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.

MERK: Ta kontakt med alle relevante sikkerhetsdatablad (MSDS) før bruk. Dampene som frembringes av laserfjerning av poly (metylmetakrylat) (PMMA) kan være giftig. Vær sikker på at laser gravør er koblet til en fungerende ventilasjonssystem. Bruk alle nødvendige sikkerhetsrutiner ved bygging av microfluidic enhet inkludert bruk av tekniske kontroller (avtrekkshette, beholder for spisse gjenstander) og personlig verneutstyr (vernebriller, ansiktsmaske, hansker, frakk, full lengde bukser, lukket-toe sko). Det er av s...

<ol>
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Mens strukturbiologiske metoder, slik som røntgen-krystallografi og NMR er fordelaktige fordi de gir ekstremt detaljerte strukturer av proteiner, disse bildene er ofte statisk. Karakteriseringen av forbigående art og svakt strukturerte domener fortsetter å være unnvikende når undersøkt ved hjelp av disse konvensjonelle metoder. Derfor, for å få dynamisk innsikt i denne typen systemer er det viktig å arbeide på raske tidsskalaer. Vi har med hell brukt Tresi-HDX-MS for å få detaljert innsikt i de konforme endr...

I løpet av de siste tiårene, har betydelige fremskritt er gjort i utviklingen av analytiske teknikker utformet for å måle proteinstruktur og dynamikk 1, 2, 3, 4. Mens Røntgenkrystallografi forblir prinsippet verktøy for bestemmelse av proteinstruktur, blir høye konsentrasjoner av protein nødvendig, og omfattende optimalisering er nødvendig for å produsere diffraksjon kvalitet krystaller. Proteiner som er vanskelig å krystallisere, slik som membranbundne og egen uordnede proteiner er klassisk blitt studert ved hjelp av hydrogen-deuterium-utveksling (HDX) NMR 5. Men i de siste tiårene, kobling av elektrospray-ionisering massespektrometri (ESI-MS) for å HDX har raskt vunnet popularitet 6, 7. Massespektrometri tilbyr en løsningtil mange av de begrensninger som følger av røntgenkrystallografi og NMR. Spesielt er svært følsom MS (nM til pM nødvendige konsentrasjoner), og det er praktisk talt ingen begrensning på proteinstørrelse. I tillegg er den høye driftssyklus av MS-analyse åpner for muligheten av å studere proteiner som de gjennomgår enzymatisk omsetning, misfolding, kompleksdannelse og andre biologisk relevante prosesser. Disse prosessene ofte forekommer på den andre millisekund til tidsskalaen og krever hurtig blanding av reagenser før analyse. Utviklingen av Time-Løst elektrospray-ionisering (Tresi) av Wilson og Konermann i 2003 tillatt reaksjoner som skal overvåkes i pseudo-sanntid ved ESI-MS. Oppsettet deres innlemmet et kapillar-blander med en trinnløst innstillbar reaksjonskammer volum 8. Anordningen består av to konsentriske kapillærer, med den indre kapillær forseglet og et hakk skåret i dens side til å tillate blanding innenfor det smale inter-kapillærtey plass fra innsnittet til den ende av den indre kapillær (typisk 2 mm). Når det anvendes på HDX eksperimenter, vil det indre kapillarrør bærer for proteinet av interesse, bærer den ytre kapillarrør merkingen D2O løsning, som deretter underkastes blanding med proteinet før inntreden i regulerbar reaksjonskammeret slik at for HDX merking forut for direkte overføring til ESI kilde. I korthet er avhengig HDX på ryggraden amid-hydrogener som gjennomgår utveksling med deuterium-atomer i oppløsning 9, 10. Utvekslingen er basekatalysert ved fysiologisk pH, sammen med syre-katalyse blir fremherskende ved pH under omkring 2,6. Kursen som er basert på fire hovedfaktorer: pH, temperatur, oppløsningsmiddel tilgjengelighet og intramolekylær hydrogenbinding. Som de tidligere to faktorer holdes konstant gjennom hele forsøket, frekvensen av utveksling, særlig ved peptidryggraden amid-posisjoner, er i første rekke dependent på proteinstruktur 11. Tett foldet regioner med omfattende og stabile hydrogenbindingsnettverk i a-helikser og p-ark vil ta opp deuterium ved vesentlig lavere priser i forhold til løkker og uordnede regioner (og noen ganger ikke i det hele tatt) 12. Dette gjør det mulig for global proteinanalyse, hvor forstyrrelsene i struktur (f.eks., Ved aggregering eller substratbindende) føre til ulik deuterium-opptaket (figur 1). Den kinetiske kapillære blanderen kan bli inkorporert i et mikrofluidplattformen som inneholder et proteolytisk kammer for lokalisering av deuterium-opptak. Dette proteolytiske kammeret opprettholdes ved lav pH for effektivt å stanse utvekslingsreaksjon, og krever en immobilisert sur protease for å fordøye protein inn i lokaliserte peptider (figur 2). Overvåking ryggrad utveksling på millisekund til andre tidsskalaer er spesielt viktig forKarakteriseringen av konformasjonelle endringer innen vanskelige å karakterisere sløyfeområdene tilgjengelige, smeltet globuler, og egen uordnede proteiner (revet) 13, 14. Alternativt kan Tresi-HDX også benyttes for å karakterisere proteiner som for øyeblikket ikke har et løst atomstrukturen gjennom metoder for røntgen-krystallografi og NMR, ved hjelp av deuterium utveksling koblet til Corex algoritme (DX-Corex) tilnærming 15, 16. Denne detaljert protokoll skal gjelde Tresi-HDX å studere tau, en IDP, både i det opprinnelige form, så vel som det er sykdomsfremkallende hyperfosforylisert tilstand. Mens nativt tau er en av de mest studerte fordrevne, er lite kjent om dets amyloidogen motstykke 13.

<table border="0" cellpadding="0" cellspacing="0" width="684">
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			<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>
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			<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>
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			<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>
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			<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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			<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>
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			<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>
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			<td height="20" style="height:20px;width:244px;">or</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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	</tbody>
</table>

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

Egen uordnede proteiner (revet) har lenge vært en utfordring for å strukturelle biologer på grunn av deres mangel på stabile sekundære strukturelementer. Hydrogen-Deuterium Exchange (HDX) målt ved hurtige tidsskalaer er unikt tilpasset for å detektere strukturer og hydrogenbindings nettverk som er kort bygde, slik at for karakterisering av transiente konformerer i native ensembler. Kobling av HDX massespektrometri gir flere viktige fordeler, blant annet høy sensitivitet, lav prøven forbruk og ingen begrensning på proteinstørrelse. Denne teknikken har avansert kraftig i de siste tiårene, inkludert muligheten til å overvåke HDX merking ganger på millisekund tidsskalaen. I tillegg, ved å inkorporere den HDX arbeidsflyten på et mikrofluidplattformen rommer en sur protease-mikroreaktor, er vi i stand til å lokalisere dynamiske egenskaper ved peptidet nivå. I denne studien, tidsoppløst elektrospray-ionisering Massespektrometri (Tresi-MS) som er koplet til HDX was brukes til å gi et detaljert bilde av gjenværende struktur i tau-protein, så vel som konformasjonelle endringer indusert ved hyperfosforylering.

Fordøyelse profiler av nativt og fosfo-tau var like, noe som gir en sekvens dekning på 77,1 og 71,7% respektivt. Deuterium opptak verdier av hvert peptid ble bestemt ved å tilpasse de observerte isotopiske fordelinger med de teoretiske fordelingene som genereres ved hjelp av en egenutviklet FORTRAN programvare. De best tilpassede fordelinger er vist (figur 3a) sammen med de tilhørende deuterium-opptaksverdier. Opptakskinetiske profiler blir så generert, og ble godt ...

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

Konformasjonelle fleksibilitet spiller en kritisk rolle i proteinfunksjon. Heri, beskriver vi bruken av tidsoppløst elektrosprayionisering massespektrometri koplet til hydrogen-deuterium-utveksling for sondering de hurtige strukturelle endringer som driver funksjon i bestilt og uordnede proteiner.

Time-løst elektrospray-ionisering Hydrogen-deuterium Veksling massespektrometri for å studere Protein struktur og dynamikk

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

Løse affinitetsrenset protein komplekser av Blå Native PAGE og Protein korrelasjon Profiling

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

Undersøkelse av protein-sekvens-struktur-dynamikk Relasjoner 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 utarbeidelse av Formalin fast prøver for peptid basert Matrix assistert Laser desorpsjon/ionisering massespektrometri Imaging arbeidsflyter

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 regulert Anstandsdame aktivitet og kartlegging Conformational endringer på Hsp33 med Hydrogen-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

Blad Spray massespektrometri: En rask Ambient ionisering teknikk å vurdere direkte metabolitter fra anlegget vev

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 av protein ubiquitination nettsteder av peptid berikelse og masse spektrometri

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 ionisering massespektrometri

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

Kvartær strukturmodellering gjennom kjemisk krysskobling av massespektrometri: Utvide 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 av cellulær lipidom eav saccharomyces cerevisiae ved hjelp av flytende kromatografi kombinert 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 Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) plattform for undersøkelse av peptidbiosyntetiske enzymer

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