在疾病的染色质蛋白质组的定量分析
1Department of Anesthesiology,David Geffen School of Medicine at UCLA, 2Department of Medicine,David Geffen School of Medicine at UCLA, 3Department of Physiology,David Geffen School of Medicine at UCLA, 4Department of Internal Medicine,Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah
Summary
允许在质谱高通量分析蛋白的表达和组织了一系列修改。结合亚细胞的分馏和疾病模型，定量质量法和生物信息学在生物系统中发现新的属性。这里描述的方法分析染色质相关蛋白在心脏疾病的设置和容易地适用于其他<em在体内</em>人类疾病的模型。
Abstract
在位于细胞核内的蛋白质组，其功能是最密切相关的基因调控。成年哺乳动物的心肌细胞的核是独特的，由于双核细胞的百分比很高，1为主的异染色质的DNA的状态，和非分割的性质的心肌细胞呈现成人细胞核在相间的永久状态。2在开发过程中的转录调控和病已经很好的研究在此器官，3-5，但仍然相对未开发的核蛋白质负责DNA的包装和表达所扮演的角色，以及如何将这些蛋白质控制转录程序的变化过程中发生的疾病。在发达国家世界，心脏疾病是男性和女性的头号死亡原因。7洞察核蛋白质合作，如何规范这种疾病的进展是至关重要的推进，目前的治疗Ø的选项是。
质谱法是解决这些问题的理想工具，因为它允许一个公正的核蛋白质组的注释和相对定量，这些蛋白质丰富的变化与疾病。虽然有过几次蛋白质组学研究哺乳动物核蛋白质复合物，8-13，直到最近，只有一个心脏的核蛋白质组研究，并认为整个细胞核，而不是研究蛋白质组的在核分舱水平15在很大程度上，这不足的工作是由于隔离心脏核的难度。心肌细胞核内发生的刚性和密集的肌动蛋白-肌球蛋白设备通过它们所连接的多个扩展名从内质网的范围内，心肌细胞的收缩改变他们的整体造型。16此外，心肌细胞线粒体40％第17卷necessita韭细胞核富集除了从其他细胞器。在这里，我们描述了一个协议，用于心肌核浓缩和进一步的分离与生物相关的车厢。此外，我们无标记定量质谱夹层这些馏分技术适合于在各种动物模型和器官系统的代谢标记是不可行的体内实验的详细方法。
Protocol
实验工作流包含七个主要步骤（ 图1）。对于任何有关的工作，将运行在质谱仪的样品，实验人员应穿白大褂，手套和头发网和照顾，以避免污染，灰尘和脱落的角质个人。 1。心同质化和核孤立小鼠心脏均化和一个完整的核颗粒中分离（ 图2）。 牺牲成年小鼠，切除心脏，在冰冷的PBS冲洗，并在冰上摇匀玻璃DOUNCE（我们?…
Representative Results
图4显示这种形式的相对定量的效用。在左侧面板是个别的单一同位素肽峰（覆盖从不同的鼠标），它已被指定为属于蛋白HMGB1（通过数据库检索标识）。每个峰值，本质上是一个给定的肽的提取离子色谱仪，来自不同的鼠标。这些团体代表三种不同的生理状态：基础，心肌肥厚，心脏衰竭，有三个生物复制为每个组。通过集成的曲线下的面积，可以计算出的相对丰度。在中间面板中?…
Discussion
核分离的两个主要方法已被先前评论：27 1是均质冻干组织中的非水溶剂中的贝伦斯技术和第二，其中在这里，我们使用的变形例，在水性的蔗糖/盐溶液均质组织随后由差分或密度梯度离心。
Subfractionation的核酸提取的组织样本，研究与原来的目标是分析组蛋白染色质已自1960年以来，28的重要工具。酸提取协议开发为实现这一目标的纯化核心的核小体的组蛋白…
Disclosures
The authors have nothing to disclose.
Acknowledgements
的Vondriska的补助金从国家心脏，肺和血液研究所，美国国立卫生研究院和Laubisch基金会在加州大学洛杉矶分校实验室的支持。 EM是收件人的詹妮弗学的布赫瓦尔德研究生奖学金，加州大学洛杉矶分校生理学，HC是收件人的美国心脏协会前博士生奖学金; MP是收件人的美国国立卫生研究院露丝基尔希斯坦博士后研究;和SF是收件人的NIH K99奖。
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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Tags
Quantitative AnalysisChromatin ProteomesDiseaseNucleusGene RegulationAdult Mammalian Cardiomyocyte NucleiBinucleated CellsHeterochromatic StateDNA PackagingTranscriptional RegulationDevelopmentDiseaseNuclear ProteinsTranscriptional ProgramsHeart DiseaseMortalityNuclear ProteomeMass SpectrometryAnnotationRelative QuantificationProtein AbundanceProteomic StudiesCardiac Nuclear ProteomeNuclear Sub Compartments
Cite This Article
Monte, E., Chen, H., Kolmakova, M., Parvatiyar, M., Vondriska, T. M., Franklin, S. Quantitative Analysis of Chromatin Proteomes in Disease. J. Vis. Exp. (70), e4294, doi:10.3791/4294 (2012).