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

Dette arbejde blev støttet af NIH / NIDCR Grant 1R01DE019796.

De præsenterede Leo-arbejdsgange muliggør den stabile isotop mærkning af protein fordøjer og syntetiske peptider. Disse tidsforløb eksperimenter (figur 1) gælder for sammenlignende og kvantitative proteomics undersøgelser samt protease forskning. Hver arbejdsproces består af to eksperimentelle trin (figur 2): A) Den tid løst sampling af de respektive 18 O-stabil isotop-kodet reaktion (protease-katalyseret peptidspaltning, protease-katalyseret carboxyl oxygen udskiftn...

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Ved at kombinere stabil isotop mærkning og høj opløsning massespektrometri på en tidsopløst måde, gør det muligt for palæo-tidsforløbet fremgangsmåde til en dynamisk analyse af dannelsen af ​​peptidprodukter. Assayet kan anvendes til at generere stabile isotopmærkede peptider til kvantitative og kvalitative proteomics undersøgelser og vurdere kinetikken hvorved proteotypic peptider genereres. Endvidere er palæo-tidsforløbet designet til at evaluere proteolytiske omsætningsveje under specifikke, fysiol...

<table border="1"><tbody><tr><td> Navn materiale </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> PepClean C-18 spin-søjler </td><td> Thermo </td><td> 89.870 </td></tr><tr><td> Opti-TOF 384 MALDI målplade </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. Materialer <table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Alpha cyano-4-hydroxykanelsyre </td><td> Sigma Aldrich </td><td> 70990-1G-F </td></tr><tr><td> Bovint serumalbumin (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> Iodacetamid (IAM) </td><td> Sigma Aldrich </td><td> 1149-5G </td></tr><tr><td> Endothelin converting enzym-1 (ECE-1) </td><td> R &amp; D Systems </td><td> 1784-ZN </td></tr><tr><td> Trypsin Gold </td><td> Promega </td><td> V5280 </td></tr><tr><td> Vand-18 O, 97 atom% 18 O </td><td> Sigma Aldrich </td><td> 329878-1G </td></tr><tr><td> Trifluoreddikesyre (TFA) </td><td> Thermo </td><td> 28.904 </td></tr><tr><td> Mass Standards Kit til Kalibrering af AB Sciex TOF / TOF instrumenter </td><td> AB Sciex </td><td> 4333604 </td></tr></tbody></table> Tabel 2. Reagenser

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

Stabile isotoper er vigtige redskaber i biologisk massespektrometri. Historisk set har 18 O-stabile isotoper omfang blevet anvendt til at undersøge de katalytiske mekanismer af proteolytiske enzymer 1-3. Med fremkomsten af massespektrometri-baseret proteomics har den enzymatisk katalyserede inkorporering af 18 O-atomer fra stabil isotopisk beriget vand blevet en populær metode til kvantitativt sammenligne protein ekspressionsniveauer (gennemgået af Fenselau og Yao 4, Miyagi og Rao 5 og Ye et al. 6). 18 O-mærkning er en enkel og billig alternativ til kemiske (f.eks iTRAQ, ICAT) og metaboliske (f.eks SILAC) mærkningsteknikker 7. Afhængigt af den anvendte protease, kan 18 O-mærkning føre til optagelse af op til to 18 O-atomer i den C-terminale carboxylgruppe af spaltningsproduktet 3 </sup&gt;. Mærkning Reaktionen kan opdeles i to uafhængige processer, peptidbindingen spaltningssteder og carboxylgruppen oxygen udskiftningsreaktion 8. I vores paleo (p rotease-en ssisted l abeling e mploying 18 O-beriget vand) tilpasning af enzymatisk 18 O-mærkning, vi udnyttet 50% 18 O-beriget vand til opnåelse af markante isotop signaturer. I kombination med høj opløsning matrix-assisteret laser desorption ionisering time-of-flight tandem-massespektrometri (MALDI-TOF/TOF MS / MS), kan de karakteristiske isotop konvolutter anvendes til at identificere spaltningsprodukter med en høj grad af specificitet. Vi tidligere har brugt palæo-metoden til at opdage og karakterisere endogene proteaser 9 og overvåge proteolytiske reaktioner 10-11. Siden paleo koder selve essensen af ​​den proteolytiske spaltning reaktion, forsøgsopstillingen er enkel og biokemisk enrichment trin spaltningsprodukter kan omgås. Paleo-metoden kan let udvides til (i) tidsforløbet eksperimenter, der overvåger dynamikken i proteolytiske spaltningsreaktioner og (ii) analyse af proteolyse i komplekse biologiske prøver, der repræsenterer fysiologiske betingelser. Palæo-tidsforløbet eksperimenter hjælpe med at identificere rate-begrænsende procestrin og reaktionsmellemprodukter i komplekse proteolytiske pathway reaktioner. Endvidere er palæo-reaktionen gør det muligt at identificere proteolytiske enzymer, såsom serin protease trypsin, der er i stand til at binde dets spaltningsprodukter og katalysere inkorporeringen af en anden 18 O-atom. Sådanne &quot;dobbelt-mærkning&quot; enzymer kan anvendes til postdigestion 18 O-mærkning, i hvilke peptider udelukkende mærket ved carboxyl oxygen udskiftningsreaktion. Vores tredje strategi udvider mærkning beskæftiger 18 O-beriget vand ud over enzymer og bruger sure pH-betingelser for at indføre 18 O-stabile isotoper skiltturer i peptider.

Vi brugte palæo-tidsforløbet workflow til dynamisk overvåge inkorporeringen af 18 O-stabile isotoper til peptid spaltningsprodukter genereret af proteolytiske enzymer. Den foreslåede metode er et alsidigt værktøj til forholdsvis studere proteolytiske processeringssteder veje til forskellige underlag og protease kombinationer. Ved at udtage proteolytiske reaktioner gentagne gange i løbet af reaktionen tilvejebringer palæo-tidsforløbet eksperiment tidsopløst snapshots af substrat og produkt mængderne...

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

Stabile isotopmærkning arbejdsgange beskæftiger<sup&gt; 18</sup&gt; O-beriget vand (Leo-arbejdsgange) er alsidige værktøjer til kvantitative og kvalitative proteomics undersøgelser. I protease-støttede (Paleo) arbejdsgange,<sup&gt; 18</sup&gt; O-atomer er indført ved proteolytisk spaltning og carboxyl oxygen udvekslingsreaktioner medieret af proteaser. I syrekatalyserede (aleo) arbejdsproces,<sup&gt; 18</sup&gt; O-atomer er indført ved carboxyl iltudvekslingen ved lav pH.

Protease-og syrekatalyseret Mærkning arbejdsgange Anvender<sup&gt; 18</sup&gt; O-beriget 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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