Name: protease- and acid-catalyzed labeling workflows employing 18o-enriched water
Uploaded: 2013-02-20T12:00:00
Description: Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Dit werk werd ondersteund door NIH / NIDCR Grant 1R01DE019796.

De gepresenteerde LEO-workflows zorgen voor de stabiele isotoop etikettering van eiwit hydrolysaten en synthetische peptiden. Deze tijdsverloop experimenten (figuur 1) zijn van toepassing op vergelijkende en kwantitatieve proteomics studies en protease onderzoek. Elke workflow bestaat uit twee experimentele stappen (figuur 2): A) De tijd opgeloste bemonstering van de respectieve 18 O-stabiele isotoop-gecodeerde reactie (protease-gekatalyseerde splitsing peptide; protease geka...

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Door het combineren van stabiele isotopen labeling en hoge resolutie massaspectrometrie in een tijdsopgeloste wijze de paleo-tijdsverloop werkwijze maakt een dynamische analyse van de vorming van peptide producten. De test kan worden gebruikt om stabiele isotoop gemerkte peptiden voor kwantitatieve en kwalitatieve proteomics studies genereren en de kinetiek waarbij proteotypic peptiden gegenereerd evalueren. Bovendien wordt paleo-tijdsverloop ontworpen om proteolytische systemen evalueren onder specifieke, fysiologisch ...

<table border="1"><tbody><tr><td> Stofnaam </td><td> Vennootschap </td><td> Catalogusnummer </td></tr><tr><td> PepClean C-18 Spin Columns </td><td> Thermo </td><td> 89870 </td></tr><tr><td> Opti-TOF 384 MALDI richtplaat </td><td> AB SCIEX </td><td> 1016629 </td></tr><tr><td> 4800 MALDI TOF / TOF </td><td> AB SCIEX </td><td></td></tr></tbody></table> Tabel 1. Materials <table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogusnummer </td></tr><tr><td> Alpha cyano-4-hydroxykaneelzuur </td><td> Sigma Aldrich </td><td> 70990-1G-F </td></tr><tr><td> Bovine serum albumine (BSA) </td><td> Sigma Aldrich </td><td> A3294-10G </td></tr><tr><td> Dithiothreitol (DTT) </td><td&gt; Acros </td><td> 16568-0050 </td></tr><tr><td> Joodaceetamide (IAM) </td><td> Sigma Aldrich </td><td> 1149-5G </td></tr><tr><td> Endotheline converterend enzym-1 (ECE-1) </td><td> R &amp; D Systems </td><td> 1784-ZN </td></tr><tr><td> Trypsine Gold </td><td> Promega </td><td> V5280 </td></tr><tr><td> Water-18 O, 97% 18 O atoom </td><td> Sigma Aldrich </td><td> 329878-1G </td></tr><tr><td> Trifluorazijnzuur (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> Massa Normen Kit voor kalibratie van AB SCIEX TOF / TOF instrumenten </td><td> AB SCIEX </td><td> 4333604 </td></tr></tbody></table> Tabel 2. Reagentia

protease- and acid-catalyzed labeling workflows employing 18o-enriched water

Stabiele isotopen zijn essentiële hulpmiddelen in biologische massaspectrometrie. Vroeger 18 O-stabiele isotopen uitgebreid gebruikt om de katalytische mechanismen van proteolytische enzymen 1-3 bestuderen. Met de komst van massaspectrometrie-based proteomics is het enzymatisch gekatalyseerde omzetting van 18 O-atomen stabiele isotoopverrijking water een populaire methode om kwantitatief vergelijken eiwitexpressieniveaus (beoordeeld door Fenselau en Yao 4, Miyagi en Rao 5 en Ye et al.. 6). 18 O-etikettering is een eenvoudige en goedkope alternatief voor chemische (bijv. iTRAQ, ICCAT) en metabole (bijv. SILAC) labelingstechnieken 7. Afhankelijk van de gebruikte protease kan 18 O-labeling leiden tot de opname van maximaal twee 18 O-atomen in de C-eindstandige carboxylgroep van de splitsing product 3 </sup&gt;. De labelingsreactie kan worden onderverdeeld in twee onafhankelijke processen, de peptidebinding splitsing en carboxyl zuurstof uitwisselingsreactie 8. In onze Paleo (p rotease-a ssisted l Abeling e mploying 18 O-verrijkt water) de aanpassing van enzymatische 18 O-etikettering, we gebruik gemaakt van 50% 18 O-verrijkt water onderscheidend isotopen opleveren. In combinatie met hoge resolutie matrix-assisted laser desorptie ionisatie time-of-flight tandem massaspectrometrie (MALDI-TOF/TOF MS / MS) kan de kenmerkende isotoop enveloppen worden gebruikt splitsingsproducten identificeren met een hoge mate van specificiteit. Wij hebben al eerder gebruik hebben gemaakt van de paleo-methodiek op te sporen en te karakteriseren endogene proteasen 9 en bewaken proteolytische reacties 10-11. Sinds Paleo codeert de essentie van de proteolytische splitsing reactie, de experimentele opstelling is eenvoudig en biochemische Enrichment stappen van splitsingsproducten kunnen worden omzeild. De Paleo-methode kan eenvoudig worden uitgebreid met (i) het tijdsverloop experimenten die de dynamiek van de proteolytische splitsing reacties en (ii) de analyse van proteolyse in complexe biologische monsters die fysiologische omstandigheden vertegenwoordigen controleren. Paleo-tijdsverloop experimenten helpen identificeren van snelheidsbeperkende processtappen en reactie-intermediairen in complexe proteolytische route reacties. Bovendien is de paleo-reactie kunnen we vaststellen proteolytische enzymen zoals de serine protease trypsine die in staat zijn splitsingproducten opnieuw binden en de opname van een tweede 18 O-atoom katalyseren. Dergelijke &quot;dubbele labeling&quot; enzymen kunnen worden gebruikt voor postdigestion 18 O-labeling, waarbij peptiden uitsluitend gelabeld door de carboxyl zuurstof uitwisselingsreactie. Onze derde strategie breidt etikettering gebruik 18 O-verrijkt water buiten enzymen en maakt gebruik van zure pH voorwaarden in te voeren 18 O-stabiele isotoop tekenperaturen in peptiden.

We gebruikten de paleo-tijdsverloop workflow voor het dynamisch toezien op de integratie van 18 O-stabiele isotopen in peptide splitsingsproducten gegenereerd door proteolytische enzymen. De gepresenteerde aanpak is een veelzijdige tool om relatief proteolytische processing paden te bestuderen voor verschillende ondergrond en protease combinaties. Door het bemonsteren proteolytische reacties herhaaldelijk over het verloop van de reactie, de paleo-tijdsverloop experiment biedt tijdsopgeloste snapshots van subs...

Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stabiele isotoop labeling workflows in dienst<sup&gt; 18</sup&gt; O-verrijkt water (LEO-workflows) zijn veelzijdige tools voor kwantitatieve en kwalitatieve proteomics studies. In protease-assisted (paleo) workflows,<sup&gt; 18</sup&gt; O-atomen die door proteolytische splitsing en carboxyl zuurstof uitwisselingsreacties gemedieerd door proteasen. In het zuur-gekatalyseerde (Aleo) workflow,<sup&gt; 18</sup&gt; O-atomen worden geïntroduceerd door carboxyl zuurstoftoevoer bij lage pH.

Protease-en Acid-gekatalyseerde Labeling Werkstromen Gebruikmakend<sup&gt; 18</sup&gt; O verrijkte Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

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Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

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