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. Rengjøring og sterilisering av SALVI Mikro <ol><li> Sterilisering av Mikro i SALVI <ol><li> Tegn 2 ml av 70% etanol, vandig løsning i en sprøyte, kobler sprøyten med innløpsenden av SALVI, og langsomt injisere 1 ml av væsken i 10 min. Fjern sprøyten ved slutten av injeksjonen. Deretter kobler innløp og utløp av SALVI bruke en polyetereterketon (PEEK) union. Alternativt bruke en sprøytepumpe til å gjennomføre samme prosedyre. For eksempel sette flyten hastigheten 100 mL / min. </li><li> Hold SALVI microc...

<ol>
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SALVI er en microfluidic grensesnitt som lar dynamisk væskeoverflaten og væske-solid grensesnitt analyse av vakuumbaserte instrumenter, for eksempel TOF-SIMS og scanning elektronmikroskopi (SEM). På grunn av bruken av små åpninger for å utsette væsken direkte i vakuum, er SALVI egnet for mange fint fokuserte spektroskopi og bildeteknikker uten noen modifikasjoner, 22 portabilitet og allsidighet MicroFluidics gjør det til en sann multimodal bildebehandling plattformen. Forskjellige funksjoner og betydn...

Hydrering er avgjørende for konstruksjonen, en konformasjon, 2 og biologisk aktivitet tre av proteiner. Proteiner uten vannmolekyler som omgir dem ville ikke ha livskraftige biologiske aktiviteter. Nærmere bestemt vannmolekyler kommuniserer med overflaten og indre struktur av proteiner, og forskjellige hydrering tilstander av proteiner gjøre slike interaksjoner tydelig. 4 Interaksjonen mellom proteiner med faste overflater er en grunnleggende fenomen med implikasjoner i nanoteknologi, biomaterialer og vev tekniske prosesser. Studier har lenge indikert at konformasjonelle forandringer kan oppstå som et protein oppstår en overflate. TOF-SIMS har blitt tenkt som den teknikk som har potensial til å studere protein-faststoffgrenseflaten. 5-7 er det viktig å forstå hydrering av proteiner på faste overflater, noe som potensielt gir en grunnleggende forståelse av mekanismen for deres struktur, konformasjon og biologiskal aktivitet. Men hovedflate analytiske teknikker er for det meste vakuumbasert og direkte applikasjoner for flyktige flytende studier er vanskelig på grunn av den hurtige fordampning av flyktig væske under vakuum miljø. Vi utviklet et vakuum kompatibel microfluidic grensesnitt, System for analyse i Liquid Vacuum Interface (SALVI), for å muliggjøre direkte observasjoner av flytende overflater og væske solid samhandling ved hjelp av time-of-flight sekundær ion massespektrometri (TOF-SIMS). 8- 11 den unike aspekter omfatter følgende: 1) deteksjonsvinduet er en åpning på 2-3 mikrometer i diameter tillater direkte avbildning av væskeoverflaten, 2) overflatespenningen brukes til å holde væsken i åpningen, og 3) er SALVI portable mellom flere analytiske plattformer. 11,12 SALVI er sammensatt av et silisiumnitrid (SiN) membran som registreringsområdet, og en microchannel fremstilt av polydimetylsiloksan (PDMS). Det er fabricated i rent rom, og fabrikasjon og nøkkelen design faktorer har blitt beskrevet i tidligere artikler og patenter. 8-12 Søknadene av TOF-SIMS som et analytisk verktøy ble demonstrert ved hjelp av en rekke vannløsninger og komplekse flytende blandinger, noen av som inneholdt nanopartikler. 13-17 Nærmere bestemt tillater SALVI væske TOF-SIMS dynamisk sondering av væske-faststoffgrenseflaten av levende biologiske systemer (dvs. biofilm), enkeltceller, og faststoff-elektrolytt-grensesnittet, åpner nye muligheter for in situ kondensert fase studier inkludert væsker ved hjelp av TOF-SIMS. Men den nåværende utforming ikke tillate gass-væske interaksjoner ennå. Dette er en retning for fremtidig utvikling. SALVI har blitt brukt til å studere det hydratiserte proteinet filmen i dette arbeidet for første gang. Fibronektin er et vanlig brukt protein dimer, som består av to nesten identiske monomerer forbundet av et par av disulfidbindinger, 18, ​​som jegs som er kjent for sin evne til å binde celler. 19,20 Det ble valgt som et modellsystem for å illustrere at det hydratiserte proteinet filmen kan være dynamisk analysert ved hjelp av SALVI flytende TOF-SIMS tilnærming. Proteinløsningen ble innført i microchannel. Etter inkubering i 12 timer, et hydratisert proteinfilm dannet på baksiden av SiN membranen. Deionisert (DI) vann ble anvendt for å skylle av kanalen etter at proteinet innledning. Informasjon ble samlet inn fra hydrerte fibronektin proteinmolekyler i SALVI microchannel bruker dynamisk TOF-SIMS. DI vann ble også undersøkt som en kontroll for å sammenligne med resultatene fra hydrert fibronektin tynn film. Det ble ikke observert tydelige forskjeller mellom hydrert protein film og DI vann. Dette arbeidet viser at protein adsorpsjon på overflaten i væsken miljøet kan studeres ved hjelp av romanen SALVI og væske TOF-SIMS tilnærming. Videoen protokollen er ment å gi teknisk veiledning for folk som er interesserti å utnytte denne nye analyseverktøy for ulike anvendelser av SALVI med TOF-SIMS og redusere unødvendige feil i væskehåndtering samt TOF-SIMS datainnsamling og analyse.

<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 arbeidet viser in situ karakterisering av protein biomolekyler i vandig løsning ved hjelp av System for analyse i Liquid Vacuum Interface (SALVI) og time-of-flight sekundær ion massespektrometri (TOF-SIMS). Den fibronektin protein Filmen ble immobilisert på silisiumnitrid (SiN) membran som danner SALVI registreringsområdet. Under TOF-SIMS-analyse, ble tre modi for analyse utført inkludert høy romlig oppløsning massespektrometri, to-dimensjonale (2D) avbildning, og dybde profilering. Massespektra ble kjøpt i både positive og negative moduser. Avionisert vann ble også analysert som en referanseprøve. Våre resultater viser at fibronektin filmen i vann har flere distinkte og sterkere vann cluster topper sammenlignet med vann alene. Karakteristiske toppene aminosyre fragmenter er også observerbare i hydrert protein TOF-SIMS spektra. Disse resultatene illustrerer at proteinmolekyl adsorpsjon på en overflate kan studeres Dynamically hjelp SALVI og TOF-SIMS i væsken miljø for første gang.

Et par representative resultater er presentert for å demonstrere fordelene med den foreslåtte protokoll. Ved hjelp av SALVI mikrofluidgrensesnittet, kan primær ionestrålen (Bi 3 +) direkte bombardere på det hydratiserte fibronektin filmen i DI-vann. Således molekyl kjemisk kartlegging av væskeoverflaten kan med hell ervervet. 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 arbeidet viser en protokoll for væskehåndtering og prøve innføring i en microchannel for in situ time-of-flight secondary ion mass spectrometry analyse av protein biomolekyler i en vandig oppløsning.

In Situ Karakterisering av Hydratiserte Proteiner i vann 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 konformasjonsendring Endringer i Proteiner løst ved Photothermal bjelkenedbøying

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 av godt definerte overflater som er Soft Landing av Mass-utvalgte 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

Vann i olje emulsjoner: Et nytt system for Montering Vannløselige Klorofyll 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 av Boron Doped Diamond elektrode Kvalitet og søknad til<em&gt; In Situ</em&gt; Endring av lokal pH ved vannelektrolyse

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

Radiomerking og Kvantifisering av Cellular Nivåer av Phosphoinositides ved HPLC-coupled 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 av Shewanella oneidensis MR1 biofilm 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 av Boehmite partikler i vann med flytende 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 partikler bindende analyse av dynamisk og Electrophoretic 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

Rask Nanoprobe Signal forsterkning av i Situ gull hydrogenion 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 avbildning av anioner illustrert av 310 Nm avdeling av 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 ...