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 arbeidet ble støttet av NIH / NIDCR Grant 1R01DE019796.

De presenterte Leo-arbeidsflyter tillate stabile isotoper merking av protein fordøyer og syntetiske peptider. Disse tidsforløpet eksperimenter (figur 1) er gjeldende for komparative og kvantitative proteomikk studier samt protease forskning. Hver arbeidsflyt består av to eksperimentelle trinn (figur 2): A) Tiden løst prøvetaking av respektive 18 O-stabil isotop-kodet reaksjon (protease-katalyserte peptid cleavage; protease-katalyserte karboksyl oksygen utveksling reaksjo...

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Ved å kombinere stabil isotop merking og høy oppløsning massespektrometri i en tid-løst måte, lar paleo-tidsforløpet metoden for en dynamisk analyse av generasjon peptid produktene. Analysen kan benyttes for å generere stabile isotopically merket peptider for kvantitative og kvalitative proteomikk studier og å evaluere kinetikken hvorved proteotypic peptider blir generert. Videre er paleo-tidsforløpet utformet for å evaluere proteolytiske trasé under spesifikke, fysiologisk relevante betingelser ex vivo.<...

<table border="1"><tbody><tr><td> Navn på materiale </td><td> Selskapet </td><td> Katalognummer </td></tr><tr><td> PepClean C-18 Spin kolonner </td><td> Thermo </td><td> 89870 </td></tr><tr><td> Opti-TOF 384 MALDI sikteplate </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> Tabell 1. Materialer <table border="1"><tbody><tr><td> Navn på reagens </td><td> Selskapet </td><td> Katalognummer </td></tr><tr><td> Alpha cyano-4-hydroxycinnamic syre </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> Ditiotreitol (DTT) </td><td&gt; Acros </td><td> 16568-0050 </td></tr><tr><td> Iodoacetamide (IAM) </td><td> Sigma Aldrich </td><td> 1149-5G </td></tr><tr><td> Endotelin-omdannende 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> Vann-18 O, 97 atom% 18 O </td><td> Sigma Aldrich </td><td> 329878-1G </td></tr><tr><td> Trifluoreddiksyre (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> Mass Standards Kit for Kalibrering av AB SCIEX TOF / TOF instrumenter </td><td> AB SCIEX </td><td> 4333604 </td></tr></tbody></table> Tabell 2. Reagenser

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

Stabile isotoper er viktige verktøy i biologisk massespektrometri. Historisk, har 18 O-stabile isotoper blitt mye brukt for å studere de katalytiske mekanismer av proteolytiske enzymer 1-3. Med bruk av massespektrometri-baserte proteomikk, har enzymatisk-katalysert inkorporering av 18 O-atomer fra stabil isotopically beriket vann blitt en populær metode for å kvantitativt sammenligne protein expression nivåer (gjennomgått av Fenselau og Yao 4, Miyagi og Rao 5 og Ye et al. 6). 18 O-merking utgjør en enkel og rimelig alternativ til kjemisk (f.eks iTRAQ, ICAT) og metabolske (f.eks SILAC) merkingsteknikker 7. Avhengig proteasen utnyttet, kan 18 O-merking resultere i inkorporering av opptil to 18 O-atomer i den C-terminale karboksylgruppe av spaltningsprodukt 3 </sup&gt;. Merking reaksjonen kan deles inn i to uavhengige prosesser, peptidbinding spalting og carboxyl oksygen utveksling 8 reaksjon. I vår Paleo (p rotease-en ssisted l abeling e mploying 18 O-beriket vann) tilpasning av enzymatisk 18 O-merking, benyttet vi 50% 18 O-beriket vann for å gi særegne isotop signaturer. I kombinasjon med høy oppløsning matrise-assistert laser desorpsjon ionisering time-of-flight tandem massespektrometri (Maldi-TOF/TOF basert masse spektrometri MS / MS), kan de karakteristiske isotop konvoluttene brukes til å identifisere spaltningsproduktene med et høyt nivå av spesifisitet. Vi tidligere har brukt Paleo-metode for å påvise og karakterisere endogene proteaser 9 og overvåke proteolytiske reaksjoner 10-11. Siden Paleo koder selve essensen av den proteolytiske spalting reaksjon, er det eksperimentelle oppsettet enkelt og biokjemiske enrichment trinn spaltningsproduktene kan omgås. Paleo-metoden kan lett utvides til (i) tidsforløp eksperimenter som overvåker dynamikken proteolytisk spaltning reaksjoner og (ii) analyse av proteolyse i komplekse biologiske prøver som representerer fysiologiske betingelser. Paleo-tidsforløpet eksperimenter hjelp til å identifisere hastighetsbegrensende behandling trinn og reaksjon mellomprodukter i komplekse proteolytiske bane reaksjoner. Videre tillater paleo-reaksjonen oss å identifisere proteolytiske enzymer som serinprotease trypsin som er i stand å binde sine spaltningsproduktene og katalysere inkorporering av en andre 18 O-atom. Slike &quot;dobbel merking&quot; enzymer kan brukes for postdigestion 18 O-merking, hvor peptidene er utelukkende merket ved karboksylsiden oksygen utveksling reaksjon. Vår tredje strategien strekker merking ansette 18 O-beriket vann utover enzymer og bruker sure pH-forhold til å innføre 18 O-stabile isotoper tegnatures inn peptider.

Vi brukte paleo-tidsforløpet arbeidsflyt for dynamisk overvåke inkorporering av 18 O-stabile isotoper til peptid spaltningsproduktene generert av proteolytiske enzymer. Den presenterte tilnærming er et allsidig verktøy til forholdsvis studere proteolytiske bearbeiding trasé for ulike underlag og proteasehemmere kombinasjoner. Ved prøvetaking proteolytiske reaksjoner flere ganger over i løpet av reaksjonen, gir paleo-tidsforløpet eksperiment Time-løst øyeblikksbilder av substrat og produkt abundances...

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 isotopen merking arbeidsflyt ansette<sup&gt; 18</sup&gt; O-beriket vann (Leo-arbeidsflyt) er allsidige verktøy for kvantitative og kvalitative proteomikk studier. I protease-assistert (Paleo) arbeidsflyter,<sup&gt; 18</sup&gt; O-atomene er introdusert av proteolytisk spalting og karboksyl oksygen utveksling reaksjoner mediert av proteaser. I syrekatalysert (Aleo) arbeidsflyt,<sup&gt; 18</sup&gt; O-atomer er introdusert av karboksyl oksygen utveksling ved lav pH.

Protease-og syrekatalysert Merking arbeidsflyter Ansette<sup&gt; 18</sup&gt; O-beriket vann

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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