Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

The authors thank Professor H. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences for providing the whole mouse tissue. The authors also gratefully acknowledge the financial assistance received from National Institutes of Health (R01GM087964), the W.M. Keck foundation, and North Carolina State University.

注：以下のプロトコルは、IR-MALDESI MSI実験を行うために必要なすべての手順について説明します。より綿密な最適化されたIR-MALDESI源の幾何学とレーザー、ステージとの同期、および質量分析計についての詳細は、別の場所で5,6見つけることができます。このプロトコルで使用される動物の組織サンプルは、施設内動物管理使用委員会（IACUC）とノースカロライナ州立大学の規則に従っ...

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上記のプロトコルは、IR-MALDESI MSI実験を行うための重要なステップについて説明します。マトリックス・アプリケーション・プロセス（第3節）は、昇華またはスプレー塗布ロボット噴霧器を用いて、MALDI MSI実験のために、典型的なマトリックス・アプリケーション・プロセスに似て約20分を取ります。また、IR-MALDESIはマトリックス結晶6への検体の分配に依存しない、氷マトリックス?...

マイクロプローブモードでの質量分析イメージング（MSI）は、試料の表面上の別個の位置でのビーム（レーザーまたはイオン）によって表面からの試料の脱離を伴います。各ラスタ点で、質量スペクトルが生成され、取得されたスペクトルは、それらが収集された元の空間位置とともに、同時に試料中の多数の分析物をマッピングするために使用することができます。質量分析の感度と特異性に結合されたイメージングのこのラベルのない方法は、MSIは、質量分析1,2の中で最も急速に進化する分野の一つとなって助けました。 マトリックス支援レーザー脱離/イオン化（MALDI）MSI分析のために使用される最も一般的なイオン化法です。しかし、有機マトリックス及びMALDIの真空要件の必要性は、再現性、サンプルスループット、および方法を用いて分析することができる試料のタイプに重大な制限をもたらします。大気圧（AP）IO数nization方法は、これらの制限3を回避するために、近年開発されてきました。これらの周囲のイオン化法は、それらの天然の状態に非常に近いと分析前の試料調製工程を簡略化環境において、生物学的試料の分析を可能にします。マトリックス支援レーザ脱離エレクトロスプレーイオン化（MALDESI）は、イオン化法4,5の例です。 IR-MALDESI実験では、氷の薄層は、エネルギー吸収マトリックスとして組織表面上に堆積されます。中赤外レーザーパルス氷マトリックスによって吸収され、水の伸縮モード共鳴を励起OHによる表面からの中性物質の脱離を容易にします。直交エレクトロの荷電液滴に中性パーティションを脱着しESI状4-6の後のイオン化されています。それが交流をするのに役立ちますので、外因性の氷マトリックスの添加は、組織内の内因性の水だけに依存よりも好ましいです異なる組織区画中の水分量の変動をカウントし、組織のイメージング実験において〜15倍7,8によって脱着6を高め、イオン存在度を改善することが示されています。 本研究では、新生児マウス全身で異なる器官全体の代謝物の分布を誘発するためにIR-MALDESI MSIを利用しています。 IR-MALDESI源の調整可能なパラメータの概要が与えられ、そして組織切片を正常に画像化のために必要なステップが示されています。

<table border="0" cellpadding="0" cellspacing="0" width="963">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:283px;">IR-MALDESI Source</td>
			<td style="width:188px;">Custom-made</td>
			<td style="width:171px;">N/A</td>
			<td style="width:323px;">Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development.</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:283px;">Q Exactive Plus&#160;</td>
			<td style="width:188px;">Thermo Scientific</td>
			<td style="width:171px;"></td>
			<td style="width:323px;">Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Water, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH365-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Methanol, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Formic Acid</td>
			<td style="width:188px;">Sigma Aldrich&#160;</td>
			<td style="width:171px;">56302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tunable mid-IR Laser</td>
			<td style="width:188px;">Opotek Inc.</td>
			<td style="width:171px;">IR Opolette</td>
			<td>Tunable 2700-3100 nm IR OPO laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Nitrogen Gas</td>
			<td style="width:188px;">Arc3 Gases</td>
			<td style="width:171px;">AG S-NI300-5.0</td>
			<td>Grade 5.0 high purity nitrogen gas cylinder (300)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">CM 1950</td>
			<td>Cryomicrotome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High Profile Microtome Blades</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td>3802123</td>
			<td>Leica DB80HS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Mounting Medium (OCT)</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">3801480</td>
			<td>Surgipath FSC 22 mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat Specimen Disc</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">14047740045</td>
			<td>40 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Glass Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:171px;">48312-003</td>
			<td>Frosted, selected, pre-cleaned</td>
		</tr>
	</tbody>
</table>

whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)

質量分析（MS）のために、周囲のイオン化源は、過去10年間に多くの関心の対象となっています。マトリックス支援レーザ脱離エレクトロスプレーイオン化（MALDESI）は、このような方法の例であり、マトリックス支援レーザー脱離/イオン化（MALDI）（ 例えば、脱着のパルス性質）及びエレクトロスプレーイオン化（ESI）（ 例えば、ソフトイオン化の特徴）合成されます。 MALDESIの主要な利点の1つは、その固有の汎用性です。 MALDESI実験では、紫外線（UV）又は赤外線（IR）レーザ共振内因性または外因性のマトリックスを励起するために使用することができます。マトリックスの選択は、分析物依存せず、励起に使用されるレーザー波長のみに依存します。 IR-MALDESI実験では、氷の薄層は、エネルギー吸収マトリックスとして試料表面上に堆積されます。 IR-MALDESIソースジオメトリは、液体試料の分析だけでなく、BIOLのための実験の統計的設計（DOE）を使用して最適化されていますogical組織標本。また、強​​固なIR-MALDESI画像源は、調整可能な中間IRレーザはコンピュータ制御XY並進ステージと高分解能質量分析計を用いて同期される場合、開発されています。カスタムグラフィカルユーザインタフェース（GUI）は、レーザの繰返し速度のユーザ選択を可能にする、ボクセル当たりのショット、試料ステージのステップサイズ、及び脱着の間の遅延とソースのイベントをスキャン数。 IR-MALDESIは、繊維と染料と生物組織切片のMSIの法医学的分析のような様々な用途に使用されてきました。内因性代謝物の組織切片内の外因性生体異物の範囲の異なる分析物の分布を測定し、この技術を用いて定量することができます。この原稿で提示プロトコルは、全身の組織切片のIR-MALDESI MSIのために必要な主な手順について説明します。

図4に示す画像は、全身の組織切片で異なる器官における代謝物の空間的な分布を示します。身体の特定の領域に固有のm / z値は、画像生成のためのバッチ処理に続いて、MSiReader PeakFinderを用いて発見されました。イメージオーバーレイツール（ 図3-4）を得られたイオンマップと氷のマトリックスの沈着の前に撮影した光学...

Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

赤外線マトリックス支援レーザー脱離エレクトロスプレーイオン化することにより、全身質量分析イメージング（IR-MALDESI）

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

The overall goal of this protocol is to describe the major steps necessary for IR-MALDESI mass spectrometry imaging of whole body tissue sections. This method can effectively answer key questions in the field of mass spectrometry imaging, such as discerning the spatial distribution of endogenous and exogenous analytes in multiple organs simultaneously. The main advantage of this technique is that it operates at atmospheric pressure and the exogenous ice matrix helps account for variations in water content in different organs.Although this protocol provides insight into a targeted whole body imaging. IR-MALDESI mass spectrometry imaging can also be applied to targeted and quantitative drug distribution analysis. The following protocol describes how to perform IR-MALDESI MSI experiments.In depth details about the optimized geometry of the IR-MALDESI source and it's synchronization with the laser, stage, and mass spectrometer can be found elsewhere. To prepare the specimen, wear protective gloves, and apply a layer of OCT mounting medium onto a 40 millimeter cryostat specimen disc. Then, gently place a frozen whole mouse specimen onto the OCT coated holder and orient it for sectioning.OCT is used solely to adhere the tissue to the specimen disc. Do not completely embed the tissue in the OCT. Place the specimen disc on the Peltier sample holder in the cryostat.Turn on the Peltier and wait 10 minutes for the sample to equilibrate. Then, place the specimen disc inside the disc holder, orient it to slice to the desired plane. Section the specimen at 20 degrees Celsius at, for instance, 25 micorons for whole body analysis.Make use of the anti-roll glass plate and use the cryostat vacuum and brushes to remove unwanted tissue debris. After the tissue is sectioned, cover the blade with a blade guard. Now, orient the frozen section on the specimen plate and thaw mount them onto pre-cleaned glass microscope slides.Bring the slides as close as possible to the sections without touching. Keep the mounted slides inside the cryostat until the IR-MALDESI calibration is complete to maintain the tissues cryopreservation. To begin, turn on the mid-IR laser and launch the laser control application.Then, choose the desired wavelength. IR-MALDESI experiments are typically performed at 2940 nanometers. Next, turn on the stage controller and launch the control program.Using the program, calibrate the stage position using the home button. Next, fill a one milliliter syringe with electrospray solvent and flush the silica capillary. Then, align the ESI emitter with the MS inlet using the source parameters, such as ESI-spot distance, spot-inlet distance, sample height, and solvent flow rate.All of which have been optimized using statistical DOE for analysis in tissue sections. Now start the electrospray and evaluate it's stability for at least 10 minutes. Monitor the total ion current, which at this point, consists solely of ambient compounds.Variation under 10%over a 10-15 minute period indicates the system is stable. After turning off the electrospray and ensuring it is off, transfer a specimen slide from the cryostat to the IR-MALDESI sample plate. Then, stand back from the ESI emitter and restart the electrospray.Close the access door of the IR-MALDESI source and purge the enclosure with dry nitrogen to prevent condensation of water on the tissue. Once the relative humidity inside the enclosure is less than 3%switch on the Peltier plate's DC power supply to about 12 volts to cool the sample plate to 9 degrees Celsius. After about 5-10 minutes, stop the nitrogen purge and open the source access door to expose the tissue to the ambient humidity.A thin layer of ice will form on the stage and mounted tissue section. If the air is dry, place a beaker of warm water inside the enclosure. Now, close the door and restart the nitrogen flow to reduce the humidity to a around 10%Adjust the nitrogen flow as needed to maintain a stable layer of ice on the sample during the experiment.Using the Rastier GUI, move the stage to the analysis position by clicking the LASER Position button. Use the adjustment diode of the laser to ensure an off tissue region will be abladed. Then, fire a test shot to calibrate the laser's offset with respect to the camera view.Click the Laser Test Fire button to update the laser position. Now, return to camera position and update the laser position in Rastier by placing the laser alignment reticle on the laser ablation spot. Then, click the ROI button and select the region of interest.Include some off tissue area to serve as control. Use this off tissue section for peak picking. The spot size of the laser on tissue is about 150 microns.A smaller diameter is possible with over sampling. Now, input the appropriate experimental parameters and name the mass spectrometer imaging file in the project name box. Then, select the appropriate laser frequency from the drop down box.Typically, use two laser pulses per voxel at 20 Hertz to completely desorb the material. Also, set the delay time to 10 milliseconds so the ions have time to reach the mass analyzer. The repetition rate should be set to the laser's maximum.Now, choose mass spectrometer parameters, such as ionization mode, electrospray voltage, solvent flow rate, capillary temperature, and the injection time in the mass spectrometer software. Once all parameters have been set, place the mass spectrometer in handshake mode for synchronization and start the mass spectrometer acquisition. Using the Rastier imaging software, verify that all steps in the checklist have been completed and then load the program.Once the RUN button becomes available, start the MSI signal acquisition. Upon completion of the imaging experiment, stop mass spectrometer acquisition and place the instrument in standby mode. Turn off the nitrogen purge to the enclosure.Turn off the stage controller. Turn off the power supply to the Peltier plate and turn off the laser. Then, open the access door.Remove the eletrospray emitter tip from the inside of the source enclosure and wearing protective gloves, remove the very hot extended metal mass spectrometer inlet. Wearing gloves and safety goggles, clean the inlet using an ultrasonic bath. First, use 15%nitric acid for 10 minutes.Then, use HPLC grade water for 10 minutes and finally, use HPLC grade methanol for 10 minutes. Dry the metal capillary inlet under a stream of nitrogen and reinsert it into the mass spectrometer for future analysis. Spatial distribution of metabolites in different organs of whole body tissue sections were analyzed using the described method.Cholesterol was observed in all tissues because of it's structural role in cell membranes. Other lipid metabolites exhibited distinct distributions within specific organs or tissue compartments. For example, some lipids, such as punitively assigned desmosterol were observed to have a specific localization in the brain.Whereas other lipids exhibited a higher distribution in the spinal cord. And other lipids were uniforming distributed across all organs except for the brain. IR-MALDESI mass spectrometry imaging is a disruptive technology that allows for the relative and absolute quantification of analytes in tissues with minimal sample preparation at atmospheric pressure.Once mastered, all of the sample preparation steps can be done in approximately 30 minutes. A key step in sample preparation is the formation of the ice matrix, which facilitates efficient desorption of tissue related material. The ice matrix also helps account for variations in water content in different organs.In tandem with this protocol, supplementary analysis, such as histological and immunohistochemical staining of the serial section can be performed.

Research

JoVE Journal

Bioengineering

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

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生物工学

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Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

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Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

非正規のアミノ酸の選択圧による 'ゴールデン' 蛍光と質量分析法と蛍光タンパク質解析工学

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While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

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ナノ製剤中のエンドトキシン検出カブトガニ リムラス ライセート (ラル) 試金を使用して

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

癌細胞培養から採取した培地における液体クロマトグラフィー-質量分析法による選択キヌレニンの同時定量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...