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.

Not: Aşağıdaki protokol IR-MALDESI MSI deneyler için gerekli tüm adımları açıklamaktadır. Derinlemesine optimize IR-MALDESI kaynağı geometrisi ve lazer sahnede ile senkronizasyon ve kütle spektrometresi ile ilgili ayrıntılar başka bir yerde 5,6 bulunabilir. Bu protokolde kullanılan hayvan doku örnekleri Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) ve North Carolina State University düzenlemelerine göre elde edilmiştir. 1. Doku Hazırlanması <ol>...

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protokol üzerinde bir IR-MALDESI MSI deneyi gerçekleştirmek için önemli adımlar açıklanır. Matris uygulama işlemi (bölüm 3), bir robot püskürtücü kullanarak kaplama sprey süblimasyon veya MALDI MSI deneyleri için tipik bir matris uygulaması işlemine benzer, yaklaşık 20 dakika alır. Ayrıca, IR-MALDESI matris kristalleri 6 içine analitlerin bölünmesi dayanmaz ve buz matrisi evrensel bakılmaksızın kitle, boyut, ya da kimyasal özelliklerinin tüm analitler için de kullanılabilir. ...

mikroprob modunda Kütle spektrometrisi görüntüleme (MSI) bir numunenin yüzeyi üzerinde ayrı ayrı konumlarda bir kiriş (lazer veya iyonlar) bir yüzeyinden numunenin geri çekilmesine içerir. Her bir tarama noktasında, bir kütle spektrumu oluşturulur ve elde edilen spektrum, toplandıkları olan uzamsal konumu ile birlikte, eş zamanlı olarak numune içinde çok sayıda analiti eşlemek için kullanılabilir. Duyarlılık ve kütle spektrometresi özgüllüğü bağlanmış görüntüleme Bu etiket serbest bir şekilde MSI kütle spektrometresi 1,2 en hızlı gelişen alanlarından biri haline gelmesine yardımcı oldu. Matris destekli lazer desorpsiyon / iyonizasyon (MALDI) MSI, analizler için en yaygın olarak kullanılan iyonizasyon yöntemidir. Bununla birlikte, bir organik matris ihtiyacı ve MALDI vakum şartları tekrarlanabilirlik, örnek hacmi ve yöntemi kullanılarak analiz edilebilir numune türleri önemli sınırlamalar getirmektedir. atmosfer basıncı (AP) io bir dizizasyon yöntemleri, bu kısıtlamaları 3 aşmak için son yıllarda geliştirilmiştir. Bu ortam iyonizasyon yöntemleri doğal duruma daha yakındır ve analiz öncesinde örnek hazırlama adımları basitleştirmek bir ortamda biyolojik numunelerin analizi için izin verir. Matris destekli lazer desorpsiyon iyonizasyon elektrosprey (MALDESI) bu tür bir iyonizasyon yöntemi, 4,5 örneğidir. IR MALDESI deneylerde, buz ince bir tabaka enerji emici matris olarak doku yüzeyi üzerine bırakılır. Bir orta-IR lazer darbeli buz matris tarafından emilir ve su modu uzanan rezonant verici OH ile yüzeyinden doğal malzemelerin geri çekilmesine kolaylaştırır. Bir dik elektropüskürtmeyle tahsil damlacıkları ve içine desorbe nötr bölümü sonrası iyonize bir ESI gibi moda 4-6 bulunmaktadır. eksojen buz matris eklenmesi bu ac yardımcı beri dokuda endojen su dayanmak tercih edilirfarklı doku bölümlerinde su miktarındaki değişimleri sayısı, ve • doku görüntüleme deneylerinde 7,8 15 misli iyon bolluğu desorpsiyon 6 geliştirmek ve iyileştirmek için gösterilmiştir. Bu çalışmada, IR-MALDESI MSI yenidoğan fare bütün vücutta değişik organlarda genelinde metabolitleri dağılımını ortaya çıkarmak için kullanmaktadır. IR-MALDESI kaynağı ayarlanabilir parametreleri bir bakış verilir ve doku kesitlerinin başarılı görüntüleme için gerekli adımlar gösterilmektedir.

<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)

kütle spektrometresi (MS) için ortam iyonizasyon kaynakları, son on yıl içinde çok ilgi konusu olmuştur. Matris destekli lazer desorpsiyon elektrosprey iyonizasyon (MALDESI) bu tür yöntemler, örnek olarak burada matris destekli lazer desorpsiyon / iyonizasyon (MALDI) (ör desorpsiyonu darbeli) VE elektrosprey iyonizasyon (ESI) (örneğin, yumuşak iyonizasyon özellikleri ) birleştirilir. MALDESI önemli avantajlarından biri, kendine özgü çok yönlülük olduğunu. MALDESI deneylerde, bir ultraviyole (UV) ya da kızıl ötesi (IR), lazer rezonant bir endojen veya eksojen matris uyarmak için kullanılabilir. matrisin seçimi bağlıdır analit değildir ve uyarma için kullanılan lazer dalga boyu sadece bağlıdır. IR MALDESI deneylerde, buz ince bir tabaka, bir enerji emici matris numunenin yüzeyi üzerinde bırakılır. IR-MALDESI kaynak geometri, istatistiksel sıvı numunelerin analizi için deneyler (DOE) tasarımı yanı sıra biol kullanılarak optimize edilmiştirogical doku örnekleri. Ayrıca, güçlü bir IR MALDESI görüntüleme kaynağı ayarlanabilir bir orta-IR lazer bilgisayar kontrollü bir XY öteleme aşamasında ve yüksek çözme gücü kütle spektrometresi ile senkronize edilir burada geliştirilmiştir. Özel bir grafik kullanıcı arayüzü (GUI) lazer tekrarlama oranı kullanıcı seçimine izin verir, voksel başına çekim, örnek aşamasının adım büyüklüğü ve desorpsiyon arasındaki gecikme ve kaynak olayları tarama sayısı. IR MALDESI bir biyolojik doku bölümlerinin elyaflar ve boyalar ve MSI adli analizler gibi uygulamalar çeşitli kullanılmıştır. endojen metabolitlerin doku bölümlerinde eksojen ksenobiyotiklerin kadar farklı analitler dağılımı ölçüldü ve bu tekniği kullanılarak belirlenebilir. Bu yazıda sunulan protokol tüm vücut doku kesitlerinde IR-MALDESI MSI için gerekli önemli adımları açıklar.

Şekil 4&#39;te gösterilmiş görüntüler tüm vücut doku bölümünde farklı organlarda metabolitlerin dağılımını göstermektedir. Vücudun belirli bölgelerine benzersiz m / z değerleri bazı görüntü üretimi için toplu işlem, ardından MSiReader PeakFinder kullanılarak bulundu. Görüntü bindirme aracı (Şekil 3-4) elde edilen iyon haritaları ile buz matriks birikimi öncesi alınan optik görüntü hizalamak için kullanı...

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.

Kızılötesi Matriks destekli lazer desorpsiyon Elektrosprey İyonizasyon tarafından tüm vücut Kütle Spektrometre Görüntüleme (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)

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