Monte Emma, Chen Haodong, Kolmakova Maria, Parvatiyar Michelle, Vondriska Thomas M, Franklin Sarah
Department of Anesthesiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
J Vis Exp. 2012 Dec 28(70):4294. doi: 10.3791/4294.
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 recently(14) 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 volume(17) 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.
细胞核中存在着与基因调控关系最为密切的蛋白质组。成年哺乳动物心肌细胞核具有独特性，这是因为双核细胞的比例很高，（1）DNA主要处于异染色质状态，且心肌细胞不进行分裂，这使得成年细胞核处于永久的间期状态。（2）在这个器官中，发育和疾病过程中的转录调控已经得到了充分研究，（3 - 5）但相对未被充分探索的是负责DNA包装和表达的核蛋白所起的作用，以及这些蛋白质如何控制疾病过程中发生的转录程序变化。（6）在发达国家，心脏病是男性和女性的首要死因。（7）深入了解核蛋白如何协同调节这种疾病的进展对于推进当前的治疗方案至关重要。质谱分析是解决这些问题的理想工具，因为它能够对核蛋白质组进行无偏注释，并对这些蛋白质的丰度如何随疾病变化进行相对定量。虽然已经有几项关于哺乳动物核蛋白复合物的蛋白质组学研究，（8 - 13）但直到最近（14）只有一项研究检查了心脏核蛋白质组，而且该研究考虑的是整个细胞核，而不是在核亚区室水平上探索蛋白质组。（15）在很大程度上，这项工作的短缺是由于分离心脏细胞核存在困难。心脏细胞核存在于一个刚性且致密的肌动蛋白 - 肌球蛋白装置中，它们通过内质网的多个延伸部分与之相连，以至于心肌细胞收缩会改变它们的整体形状（16）。此外，心肌细胞体积的40%是线粒体（17），这就需要将细胞核与其他细胞器分开进行富集。在这里，我们描述了一种用于心脏核富集以及进一步分离为具有生物学相关性的区室的方案。此外，我们详细介绍了对这些组分进行无标记定量质谱分析的方法——这些技术适用于各种动物模型和器官系统的体内实验，在这些实验中代谢标记不可行。
