Name: time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics
Uploaded: 2017-04-17T12:30:00
Description: Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

We gratefully acknowledge Dr. Markus Zweckstetter for providing the pdb coordinate file for the 'native' tau ensemble predicted from his NMR work, with contributed analysis tools provided by Dr. Adnan Sljoka. Funding for this work was provided by the Natural Science and Engineering Research Council of Canada (NSERC) ENGAGE Grant program.

BEMÆRK: Venligst høre alle relevante materiale sikkerhedsdatablade (MSDS) før brug. Gassen, laserablation af poly (methylmethacrylat) (PMMA) kan være giftige. Vær sikker på, at laseren gravør er sluttet til en fungerende ventilationssystem. Brug alle passende sikkerhedsforanstaltninger, når bygningen mikrofluidapparatet herunder anvendelse af tekniske kontroller (stinkskab, kanylebøtte) og personlige værnemidler (sikkerhedsbriller, ansigtsmaske, handsker, kittel, fuld længde bukser, lukket-toe sko). Det er yd...

<ol>
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Mens strukturelle biologi fremgangsmåder såsom røntgenkrystallografi og NMR er fordelagtige, fordi de giver ekstremt detaljerede strukturer af proteiner, disse billeder er ofte statisk. Karakteriseringen af ​​transiente arter og svagt strukturerede domæner fortsat undvigende når undersøgt ved disse konventionelle fremgangsmåder. Derfor, for at få dynamiske indsigt på disse typer af systemer er det vigtigt at arbejde på hurtige tidsskalaer. Vi har med succes anvendt Tresi-HDX-MS for at få detaljerede indbl...

I de sidste adskillige årtier har betydelige fremskridt er gjort i udvikling af analytiske metoder, der skal måle proteinstruktur og dynamik 1, 2, 3, 4. Mens røntgenkrystallografi forbliver princippet værktøj til bestemmelse proteinstruktur der høje koncentrationer af protein nødvendig og omfattende optimering er nødvendig for at fremstille krystaller af diffraktionskvalitet. Proteiner, som er vanskelige at krystallisere, såsom membranassocierede og uløseligt uordnede proteiner er klassisk blevet undersøgt af hydrogen-deuterium udveksling (HDX) NMR 5. Men i de seneste årtier, kobling af elektrospray-massespektrometri (ESI-MS) til HDX har hurtigt vundet popularitet 6, 7. Massespektrometri tilbyder en løsningtil mange af de begrænsninger, der er forbundet med røntgenkrystallografi og NMR. Især MS er meget følsom (nM til uM nødvendige koncentrationer), og der er næsten ingen grænse for protein størrelse. Desuden høj arbejdscyklus på MS-analyse giver mulighed for at studere proteiner som de undergår enzymatisk omsætning, misfoldning, kompleksdannelse og andre biologisk relevante processer. Disse processer opstår ofte på millisekund til anden tidsskala og kræver hurtig blanding af reagenser inden analyse. Udviklingen af ​​tidsopløst elektrosprayionisering (Tresi) af Wilson og Konermann i 2003 tilladt reaktioner, der skal overvåges i pseudo-realtid ved ESI-MS. Deres setup inkorporeret en kapillær mixer med en kontinuerligt regulerbar reaktion kammervolumen 8. Anordningen består af to koncentriske kapillærer, med den indre kapillar forseglet og et hak skåret i siden for at muliggøre blanding inden for det snævre inter-kapillary plads fra indhakket til enden af ​​den indre kapillar (typisk 2 mm). Ved påføring på HDX eksperimenter, den indre kapillar bærer proteinet af interesse, den ydre kapillar bærer mærkningen D2O opløsning, som derefter undergår blanding med proteinet før indtastning justerbare reaktionskammer tillader HDX mærkning forud for direkte overførsel til ESI kilde. Kort fortalt HDX afhængig af backbone amide hydrogener undergår udveksling med deuteriumatomer i opløsning 9, 10. Udvekslingen er basekatalyseret ved fysiologisk pH, med syre-katalyse bliver fremherskende ved pH under ca. 2,6. Den vekselkurs er baseret på fire hovedfaktorer: pH, temperatur, opløsningsmiddel tilgængelighed og intramolekylær hydrogenbinding. Som de tidligere to forhold holdes konstant under hele forsøget, den vekselkurs, især ved peptidskeletmodifikationer amid stillinger, primært dependent på proteinstruktur 11. Stramt foldet regioner med omfattende, stabile hydrogenbindende netværk i a-helixer og p-sheets vil tage op deuterium ved væsentligt langsommere hastigheder sammenlignet med løkker og uordnede områder (og undertiden slet ikke) 12. Dette giver mulighed for global proteinanalyse, hvor forstyrrelser i struktur (f.eks., Upon aggregering eller substratbinding) medføre afvigende deuterium optagelse (figur 1). Den kinetiske kapillære mixer kan inkorporeres i en mikrofluid platform med et proteolytisk kammer til lokalisering af deuterium-optagelse. Denne proteolytiske kammer holdes ved lav pH for effektivt standse udskiftningsreaktion, og kræver en immobiliseret sur protease for at fordøje proteinet i lokaliserede peptider (figur 2). Overvågning rygrad udveksling på millisekund til anden tidsskalaer er især vigtigt for dekarakterisering af konformationsændringer inden vanskeligt at karakterisere sløjferegioner, smeltede kugler og uløseligt uordnede proteiner (IDP) 13, 14. Alternativt kan Tresi-HDX også anvendes til at karakterisere proteiner, som i øjeblikket ikke har en løst atomare struktur gennem fremgangsmåderne ifølge røntgenkrystallografi og NMR under anvendelse af deuterium udveksling koblet til Corex algoritme (DX-Corex) tilgang 15, 16. Denne detaljerede protokol vil gælde Tresi-HDX at studere tau, en IDP, i både det oprindelige form såvel som det er patogene hyperphosphoryleret tilstand. Mens indfødte tau er en af de mest godt studerede internt fordrevne, der er lidt kendt om dens amyloidogent modstykke 13.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:244px;">Poly(methyl methacrylate) or PMMA</td>
			<td style="width:136px;">Professional Plastics</td>
			<td style="width:145px;">SACR.250CCP</td>
			<td style="width:159px;">8.9 cm x 3.8 cm x 0.6 cm</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Fused Silica Glass Capillary</td>
			<td style="width:136px;">Polymicro Technologies</td>
			<td style="width:145px;">106815-0018</td>
			<td style="width:159px;">ID: 75&#181;m, OD: 150&#181;m</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Metal Capillaries</td>
			<td style="width:136px;">McMaster-Carr</td>
			<td style="width:145px;">28 ga &#8211; 89875K97 
			30 ga&#160; - 89875K99</td>
			<td style="width:159px;"></td>
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		<tr height="52">
			<td height="52" style="height:52px;width:244px;">Fluorinated Ethylene Propylene (FEP) Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">1477 
			1548</td>
			<td style="width:159px;">ID: 0.007&#8221;, OD: 1/16&#8221; 
			ID: 0.020&#8221;, OD: 1/16&#8221;</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Standard Polymer Tubing Cutter</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">A-327</td>
			<td style="width:159px;">for 1/16&#8221; and 1/8&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micro Static Mixing Tee</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">M-540</td>
			<td style="width:159px;">for 1/16&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">or</td>
			<td style="width:136px;"></td>
			<td style="width:145px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Stainless Steel Tee, 0.25mm Bore</td>
			<td style="width:136px;">Valco Instruments Co., Inc. (VICI)</td>
			<td style="width:145px;">ZT1C</td>
			<td style="width:159px;">for 1/16&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PEEK Tee for 1/16&#8221; OD Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">P-727</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">10-32 Female to Female Luer</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">P-659</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">10-32 PEEK Double-Winged Nut</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">F-300</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ferrule for 1/16&#8221; OD Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">F-142</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">100 Series Rotary Tool</td>
			<td style="width:136px;">Dremel</td>
			<td style="width:145px;">F013010001</td>
			<td style="width:159px;"></td>
		</tr>
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			<td height="20" style="height:20px;width:244px;">Cut-Off Discs</td>
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	</tbody>
</table>

time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics

Intrinsisk uordnede proteiner (IDP) har længe været en udfordring at strukturelle biologer på grund af deres mangel på stabile sekundære strukturelementer. Hydrogen-Deuterium Exchange (HDX) målt ved hurtige tidsskalaer er unikt egnet til at detektere strukturer og hydrogenbindende netværk, der er kort befolkede, hvilket muliggør karakteriseringen af ​​forbigående konformere i native ensembler. Kobling af HDX til massespektrometri tilbyder flere vigtige fordele, herunder høj følsomhed, lavt prøve og ingen begrænsning på protein størrelse. Denne teknik har avancerede kraftigt i de sidste mange årtier, herunder evnen til at overvåge HDX mærkningsordninger gange på millisekund tidsskala. Desuden ved at inkorporere HDX arbejdsgang på en mikrofluid platform huser en sur protease mikroreaktor, er vi i stand til at lokalisere dynamiske egenskaber på peptidet niveau. I denne undersøgelse, tidsopløst elektrosprayionisering massespektrometri (Tresi-MS) koblet til HDX was bruges til at give et detaljeret billede af tilbageværende struktur i tau-protein, såvel som de konformationelle forskydninger induceret ved hyperphosphorylering.

Fordøjelse profiler af nativt og phospho-tau var ens, hvilket giver en sekvens dækning på 77,1 og 71,7% henholdsvis. Deuterium uptake værdier af hvert peptid blev bestemt ved at tilpasse observerede isotopiske fordelinger med de teoretiske fordelinger genereret anvendelse af en in-house udviklet FORTRAN software. Den bedst tilpassede fordelinger er vist (figur 3a) sammen med de tilhørende deuterium optagelsesværdier. Optagelse kinetiske profiler er derefter generer...

Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Konformationel fleksibilitet spiller en afgørende rolle i protein funktion. Heri, beskriver vi anvendelsen af ​​tidsopløst elektrospray massespektrometri koblet til hydrogen-deuterium gengæld for probing de hurtige strukturændringer, som driver funktion i ordnede og uordnede proteiner.

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Research

JoVE Journal

Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Løsning affinitetsrensede proteinkomplekser ved Blå Native PAGE og protein Korrelation Profilering

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

Undersøgelse Proteinsekvens-struktur-dynamik Forhold med Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

Optimal forberedelse af Formalin fast prøver for peptid baseret Matrix Assisted Laser Desorption/Ionisation massespektrometri Imaging arbejdsprocesser

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Definere Hsp33's Redox-regulerede anstandsdame aktivitet og kortlægning konformationelle ændringer på Hsp33 ved hjælp af brint-deuterium Exchange massespektrometri

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Leaf Spray massespektrometri: En hurtig omgivende ionisering teknik til direkte vurdere metabolitter fra plantevæv

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Påvisning af proteinubiquitinationssteder ved peptidberigelse og massespektrometri

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Prøve forberedelse til Sonde Elektrospray Ionization Mass Spektrometri

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports

Kvaternær strukturmodellering gennem kemisk tværbinding af massespektrometri: Udvidelse af TX-MS Jupyter-rapporter

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Kvantitativ analyse af cellulære lipidom af Saccharomyces Cerevisiae Ved hjælp af flydende kromatografi kombineret med tandem massespektrometri

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

En platform for hydrogendeuteriumudvekslingsmassespektrometri (HDX-MS) til undersøgelse af peptidbiosyntetiske enzymer

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...