Name: maldi-mass spectrometric imaging for the investigation of metabolites in medicago truncatula root nodules
Uploaded: 2014-03-05T17:30:00
Description: Watch this Scientific Journal Video about MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules at JoVE.com

Forfatterne vil gerne anerkende Dr. Jean-Michel ane på Institut for Agronomi på UW-Madison for at give Medicago truncatula prøver. Dette arbejde blev støttet delvist af midler fra National Science Foundation (NSF) tilskud CHE-0.957.784, University of Wisconsin Graduate School og Wisconsin Alumni Research Foundation (WARF) og Romnes Faculty Research Fellowship program (LL). EG anerkender en NSF Graduate Research Fellowship (DGE-1.256.259).

1.. Instrumentering <ol><li> MALDI-TOF/TOF MSI. Brug et massespektrometer udstyret med et MALDI kilde til analyse af små molekyler (se tabel of Materials / udstyr). Udfør opkøb i positiv eller negativ ion-mode afhængigt af analysand. Angiv en masse afstanden af interesse og indsamle 500 laser shots / plet på 50 um intervaller i både x-og y-dimensioner hen over overfladen af prøven for at generere ion billeder. Raster bredde og antal laserskud kan justeres for at opnå højere rumlig opløsning og maksi...

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Som diskuteret ovenfor, prøveforberedelse er den mest afgørende skridt i MSI workflow. Embedding vævet ujævnt vil forårsage skæring at være vanskeligt eller ikke er mulig i nogle tilfælde. Afsnittet størrelse og tilstrækkelig Ekvilibreringstiden er afgørende for at opretholde vævet integritet og undgå foldning og tårer. Udvælgelse af matrix og anvendelse teknik vil spille en rolle i at bestemme typerne af analytter skal påvises, den rumlig opløsning, og reproducerbarhed af resultaterne. Ved hjælp af en...

Den voksende inden for metabolomics har mange vigtige biologiske applikationer, herunder biomarkører, decifrere metaboliske veje i planter og andre biologiske systemer, og toksikologi profilering 4,6-10. En stor teknisk udfordring, når man studerer biologiske systemer er at studere Metabolomic veje uden at forstyrre dem 11. MALDI-MSI tillader direkte analyse af intakte væv, der muliggør sensitiv påvisning af analytter i enkelte organer 12,13 og endda enkelte celler 14,15. Prøveforberedelse er et afgørende skridt i at producere reproducerbare og pålidelige massespektrale billeder. Kvaliteten af ​​billederne i høj grad afhænger af faktorer såsom væv indstøbningsmedium, skivetykkelse, MALDI matrix og matrix ansøgning teknik. Til programmer til billedbehandling, ideel godstykkelse er bredden af ​​en celle (8-20 um afhængig af prøvetype). MALDI kræver aflejring af en organisk, crystalline matrix sammensatte, typisk en svag syre, på prøven for at hjælpe analysanden ablation og ionisering. 16 forskellige matricer giver forskellige signalintensiteter, interfererende ioner og ionisering effektivitetsgevinster af forskellige klasser af forbindelser. Matrixen påføringsteknikken spiller også en rolle i kvaliteten af ​​massespektrale billeder og forskellige teknikker er egnede til forskellige klasser af analytter. Tre matrix påføringsmetoder præsenteres i denne protokol: airbrush, automatisk sprøjte, og sublimering. Airbrush matrix ansøgning er blevet meget udbredt i MALDI billeddannelse. Fordelen airbrush matrix anvendelse er, at det er relativt hurtigt og nemt. Men kvaliteten af airbrush matrix ansøgning i høj grad afhænger af dygtighed af brugeren og har tendens til at være mindre reproducerbar og forårsage diffusion af analytter, især små molekyler 17. Automatiske sprøjtende systemer har lignende mekanik til airbrush matrix ansøgning, men HAVe blevet udviklet til at fjerne variation ses med manuel airbrush ansøgning, hvilket gør spray mere reproducerbare. Denne metode kan nogle gange være mere tidskrævende end traditionel airbrush matrix ansøgning. Både manuel airbrush og automatiske sprøjtende systemer er solvent baserede matrix anvendelsesmetoder. Sublimation er en tør matrix ansøgning teknik, der bliver mere og mere populært for massespektrumdata billeddannelse af metabolitter og små molekyler, fordi det mindsker analyt diffusion, men mangler det opløsningsmidlet nødvendigt at ekstrahere og observere højere masse forbindelser 18. Trygge identifikation af metabolitter kræver typisk nøjagtige massemålinger at opnå formodede identifikationer efterfulgt af tandem masse (MS / MS) eksperimenter til validering, med MS / MS spektre blive sammenlignet med standarder, litteratur eller teoretiske spektre. I denne protokol høj opløsning (masse opløsningsevne på 60.000 ved m / z 400), væskekromatografi (LC)-MS er koblet til MALDI-MSI at opnå både geografisk information og tillidsfulde identifikationer af endogene metabolitter, ved hjælp af Medicago truncatula rødder og rodknolde, som det biologiske system. MS / MS eksperimenter kan udføres direkte på væv med MALDI-MSI eller på vævsekstrakter med LC-MS og anvendes til validering af metabolit identifikationer. Denne protokol giver en enkel metode til at kortlægge endogene metabolitter i M. truncatula, som kan tilpasses og anvendes til MSI af små molekyler i forskellige vævstyper og biologiske systemer.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Gelatin</td>
			<td style="width:181px;">Difco</td>
			<td style="width:163px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=214340">214340</a></td>
			<td style="width:611px;">heat to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat- HM 550</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:163px;">956564A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">indium tin oxide (ITO)-coated glass slides&#160;</td>
			<td>Delta Technologies</td>
			<td>CB-90IN-S107</td>
			<td>25-75-0.8 mm (width-length-thickness)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,5-Dihydroxybenzoic acid (DHB) matrix&#160;</td>
			<td>ICN Biomedicals</td>
			<td><a href="http://www.labsource.com/Catalog/Item.aspx?ItemID=1787195">PI90033</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Airbrush</td>
			<td>Paasche Airbrush Company</td>
			<td>TG-100D</td>
			<td>coupled with 75 ml steel container</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Automatic matrix sprayer system- TM-Sprayer</td>
			<td>HTX Technologies, LLC</td>
			<td style="width:163px;">HTX.TMSP.H021-U</td>
			<td>Specific start-up and shut-down instructions will be given when the instrument is installed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sublimation apparatus&#160;</td>
			<td>Chemglass Life Science</td>
			<td style="width:163px;"><a href="http://www.chemglass.com/product_view.asp?pnr=CG-3038">CG-3038-01</a></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vaccum pump- Alcatel 2008 A</td>
			<td>Ideal Vacuum Products</td>
			<td>P10976</td>
			<td>Ultimate Pressure = 1x10-4 Torr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultrafleXtreme MALDI-TOF/TOF</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td style="width:163px;">276601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">FlexImaging</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td>269841</td>
			<td>One example of &#34;vender specific software&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">MALDI LTQ Orbitrap</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMASZ</td>
			<td>High resolution MALDI instrument for accurate mass measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Q Exactive</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMAZR</td>
			<td>High resolution LC-MS instrument for accurate mass measurements</td>
		</tr>
	</tbody>
</table>

maldi-mass spectrometric imaging for the investigation of metabolites in medicago truncatula root nodules

De fleste teknikker, der anvendes til at studere små molekyler, såsom lægemidler eller endogene metabolitter, beskæftiger vævsekstrakter som kræver homogenisering af vævet af interesse, der potentielt kan forårsage ændringer i de metaboliske veje blive undersøgt 1. Massespektrometrisk imaging (MSI) er et kraftfuldt analytisk redskab, der kan give geografisk information af analytter i intakte skiver af biologiske vævsprøver 1-5. Denne teknik er blevet brugt i udstrakt grad til at studere forskellige typer af forbindelser, herunder proteiner, peptider, lipider, og små molekyler, såsom endogene metabolitter. Med matrix-assisteret laser desorption / ionisering (MALDI)-MSI, kan rumlige fordelinger af flere metabolitter samtidigt opdaget. Heri er en metode udviklet specielt til at gennemføre målrettede metabolomics MSI eksperimenter på bælgplanter rødder og rodknolde præsenteret som kan afsløre indsigt i de biologiske processer, der finder sted. Denmetode præsenteres her viser en typisk MSI workflow, fra prøveforberedelse til billede erhvervelse, og fokuserer på matricen ansøgning trin, viser flere matrix ansøgning, som er nyttige til påvisning af små molekyler. Når MS billederne er genereret, er analyse og identifikation af metabolitter af interesse diskuteret og demonstreret. Standardarbejdsgangen præsenteret her, kan let modificeres til forskellige vævstyper, molekylære arter og instrumentering.

En eksperimentel oversigt over MSI er vist i figur 1. Ved selve begyndelsen af ​​eksperimentet prøveforberedelse er et kritisk trin. Knuder er trimmet fra planten rod og indlejret i gelatine. Vævet skal trykkes fladt mod kryostat kop, med ingen bobler, mens det bliver frosset, hvilket vil sikre en lettere og korrekt tilpasning af vævet, mens den bliver sektioneret. Når væv bliver skåret, er det vigtigt at skære vævet ved korrekt tykkelse for tynd sektioner vil rive, ødelægger vævet integr...

Watch this Scientific Journal Video about MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules at JoVE.com

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Massespektrometrisk imaging (MSI) er et kraftfuldt værktøj, der kan bruges til at opdage og identificere forskellige kemiske stoffer i intakte væv, bevare de forbindelser i deres oprindelige miljøer, som kan give ny indsigt i biologiske processer. Heri beskrives en MSI metode udviklet til analyse af små molekyler er beskrevet.

MALDI-Massespektrometrisk Imaging for undersøgelse af metabolitter i<em&gt; Medicago truncatula</em&gt; rodknolde

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Research

JoVE Journal

Biology

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

Den Ex vivo Isoleret Skeletal mikrokardannelse Forberedelse til undersøgelse af vaskulær reaktivitet

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Levende Imaging af Apoptotisk Cell Clearance under<em&gt; Drosophila</em&gt; Embryogenese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Purification of Transcripts and Metabolites from Drosophila Heads

Purification of Transcripts and Metabolites from Drosophila Heads

Oprensning af udskrifter og metabolitter fra<em&gt; Drosophila</em&gt; Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synkronisering af<em&gt; Caulobacter Crescentus</em&gt; Til undersøgelse af bakteriecellen Cycle

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Kontraktilitet Målinger på isolerede papillærmuskler for undersøgelse af Cardiac inotropi i mus

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

MRM Microcoil Performance Kalibrering og brug demonstreret på Medicago truncatula Rødder på 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Prøveforberedelse til metabolisk profilering ved hjælp af MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

Divergens af Root Microbiota i forskellige levesteder baseret på vægtede korrelationsnetværk

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Isolering af hjerte- og vaskulære glatte muskelceller fra voksne, unge, larve- og embryonale zebrafisk til elektrofysiologiske undersøgelser

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...