En Protein Forberedelse Metode til High-throughput Identifikation af proteiner Samspil med en Nuclear Cofactor Brug LC-MS / MS-analyse
Summary
Vi har etableret en fremgangsmåde til oprensning af coregulatoriske interaktion proteiner ved anvendelse af LC-MS / MS-system.
Abstract
Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.
Introduction
Protein-protein interaktioner spiller en vigtig rolle i mange biologiske funktioner. Som sådan er disse interaktioner blevet impliceret i signaltransduktion; protein transport over cellemembraner; cellemetabolisme; og adskillige nukleare processer, herunder DNA-replikation, DNA beskadigelse reparation, rekombination og transkription 1, 2, 3, 4. Identifikation af proteiner involveret i disse interaktioner er derfor afgørende for at fremme vores forståelse af disse cellulære processer.
Immunpræcipitering (IP) er en valideret teknik, der anvendes til at analysere protein-protein-interaktioner. For at lette identifikationen af co-immunpræcipiteret proteiner, er massespektrometri ofte brugt 5, 6, 7, 8,9. Ved at målrette en kendt medlem af et proteinkompleks med et antistof, er det muligt at isolere protein-kompleks og efterfølgende identificere sine ukendte komponenter via massespektrometrianalyse. ARIP4 (androgen receptor-interagerende protein 4), en transskriptionel coregulator, interagerer med nukleare receptorproteiner for at aktivere eller undertrykke sit mål initiativtagere i en kontekst-afhængig måde 9, 10. For bedre at forstå de transkriptionelle reguleringsmekanismer for disse nukleare faktorer, beskriver vi en omfattende metode til at rense og identificere ARIP4 interagerende proteiner ved hjælp af LC-MS / MS-system.
Protocol
Representative Results
Discussion
Effektiv transfektion er afgørende for at opnå et vellykket resultat med denne protokol. Derfor anbefaler vi at bruge Western blot-analyse for at bestemme de immunopræcipiterede FLAG-mærkede protein niveauer. Dette trin giver brugerne mulighed for at kontrollere, at deres protein af interesse er korrekt overudtrykt og desuden, at IP blev udført med succes. FLAG-mærket protein niveauer bør også kontrolleres forud for massespektrometri analyse.
En mock prøve bør anvendes som en negat…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), the Takeda Science Foundation (HT), and by a Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) to HT.
Materials
| Lipofectamine 2000 Transfection Reagent | Thermo Fisher Scientific | 11668019 | |
| Protease Inhibitor Cocktail (EDTA free) (100x) | nacalai tesque | 03969-21 | |
| ANTI-FLAG M2 Affinity Gel | Sigma-Aldrich | A2220 | |
| Micro Bio-Spin Columns | BIO-RAD | 732-6204 |
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