रोग में Chromatin Proteomes के मात्रात्मक विश्लेषण
Summary
मास स्पेक्ट्रोमेट्री अग्रिम में प्रोटीन अभिव्यक्ति और ऊतकों की एक मेजबान में संशोधन के उच्च throughput विश्लेषण की अनुमति दी है. Subcellular fractionation और रोग मॉडल, मात्रात्मक जन स्पेक्ट्रोमेट्री और जैव सूचना विज्ञान जैविक प्रणालियों में नए गुण प्रकट कर सकते हैं के साथ संयुक्त है. यहाँ वर्णित विधि हृदय रोग की सेटिंग में chromatin जुड़े प्रोटीन का विश्लेषण करता है और आसानी से दूसरे के लिए लागू है<em> Vivo में</em> मानव रोग के मॉडल.
Abstract
In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.
Protocol
Representative Results
Discussion
पहले परमाणु अलगाव के लिए दो मुख्य तरीकों की समीक्षा की गई है: एक 27 Behrens एक गैर जलीय विलायक में lyophilized ऊतक homogenizing की तकनीक और दूसरा, एक संशोधन जिनमें से हम यहाँ का उपयोग एक जलीय sucrose / नमक के घोल में ऊतक homogenizing की ह?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
प्रयोगशाला Vondriska NIH और UCLA में Laubisch बंदोबस्ती के राष्ट्रीय हृदय, फेफड़े और रक्त संस्थान से अनुदान द्वारा समर्थित है. EM जेनिफर एस UCLA में फिजियोलॉजी में Buchwald ग्रेजुएट फैलोशिप के प्राप्तकर्ता है, कोर्ट एक अमेरिकन हार्ट एसोसिएशन के पूर्व डॉक्टरेट फैलोशिप के प्राप्तकर्ता है, सांसद एक NIH रुथ Kirschstein पोस्ट डॉक्टरेट फैलोशिप के प्राप्तकर्ता है, और एस एफ के प्राप्तकर्ता है एक NIH K99 पुरस्कार से सम्मानित किया गया.
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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