Kvantitativ analyse af kromatin Proteomes i Disease
Summary
Fremskridt inden massespektrometri har tilladt high throughput analyse af proteinekspression og ændringen i en lang række væv. Kombineret med subcellulær fraktionering og sygdomsmodeller, kvantitativ massespektrometri og bioinformatik kan afsløre nye egenskaber i biologiske systemer. Fremgangsmåden beskrevet heri analyseres chromatin-associerede proteiner i tilfælde af hjertesygdomme og let anvendes til andre<em> In vivo</em> Modeller af sygdomme hos mennesker.
Abstract
I kernen bor de proteomes hvis funktioner er mest tæt knyttet til genregulering. Voksent pattedyr cardiomyocyte kerner er unik på grund af den høje procentdel af binucleated celler, 1 den overvejende heterochromatic tilstand af DNA'et, og den ikke-delende art cardiomyocyte som gør voksne kerner i en permanent tilstand af interfase. 2 Transkriptionel regulering under udvikling og sygdom er blevet godt undersøgt i dette organ, 3-5, men hvad forbliver relativt uudforsket, er den rolle, som de nukleare proteiner, der er ansvarlige for DNA emballage og udtryk, og hvordan disse proteiner styre ændringer i transkriptionelle programmer, der opstår under sygdom. 6 I den udviklede verden, hjertesygdom er den største årsag til dødelighed for både mænd og kvinder. 7 indsigt i, hvordan kerneproteiner samarbejder om at regulere udviklingen af denne sygdom er afgørende for at fremme den nuværende behandling oULIGHEDER.
Massespektrometri er det ideelle redskab til at løse disse spørgsmål, da det giver mulighed for en fordomsfri annotation af det nukleare proteomanalyse og relativ kvantificering for hvordan overflod af disse proteiner ændringer med sygdom. Mens der har været flere proteom undersøgelser for mammale nukleare proteinkomplekser, 8-13 indtil for nylig 14 har der kun været én undersøgelse undersøger hjertets nukleare proteom, og den fandt hele kerne, snarere end at udforske proteomet på niveau med nukleare sub rum . 15. I stor del, dette arbejdsmangel skyldes vanskeligheden ved at isolere hjerte kerner. Cardiac kerner forekommer inden for et stift og tæt actin-myosin apparatur, hvortil de er forbundet via flere udvidelser fra det endoplasmatiske reticulum, i det omfang, myocyt sammentrækning ændrer deres samlede form. Seksten Derudover kardiomyocytter er 40% mitokondrier vol. 17, som necessitaTES berigelse af kernen bortset fra de andre organeller. Her beskriver vi en protokol for hjerte-nukleare berigelse og yderligere fraktionering i biologisk relevante rum. Endvidere har vi detalje metoder til label-fri kvantitativ massespektrometrisk dissektion af disse fraktioner-teknikker modtagelige for in vivo eksperimenter i forskellige dyremodeller og organsystemer hvor metabolisk mærkning ikke er mulig.
Protocol
Representative Results
Discussion
To primære fremgangsmåder til nuklear isolation er gennemgået tidligere: 27 ene er den Behrens teknik homogenisering lyofiliseret væv i et ikke-vandigt opløsningsmiddel, og den anden, en modifikation, som vi bruger her, for at homogenisere vævet i en vandig saccharose / saltopløsning efterfulgt ved differentiel eller densitet-gradientcentrifugering.
Subfraktionering af kernerne ved syreekstraktion på vævsprøver er et vigtigt redskab til undersøgelse af kromatin, som har…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Den Vondriska lab er støttet af tilskud fra National Heart, Lung og Blood Institute of NIH og Laubisch Endowment på UCLA. EM er modtager af Jennifer S. Buchwald Graduate Fellowship i fysiologi ved UCLA, HC er modtageren af en American Heart Association Pre-ph.d Fellowship, MP er modtageren af en NIH Ruth Kirschstein Post-ph.d. stipendium, og SF er modtageren af en NIH K99 Award.
Materials
| Name of the reagent | Company | Catalogue number |
| Dulbeco Modified Eagle Medium | Invitrogen | 11965 |
| Protease pellet | Roche | 04 693 159 001 |
| 100 μm strainer | BD Falcon | 352360 |
| Ultracut ultramicrotome | Reichert | |
| 100CX Transmission Electron Microscope |
JEOL USA, Inc. | |
| Oriole | BioRad | 161-0496 |
| Histone H2A antibody | Santa Cruz | sc-8648 |
| Nucleoporin p62 antibody | BD Biosciences | 610498 |
| Adenine nucleotide transporter antibody | Santa Cruz | sc-9299 |
| BiP antibody | Santa Cruz | sc-1050 |
| Tubulin antibody | Sigma | T1568 |
| Histone H3 antibody | Abcam | ab1791 |
| Fibrillarin antibody | Cell Signaling | C12C3 |
| SNRP70 antibody | Abcam | ab51266 |
| E2F-1 antibody | Thermo Fisher | MS-879 |
| Retinoblastoma antibody | BD Biosciences | 554136 |
| Hypoxia inducible factor-1 antibody | Novus Biologicals | NB100-469 |
| BCA protein assay | Thermo Scientific | 23227 |
| Reverse phase column | New Objective | PFC7515-B14-10 |
| BioWorks Browser | Thermo Scientific |
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