Name: in situ characterization of hydrated proteins in water by salvi and tof-sims
Uploaded: 2016-02-15T15:00:00
Description: Watch this Scientific Journal Video about In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS at JoVE.com

We are grateful to the Pacific Northwest National Laboratory (PNNL) Chemical Imaging Initiative-Laboratory Directed Research and Development (CII-LDRD) and Materials Synthesis and Simulation across Scales (MS3) Initiative LDRD fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) Science Themed Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Mr. Xiao Sui, Mr. Yuanzhao Ding, and Ms. Juan Yao for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

1. Rengøring og sterilisering af SALVI Microchannel <ol><li> Sterilisering af mikrokanalen i SALVI <ol><li> Tegn 2 ml 70% ethanol vandig opløsning ind i en sprøjte, tilslut sprøjten med indløbet slutningen af ​​SALVI, og langsomt tilføre 1 ml af væsken i 10 min. Fjern sprøjten i slutningen af ​​injektionen. Dernæst tilslutte ind- og udløb af SALVI bruge en polyetheretherketon (PEEK) union. Alternativt, brug en sprøjtepumpe til at gennemføre den samme procedure. For eksempel indstille strømningshas...

<ol>
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SALVI er en mikrofluid interface, der tillader dynamisk væskeoverflade og væske-faststof-grænsefladen analyse ved vakuum baserede instrumenter, såsom ToF-SIMS og scanningselektronmikroskopi (SEM). På grund af brugen af små åbninger for at eksponere væske direkte i vakuum, SALVI er velegnet til mange fint fokuserede spektroskopi og billedbehandling teknikker uden ændringer; 22 portabilitet og alsidighed mikrofluidik gør det til en sand multimodal imaging platform. Tydelige funktioner og betydning af ...

Hydrering er afgørende for strukturen, 1 konformation, 2 og biologisk aktivitet 3 af proteiner. Proteiner uden vandmolekyler omgiver dem ville ikke have levedygtige biologiske aktiviteter. Specifikt vandmolekyler interagerer med overfladen og interne struktur af proteiner, og forskellige hydrering tilstande af proteiner gør sådanne interaktioner forskellige. 4 Interaktionen af proteiner med faste overflader er et grundlæggende fænomen med implikationer i nanoteknologi, biomaterialer og vævsmanipulering processer. Undersøgelser har længe indikeret, at der kan forekomme konformationelle ændringer som et protein møder en overflade. ToF-SIMS er blevet planlagt som den teknik, der har potentiale til at studere protein-faststof-grænsefladen. 5-7 Det er vigtigt at forstå hydreringen af proteiner på faste overflader, hvilket potentielt giver en grundlæggende forståelse af mekanismen i deres struktur, kropsbygning, og biologiskal aktivitet. Men større overflade analyseteknikker er for det meste vakuum-baserede og direkte ansøgninger for flygtige væskeformige undersøgelser er vanskelig på grund af den hurtige fordampning af flygtige væske under vakuum miljø. Vi udviklede et vakuum kompatibel mikrofluid interface, System til analyse på Liquid Vacuum Interface (SALVI), for at muliggøre direkte observationer af flydende overflader og flydende og faste stoffer interaktioner ved hjælp af time-of-flight sekundær ion massespektrometri (ToF-SIMS). 8- 11 de unikke aspekter omfatter følgende: 1) vinduet påvisning er en åbning på 2-3 um i diameter muliggør direkte afbildning af væskeoverfladen, 2) overfladespænding anvendes til at holde væsken inden i åbningen, og 3) SALVI er bærbare mellem flere analytiske platforme. 11,12 SALVI er sammensat af en siliciumnitrid (SiN) membran som detekteringsområdet og en mikrokanal lavet af polydimethylsiloxan (PDMS). Det er fabricated i rene rum, og fremstilling og centrale design faktorer er blevet beskrevet i tidligere artikler og patenter. 8-12 Ansøgningerne fra ToF-SIMS som et analytisk værktøj blev demonstreret ved hjælp af forskellige vandige opløsninger og komplekse flydende blandinger, nogle af som indeholdt nanopartikler. 13-17 Konkret SALVI flydende ToF-SIMS tillader dynamisk sondering af væske-faststof-grænsefladen af levende biologiske systemer (dvs. biofilm), enkelte celler, og faststof-elektrolyt interface, åbner nye muligheder for in situ kondenseret fase undersøgelser, herunder væsker ved hjælp ToF-SIMS. Men den nuværende udformning ikke tillader gas-væske interaktioner endnu. Dette er en retning for den fremtidige udvikling. SALVI er blevet anvendt til at undersøge den hydratiserede protein film i dette arbejde for første gang. Fibronectin er et almindeligt anvendt protein dimer, der består af to næsten identiske monomerer forbundet af et par disulfidbindinger, 18, ​​som is kendt for sin evne til at binde celler. 19,20 Det blev valgt som et modelsystem til at illustrere, at det hydratiserede proteinfilm kan dynamisk probes under anvendelse af SALVI flydende ToF-SIMS tilgang. Proteinopløsningen blev indført i mikrokanal. Efter inkubering i 12 timer blev en hydreret protein film dannet på bagsiden af ​​SiN membranen. Deioniseret (DI) vand blev anvendt til at skylle kanalen efter protein introduktion. Der blev indsamlet fra hydrerede fibronektin proteinmolekyler i SALVI mikrokanalplade bruge dynamisk ToF-SIMS. DI vand blev også undersøgt som en kontrol til at sammenligne med resultater opnået fra hydreret fibronektin tynd film. Der blev observeret tydelige forskelle mellem den hydratiserede protein film og DI vand. Dette arbejde viser, at protein adsorption på overfladen i den flydende miljø kan studeres ved hjælp af romanen SALVI og flydende ToF-SIMS tilgang. Videoen protokollen er beregnet til at give teknisk vejledning for folk, der er interesseredei at udnytte dette nye analytisk værktøj til forskellige anvendelser af SALVI med ToF-SIMS og reducere unødvendige fejl i væskehåndtering samt ToF-SIMS indsamling og analyse af data.

<table border="0" cellpadding="0" cellspacing="0" width="1170">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">ToF-SIMS</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:121px;">TOF.SIMS 5</td>
			<td style="width:400px;">Resolution: &#62; 10,000 m/&#916;m for mass resolution; &#62; 4,000 m/&#916;m for high spatial resolution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">System for Analysis at the Liquid Vacuum Interface (SALVI)</td>
			<td style="width:260px;">Pacific Northwest National Laboratory</td>
			<td style="width:121px;">N/A</td>
			<td style="width:400px;">SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum-based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">PEEK Union</td>
			<td style="width:260px;">Valco</td>
			<td style="width:121px;">ZU1TPK</td>
			<td>for connecting the inlet and outlet of SALVI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">5 Axes Sample Stage</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:121px;">N/A</td>
			<td>Stage is self-made for mounting SALVI in ToF-SIMS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Barnstead Nanopure Water Purification System</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">D11921</td>
			<td>ROpure LP Reverse Osmosis filtration module (D2716)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:121px;">309659</td>
			<td style="width:400px;">1 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Pipette</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">21-377-821</td>
			<td>Range: 100 to 1000 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Pipette Tip</td>
			<td style="width:260px;">Neptune</td>
			<td style="width:121px;">2112.96.BS</td>
			<td>1000 &#181;L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Centrifuge Tube</td>
			<td style="width:260px;">Corning</td>
			<td style="width:121px;">430791</td>
			<td>15 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Fibronectin</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:121px;">F1141</td>
			<td>1 mg/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Ethanol</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">S25310A</td>
			<td>95% Denatured</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Gibco PBS</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">10010-023</td>
			<td>pH 7.4</td>
		</tr>
	</tbody>
</table>

in situ characterization of hydrated proteins in water by salvi and tof-sims

Dette arbejde viser in situ karakterisering af protein biomolekyler i den vandige opløsning ved hjælp af systemet for analyse på Liquid Vacuum Interface (SALVI) og time-of-flight sekundær ion massespektrometri (ToF-SIMS). Den fibronectin protein film blev immobiliseret på siliciumnitrid (SiN) membran, der danner SALVI detekteringsområdet. Under ToF-SIMS-analyse, blev tre former for analyse udført herunder høj rumlig opløsning massespektrometri, todimensional (2D) billedbehandling, og dybde profilering. Massespektre blev opnået i både positive og negative tilstande. Deioniseret vand blev også analyseret som en referenceprøve. Vores resultater viser, at fibronectin film i vand har flere særskilte og stærkere vand klynge toppe sammenlignet med vand alene. Karakteristiske toppe af aminosyre fragmenter er også observeres i hydreret protein ToF-SIMS spektre. Disse resultater illustrerer, at proteinmolekyle adsorption på en overflade kan studeres Dynamically bruger SALVI og ToF-SIMS i flydende miljø for første gang.

Et par repræsentative resultater præsenteres for at demonstrere fordelene ved den foreslåede protokol. Ved at bruge SALVI mikrofluid interface, kan den primære ionstråle (Bi 3 +) direkte bombardere på det hydratiserede fibronectin film i DI vand. Således molekylære kemiske kortlægning af væskeoverfladen held kan erhverves. Figur 1a og 1b viser den positive ToF-S...

Watch this Scientific Journal Video about In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS at JoVE.com

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Dette arbejde viser en protokol til flydende håndtering og indføring af prøve til en mikrokanal til in situ time-of-flight sekundær ion massespektrometri-analyse af biomolekyler i en vandig opløsning.

In situ karakterisering af Hydratformerne Proteiner i vand ved Salvi og ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

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JoVE Journal

Chemistry

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Submillisecond konformationsændringer i Proteiner løst ved Photothermal Beam Nedbøjning

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

In Situ SIMS og IR-spektroskopi veldefinerede Overflader Udarbejdet af Soft Landing af Mass udvalgte ioner

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Vand i olie Emulsioner: Et nyt system til samling Vandopløselige klorofyl-bindende proteiner med Hydrofobe Pigmenter

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Vurdering af bordoteret Diamond Elektrode Kvalitet og Ansøgning til<em&gt; In Situ</em&gt; Ændring af lokal pH ved vandelektrolyse

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Radioaktiv mærkning og kvantificering af cellulære niveauer af phosphoinositider af højtryksvæskekromatografi-koblede Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

In Situ Karakterisering af Shewanella oneidensis MR1 biofilm af SALVI og ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In Situ Karakterisering af Boehmite partikler i vandet ved hjælp af flydende SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

DNA-magnetiske partikel bindende analyse af dynamiske og elektroforese lysspredning

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

Hurtig Nanoprobe Signal forstærkning af In Situ guld nanopartikel syntese

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of FÃƒÆ’Ã‚Â¢Ãƒâ€¹Ã¢â‚¬Â '

Photoelectron billeddannelse af anioner illustreret ved 310 Nm udstationering af F−

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...