nih P01 HL112730 (抹布, JVE), nih R01 HL132075 (抹布, JVE), 芭芭拉史翠珊妇女心脏中心 (抹布, JVE), 桃乐茜和 e. 菲利普在分子心脏病学 (抹布) 的里昂椅子, 艾丽丝在妇女的心脏健康 (JVE) 和捷克科学院的椅子机构支持 RVO: 68081715 (MS)。

所有动物研究都是按照机构指南进行的, 并由雪松-西奈医学中心的机构动物护理和使用委员会批准。
1. Langendorff 灌注小鼠心脏
<ol><li>
小鼠心脏缺血再灌注 Langendorff 灌注的研究
	<ol>
<li>管理腹腔戊巴比妥钠70毫克/千克的成年小鼠 (8 周岁, 男性, C57BL6/j)。确认深部麻醉, 缺乏撤退到脚趾捏。</li>
		<li>Anticoagulate 腹腔肝素 500 U/千克。</li>
		<li>通过胸骨切口打开胸腔。打开隔膜, 沿肋边界切开至腋前轴。然后, 将前腋轴切开至前肢, 完全打开胸腔笼。之后, 视觉化的心脏, 钳位上升主动脉与钳和快速消费心脏。死亡是由于放血。将心脏放在冷的克雷布斯 (氯化钠6.9 克/升, 氯化钾0.35 克/升,NaHCO 3 2.1 克/升,MgSO 2 PO 4 0.16 克/升, , 4 克/升, 葡萄糖0.141 克/升和 CaCl2 2 克/升)。</li>
		<li>Cannulate 主动脉与钝尖针和灌注心脏 retrogradely 克雷布斯溶液在恒定压力模式。</li>
		<li>稳定期15分钟后, 心脏与全球无流性缺血30分钟, 其次再灌注30分钟。</li>
		<li>收获心后15分钟基线灌注后, 30 分钟缺血, 或30分钟再灌注后。修剪心房, 并在液氮中对组织进行冷冻。</li>
		<li>存储在-80 °c, 直到使用</li>
	</ol>
</li>
</ol>2. 组织均匀化、增溶和蔗糖密度梯度沉淀
<ol><li>
渐变准备
	<ol>
<li>准备2溶液 (15% 和 50%) 的100毫升蔗糖, 混合4毫升氯化钠 2.5 M; 0.8 毫升氯化镁2 1.25 M;1毫升三盐酸1米, pH 7.5;15克或50克蔗糖 (分别为15% 和 50%);超纯水达到100毫升;和二甲苯蓝 (0.02 毫克/毫升) 来着色15% 溶液</li>
		<li>过滤每个解决方案 (0.22 µm 过滤器)</li>
		<li>使用梯度的一天, 准备40毫升的每一个蔗糖溶液, 并添加放线菌酮到每个溶液, 以达到最终浓度的100µg/毫升<ol>
<li>使用渐变制作器来准备15% 和50% 蔗糖溶液的混合物</li>
			<li>用5毫升的15% 蔗糖梯度填充左侧隔间</li>
			<li>用50% 蔗糖梯度填充合适的隔间</li>
			<li>打开水龙头, 用磁力搅拌器将两个小隔间搅拌在泵上开关</li>
			<li>使用连接到第二个水龙头的管, 使混合蔗糖溶液流动在超离心管与薄壁。最终体积将约10毫升。</li>
		</ol>
</li>
		<li>保持梯度在4°c (一夜之间, 如果需要, 但不长)</li>
	</ol>
</li>
	<li>
心脏组织的均匀化
	<ol>
<li>融汇新鲜或冷冻心脏与 polytron 在过滤的溶解缓冲 (被改进为蔗糖梯度分馏) 包含100毫米氯化钾, 20 毫米三 pH 7.5, 5 毫米氯化镁2, 0.4% NP-40。包括放线菌酮100µg/毫升, 以维持 polysome 结构, 0.1 U RNase 抑制剂和蛋白酶抑制剂鸡尾酒 (1 片剂50毫升)。</li>
		<li>在冰上孵化15分钟的裂解液。</li>
		<li>离心机在 1.8万 x g 15 分钟在4°c 去除不溶性的材料并且收集上清。</li>
		<li>保留50µL 蛋白质测定、rna 分离和 rna 完整性分析 (见图 2)。</li>
		<li>将上清 (500–100µL) 层在梯度上, 平衡管与溶解缓冲。</li>
	</ol>
</li>
	<li>
沉淀和馏分的收集
	<ol>
<li>执行离心120分钟在 3.7万 rpm 在4°c 在摆动斗转子。根据制造商, 这对应于 22.8万 x g在最大半径。</li>
		<li>收集每一个梯度为17个分数 (大约600µL 每分数) 在连续监测吸收率在 254 nm (OD254)。</li>
		<li>按如下步骤对分数收集器进行编程:<ol>
<li>丢弃前3分钟的收集 (与在渐变前的管子中所含的液体相对应)</li>
			<li>从4到19分钟, 收集的分数以1毫升/分钟的速度在17个分数。</li>
		</ol>
</li>
		<li>把分数放在冰上, 一旦收集。如果没有立即处理, 请将样品冷冻-80 摄氏度。如图 1所示, 一个典型的 UV 密度对渐变的跟踪。</li>
	</ol>
</li>
</ol>3. Polysome 和 Nontranslating 馏分中基因的分离与分析
<ol><li>
mRNA 的提取
	<ol>
<li>解冻在冰上的分数, 如果他们收集后闪光冷冻</li>
		<li>将每个分数的200µL 转移到一个新鲜的管子上 注意: 在这个步骤中, 两个或多个连续的分数可以汇集在一起。在分析 OD254图的基础上, polysome 和非 polysome 分数不混合时, 应仔细进行。</li>
		<li>将 10 ng 荧光素酶 RNA 作为内部控制添加到每个样品中, 以达到规范化目的。 注: 荧光素酶底漆应作为内部控制, 以规范化的结果。</li>
		<li>添加700µL RNA 萃取试剂 (苯酚和异硫氰酸胍单相溶液; 见材料表) 到每管。混合好, 并孵化管5分钟的 RT (室温)。</li>
		<li>十五年代加入140µL 氯仿和涡流. 在 RT 中孵育2-3 分钟。</li>
		<li>离心样品15分钟在 1.2万 x g在4°c。</li>
		<li>小心地将上水相转移到新管上。</li>
		<li>由于分数的 RNA 含量不高, 增加10µg 糖原作为载体对每管。</li>
		<li>每管加350µL 异丙醇。拌匀, 在-20 摄氏度孵育1小时, 以提高产量。</li>
		<li>离心机为15分钟在 1.2万 x g在4°c。</li>
		<li>丢弃上清液, 加入700µL 乙醇75% 清洗 RNA 颗粒。</li>
		<li>离心机为5分钟在 7500 x g在4°c。</li>
		<li>去除乙醇, 让颗粒空气干燥10–15分钟。 注: 过度干燥的 RNA 颗粒将防止其在水中的完全溶解。</li>
		<li>并用重悬 RNA 颗粒在 50 ul RNase 无超纯 H2O。</li>
		<li>在55摄氏度孵化样品10分钟。</li>
		<li>使用每个样本的 2 ul, 以测量提取的 RNA 的质量和数量, 并存储其余的-80 °c。</li>
	</ol>
</li>
	<li>
反转转录和 qPCR
	<ol>
<li>使用 4 ul 的反向转录 cDNA 合成试剂盒 (见材料表) 的 20 ul 反应。根据样品的数量准备 supermix。将所需体积转移到每管, 并将输入 RNA 的 5 ul 添加到每个管中。这个体积可以被纠正, 以适用于 Taq 聚合酶工作条件的范围。</li>
		<li>在热循环仪中孵化管, 由制造商推荐的以下程序: 5 分钟在25°c, 20 分钟反向转录为20分钟在46°c, 并且逆转录酶失活为1分钟在95°c。</li>
		<li>运行 qPCR 与优化程序为每对引物。 注意: 要检测某些基因在分数中的存在, 应使用来自每个分数 (分离 RNA) 的相等体积来进行反向转录。</li>
	</ol>
</li>
	<li>
结果分析
	<ol>
<li>应用感兴趣基因和荧光素酶的 ct 值计算三角洲 ct 值</li>
		<li>将每个分数三角洲 Ct 值表示为所有分数中包含的总 mrna 值的百分比, 以可视化不同分数的 mrna 分布情况 (表 1)</li>
	</ol>
</li>
</ol>4. Polysome 馏分和 Nontranslating 馏分中 miRNAs 的分离与分析
<ol><li>
mRNA 和 miRNA 的提取 注意: 本协议遵循制造商的说明, 并进行了一些更改。<ol>
<li>解冻糖原和控制钉。保持冰块。</li>
		<li>添加700µL miRNA 萃取试剂到200µL 的汇集样品。</li>
		<li>添加1µg/µL 糖原, 并融汇它使用吸管。在室温下孵化5分钟。</li>
		<li>添加3.5 µL 1.6 x 108钉入控制和混合。</li>
		<li>添加200µL 氯仿和涡旋至少15秒。</li>
		<li>室温下孵育3分钟。</li>
		<li>离心机在 1.2万 x g 15 分钟在4摄氏度。</li>
		<li>使用吸管, 转移到上清到一个新的1.5 毫升管。丢弃中、下阶段。</li>
		<li>将1.5x 容积的100% 乙醇添加到上清液中并混合。</li>
		<li>将此混合物的700µL 转移到 miRNA 萃取塔上, 并在十五年代在 RT 上全速离心, 并丢弃流经;使用任何剩余的示例重复此步骤。</li>
		<li>将700µL 的缓冲1添加到该柱上, 并在十五年代在 RT 上全速离心;放弃流程。</li>
		<li>将500µL 的缓冲2添加到该柱上, 并在十五年代在 RT 上全速离心;放弃流程。</li>
		<li>在该柱上加入500µL 80% 乙醇, 并以全速将其离心为2分钟;放弃流程。</li>
		<li>将该列转移到新的2毫升名额管, 打开盖子, 并将其在 RT 上离心, 全速运行5分钟;放弃流程。</li>
		<li>添加16µL 的 RNAse 水到柱和离心机在全速1分钟的 RT. 保持洗脱液在冰上或存储在-80 °c, 以供将来分析。 注: 每个样品的两个微升可用于 OD 分析。</li>
	</ol>
</li>
	<li>
反向转录
	<ol>
<li>准备冰块。对于每20µL rt-pcr 反应, 添加4µL 的 miRNA rt 缓冲器, 2 µL 的核酸, 9 µL 的总 RNA, 2 µL 的酶和3µL 无 RNAse 水;混合它, 并简单地旋转下来。</li>
		<li>设置热循环仪如下: 37°c 为60分钟; 95°c 为5分钟; 4°c 举行。</li>
		<li>从热循环仪中取出管子, 加入200µL 的 RNAse 水。贮存在-20 摄氏度或进行实时 PCR。</li>
	</ol>
</li>
	<li>
实时 PCR 注: 本协议遵循96井板格式。在冰上准备反应混合物。<ol>
<li>根据协议所接受的范围, 准备 PCR 反应组合并添加 cDNA (从 miRNAs 以上)。多反应可以分批准备。</li>
		<li>在每个井中添加25µL 的 PCR 混合 + cDNA。</li>
		<li>用光学板密封密封板。</li>
		<li>在室温下用 1000 x g离心板1分钟。</li>
		<li>程序实时循环仪如下: 初始活化步骤在95°c 为15分钟; 3步循环: 变性在94°c 十五年代;退火在55°c 三十年代;延长在70°c 三十年代 (执行荧光数据收集);增加40个周期; 熔化曲线根据循环仪缺省。</li>
	</ol>
</li>
	<li>
比较分析
	<ol>
<li>规范化示例 (见表 2):<ol>
<li>查找 SCM 最小值。</li>
			<li>将所有 SCM 值规范化到最低限度。</li>
			<li>从 miRNAs 和参考基因 (REF) 的 Ct 值中减去此值。</li>
			<li>使用 2ΔCt公式分析数据。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. 蛋白质组分析
<ol><li>
polysome 中蛋白质的提取
	<ol>
<li>将所收集的蔗糖馏分组合成三个最终馏分。将分数标记为重(高效率 polysomes 在汇集的组分 4, 5, 6 和7以蔗糖的最高的集中; 梯度管的底部),光(低效率 polysomes 在汇集的分数 8, 9, 10 和 11; 中间的梯度管) 和非平移(汇集分数 12, 13, 14 和 15, 蔗糖浓度最低; 梯度管顶部)。对汇集的分数进行蛋白质检测。</li>
		<li>准备100% 股乙酸酸 (TCA), 并将其储存在4摄氏度在黑暗中。混合0.5 毫升的样品与60µL 100% TCA 和孵化在-20 °c 过夜在黑暗中。 注: 使用1.5 毫升低蛋白保留管 (见材料表)。</li>
		<li>夜间孵化后, 在冰上解冻样品, 离心机在 1.5万 x g, 4 °c 为30分钟。 注: 样品中总蛋白的100µg 在管底提供可见蛋白颗粒。</li>
		<li>预冷丙酮在-20 °c 冰箱。将0.5 毫升的冷丙酮添加到蛋白质颗粒和离心机 1.5万 x g, 4 °c 15 分钟。重复此步骤两次。</li>
		<li>空气干燥颗粒。并用重悬360µL 50 毫米三盐酸缓冲液中的颗粒 (pH 8)。 注: 如果需要, 使用脉冲 sonicator 并用重悬颗粒。</li>
		<li>添加40µL 0.1 米 dithiothreitol (最后浓度10毫米) 和孵化在55°c 在振动筛为45分钟. 添加50µL 0.15 米 iodoacetamide (最终浓度17毫米) 和在室温下孵化, 在黑暗中30分钟。</li>
		<li>准备胰蛋白酶溶液: 并用重悬20µg 的胰蛋白酶在100µL 50 毫米碳酸氢铵。在 1:50 (w/w) 胰蛋白酶中添加相应的胰蛋白酶溶液体积, 以确保 pH 值为 8, 并在37摄氏度的温度下在振动筛上孵育。 注: 添加1米碳酸氢铵, 使 pH 值为8。</li>
		<li>胰蛋白酶消化后, 冷却试样, 离心机在台式离心机上, 加入10% 甲酸对 pH 2–3, 以淬火胰蛋白酶活性, 并进行样品脱盐。 注: 使用µ洗脱板进行样品脱盐。</li>
		<li>在96井板井中, 用200µL 甲醇, 用真空三倍和200µL 0.1% 甲酸三次调理。用200µL 0.1% 甲酸三倍的样品进行试样清洗。洗脱样品与100µL 50% 乙腈/0.1% 甲酸两次到低蛋白保留0.5 毫升管。 注: 洗脱的样品首先使用引力, 其次是低真空。</li>
		<li>蒸发样品洗脱液干燥使用选矿厂, 或者直接进行质谱分析或存储在-80 °c, 直到使用。</li>
	</ol>
</li>
	<li>
质谱分析
	<ol>
<li>在50–100µL 中重建每个颗粒 (包括胰蛋白酶肽), 并向质谱仪注入1µL。 注: 优化重构和注入量以获得最大强度 LC/ms/毫秒信号。在我们的情况下, 最大信号 (总离子电流;TIC) 在扫描的心脏是在范围1E8 到2E9。</li>
		<li>使用陷印柱 C18 (300 µm id x 5 毫米, 5 µm, 100Å) 后, 使用 C18 柱 (75 µm 250 毫米, 2 µm, 100Å) 的肽分离, 流速设置为 300 nL/分钟. 将纳米源毛细管温度设置为275摄氏度, 喷雾电压为2伏。</li>
		<li>应用线性梯度 5–35% b 为90分钟, 35–95% b 为3分钟, 持有在 95% b 为7分钟和平衡在 5% b 为 25 min (a: 0.1% 甲酸/水和 B: 0.1% 甲酸在乙腈)。利用 CID 模式获取每 MS1 扫描的15种最高强度离子的 MS2 谱。使用3质谱复制为每个样品分析。 注: 优化和使用适用于特定质谱仪样品的实验条件和参数。第5.2 节所述的条件/参数。在实验室中得到了应用和优化。</li>
	</ol>
</li>
	<li>
蛋白质鉴定/定量
	<ol>
<li>在质谱分析后, 使用 MSConvert 软件 (http://proteowizard.sourceforge.net/tools/msconvert.html) 将原始光谱文件转换为兼容的 mzXML 文件。</li>
		<li>使用搜索条件将转换后的文件提交到 SEQUEST 搜索引擎,例如, UniProtKB/瑞士-波特鼠标数据库 (最新的规范化审查);半酶消化用胰蛋白酶 (在 KR/-之后);与2错过了分裂;前体质量范围: 400 到 4500;静态修改: carbamidomethyl (C) 57.021465;肽质量耐受性:50 ppm;片段质量类型: monoisotopic。 注: 可使用其他蛋白质数据库搜索引擎和数据管道。如果使用数据库 (如鼠), 它比鼠标和人的数据库小, 则可能需要搜索鼠标 (和/或人) 数据库以增加蛋白质组覆盖率。在这种情况下, 将需要删除蛋白质名称中的冗余。</li>
		<li>执行搜索后分析。设置用于查看数据的筛选器。例如, 将最小蛋白质概率 (蛋白质阈值) 设置为95% 或 99%,即,只有统计分析意味着在样本中存在95% 或99% 概率的蛋白质才会显示出来。设置用于肽阈值的筛选器 (例如, 95%),即在确定光谱是否识别肽时将使用的最小概率。选择将决定蛋白质是否存在的最小肽数量 (推荐2肽作为蛋白质鉴定的最低值)。</li>
		<li>评估确定的蛋白质质量/数量。确保 proteotypic 肽用于定量。如果异构体被确定, 确保它是根据观察的胰蛋白酶肽组成的氨基酸序列特有的特定异构体。任何 "类似的" 或假设的蛋白质应该进行比较, 看看观察到的肽是否对应于已知的蛋白质。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

通过分析特定 mRNA 或整个转录6、7的平移状态, Polysome 剖面分析允许对蛋白质的翻译进行研究。当需要对本地翻译进行研究时, 如突触体8, 也有很大的帮助。传统上, 这种方法包括分离单和核糖体和相关的基因的蔗糖梯度, 可以结合基因组或蛋白质蛋白技术, 以获得预期结果6,9。例如, 我们小组最近进行的一项研究揭示了在体外循环患者心脏中执行的转录后调控机制, 它模仿缺血/再灌注损伤。利用 polysome 剖面分析, 我们在体外循环术后的心脏核编码线粒体蛋白的翻译增加了2。
这里提出的动态蛋白质组 polyribosomal 分析方法可以揭示与核糖体相关的基因种群的变化以及正在积极翻译的蛋白质。由于基因的翻译部分受 miRNAs 的制约, 这些分数中的 miRNAs 分析可以揭示更多的洞察力。事实上, 一些研究已经修改了这一方法, 并证明它是一个适当和可靠的方法来调查10,11的 miRNA 模式。
在过去十年中, 许多研究都在探讨 miRNAs 的作用, 考虑到它们在各种生物功能12、13中的重要性。由于 miRNAs 通过与对应基因的基配对来标记它们的退化, 因此进行了 polysome 剖面分析, 表明 miRNAs 实际上是在 polysome 分数中找到的, 目标是翻译基因14,15,16. miR-21 是一个很好的例子, 它与正常细胞中的低压迫和弱 polysomes 结合有关, 而在癌细胞中, 与 polysomes 的关联增加, 压制作用更强17。
本研究利用 miRNAs 的 ct 值 (miRNA)、峰值控制 (SCM) 的 ct 值, 对样本中稳定的参考基因或 miRNA (REF) ct 值进行实时 PCR 分析。第一步是规范化的 ct 值的语言和裁判的 ct 值的单片机。然后有第二步规范化的教学语言对裁判和结果值被用来计算 2ΔCt公式。这些值或折叠更改值可用于显示组之间的差异。
由于蛋白质的提取是很难从 polysome 的分数, 大部分的研究迄今进行, 已使用的分数直接用于西方印迹分析。它们简单地沉积每一个分数的蛋白质含量, 并将其与 sds 加载缓冲器混合使用, 用于 sds 页分析8,18。在这里, 我们开发了一种方法, 使我们不仅提取基因和 miRNAs 后分馏, 而且获得高品质的肽和蛋白质进行质谱分析。
这种方法可以进一步扩大, 通过积极测量新合成的蛋白质使用代谢标签, 如双正交氨基酸同源, 如 azidohomoalanine (哈) 为蛋氨酸19,20。哈得公司随后点击化学允许加入生物素标签, 然后链亲和素纯化所有的蛋白质, 纳入哈。这就允许对新合成的蛋白质--动态蛋白质组的另一部分--进行明确的鉴定。检测 miRNAs, 在翻译和 nontranslating 分数之间重新分配的反应刺激 (这里与我/R) 允许审讯的动态调节蛋白质的翻译。然而, 在核糖体束缚 mRNA 附近招募 miRNAs 的因素仍有待确定和系统地研究。
在我们的研究中, 除了 TCA 沉淀, 我们还测试了其他三种蛋白质提取方法从 polysome 分数: i) FASP (过滤器辅助样品制备)21, ii) 丙酮沉淀, iii) 氯仿/甲醇沉淀。根据对高浓度蔗糖 (最多 50%) 和质谱鉴定的蛋白质数量 (图 7和表 3) 处理分数的能力, 对方法的适用性进行了评价。
对于 FASP 方法, 我们遵循了 FASP 蛋白消化试剂盒给出的样品制备协议, 它采用了一个相对分子质量切断 30 kDa 的超滤装置。该过滤装置作为一种工具, 用于洗涤剂去除, 缓冲交换, 蛋白质消化和肽洗脱能力保留高分子量的物质 (蛋白质和 DNA), 否则会干扰随后的肽分离21. 简单地说, 该样品与含尿素缓冲液混合, 并在单元自旋过滤器上加载, iodoacetamide 溶液、胰蛋白酶溶液和氯化钠溶液 (洗脱) 随后加入。用 TFA、淡化和贮存的方法对含有消化肽的最终滤液进行了酸化。然而, 使用这种方法, 沉淀的蔗糖的大部分在过滤器的顶部稳步积累, 使离心和洗涤步骤困难。
对于丙酮沉淀和氯仿/甲醇沉淀, 需要多量的溶剂来沉淀蛋白质从一个单一的样本量。这限制了在1.5 毫升管 (蛋白质低保留管) 进行降水时应用的试样体积大小。同时, 在两种方法沉淀后, 在管子底部积累的蔗糖粘性污泥, 在丙酮沉淀和氯仿/甲醇沉淀后, 在管底和液体界面上没有可见的小球,分别。
表 3和图 7显示了四种蛋白质提取方法的比较, 我们用来优化我们的实验工作流程。每个方法所给出的红色数字 (图 7) 代表了质谱识别的蛋白质数量 (见5.2 和5.3 节)。虽然每个方法使用的样本量是不同的, 氯仿/甲醇和 TCA 降水导致了最高数量的蛋白质鉴定。然而, 由于上面提到的缺点 (样品容量限制和蔗糖沉淀), 我们选择了 TCA 沉淀为以后实验。
为了找到样品处理的最佳条件, 在不同温度下和 tca 浓度22、23、24进行了 tca 沉淀。因此, 我们执行了一个降水与 10.7% TCA 在4°c 和-20 °c 并且进一步沉淀了上清液与另外60µL 的 tca (最后 19.4% tca) 在药丸以后收集了。这导致了四粒, 分析分离的质谱。此外, 我们重复了两次协议, 以确保获得的结果是可重现的。在图 7 (红色的数字) 中, 工作流和在4°c 和-20 °c 条件下的颗粒中所确定的蛋白质数量。虽然分析从上清液获得的药丸产生了另外数量蛋白质 (85 和60在4°c; 81 和55在-20 °c);大多数蛋白质已经被确定在第一粒, 因此, 我们选择使用 10.7% TCA 的所有后续实验。虽然氯仿/甲醇沉淀导致了与 tca 沉淀相比较的蛋白质的数量相似, 但我们更喜欢 tca 沉淀, 因为有几个优点: i) 需要少量的溶剂沉淀蛋白质 (60 µL 的 tcavs 1.28 毫升氯仿/甲醇/水) 允许使用较大的样本量, 如果需要, 而方便地使用 1.5 mL 管沉淀, ii) 在管底部的颗粒可见性, 和 iii) 不积累蔗糖在沉淀步骤。
尽管提供了关于平移状态的深入功能信息, 但这种方法的一个主要缺点是需要新的和大型的起始材料。许多研究试图通过小体积的细胞或组织来优化条件, 或者添加步骤来提高提取 RNA 和蛋白质的效率25。然而, 如果需要的话, 在相同实验条件下从不同的动物身上获得的组织可以被拉到一起。
最后, 该协议允许同时分析从 polysome 分析获得的分数的 mRNA、miRNA 和蛋白质, 以研究参与缺血/再灌注的调控机制。然而, 需要进一步的研究, 以了解翻译基因是在缺血或再灌注后, 如果他们将关闭后, 消除压力。

心脏以动态的方式对缺血 (I) 和再灌注 (R) 的损伤进行反应。然而, 在反应过程中, 对蛋白质合成的急性变化没有什么洞察力。为了解决这一问题, 我们利用 polysome 分析1的成熟方法来确定蛋白质丰度的变化, 反映核糖体和转化调节因子从细胞质到 polysomes 的再分配, 以及增加新合成的蛋白质 (NSPs)。在 I/R 的设置中, 新蛋白合成的增加发生在与新基因2的转录不一致的时间范围内;此外, 在 mRNA 表达水平和蛋白质丰度之间的不一致报告了3。基于这些原因, 我们选择分析蛋白质翻译所反映的动态蛋白质组的变化。为了做到这一点, 我们量化的 polysome 分数 mRNA, 并分析蛋白质组成的 polysome 分数。最后, 由于 microRNAs (大鹏湾) 调节基因的可用性, 并能干扰蛋白质翻译4、5的效率, 我们研究了大鹏湾在 polysome 馏分中的分布情况, 着重于对我/R 的响应。
我们选择使用孤立的小鼠 Langendorff 灌注模型和收获组织在基础条件下连续灌注, 30 分钟的全球无流性缺血后, 30 分钟的缺血后30分钟再灌注。然后, 我们可溶性的心脏组织和分离的 polysomes 在蔗糖梯度, 其次是蛋白质组分析和选择性检测基因和 miRNAs 的 PCR 和微阵列, 分别。这种方法的组合代表了一个强大的方法来了解动态蛋白质组, 使同时检测 mRNA, miRNA 和 NSPs, 以及调节蛋白, miRNA 和 mRNA 之间的 nontranslating 分数之间的重新分配,低效率的 polysomes 和高效的 polysomes (见图 1)。通过进一步分析诸如 eIF2α或 mTOR 等关键调控因素的磷酸化, 可以进一步深入研究这一过程的动态调节。现在详细描述了这些单独的步骤。

<table>
	<tbody>
		<tr>
			<td>Pentobarbital</td>
			<td>Vortech Phamaceuticals</td>
			<td>9373</td>
			<td>for euthansia</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent</td>
			<td>103424</td>
			<td>used in langendorff preparation</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fine Science Tools</td>
			<td>91110-10</td>
			<td>used to hang the heart</td>
		</tr>
		<tr>
			<td>Langendorff system</td>
			<td>Radnoti + home made</td>
			<td>n/a</td>
			<td>A &#39;four heart&#39; system consisting of custom blown glass, tubing and water baths</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653-5KG</td>
			<td>Krebs buffer and Sucrose gradient</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>Krebs buffer and Lysis buffer</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P-5504</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7774-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G5767</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Sucrose powder</td>
			<td>Sigma</td>
			<td>S0389-1KG</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>208337</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Xylene Cyanole</td>
			<td>Sigma</td>
			<td>X-4126</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-aldrich</td>
			<td>239763</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies</td>
			<td>C00019</td>
			<td>RNAse inhibitor for Lysis buffer</td>
		</tr>
		<tr>
			<td>Igepal CA-360 (NP40)</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail tablets, EDTA free</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>LE-80K</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Beckman</td>
			<td>SW41</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Biologic LP (pump)</td>
			<td>Biorad</td>
			<td>731-8300</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>BioFrac</td>
			<td>Biorad</td>
			<td>741-0002</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube</td>
			<td>Sigma</td>
			<td>Z666513-100EA</td>
			<td>Gradient fraction and RNA extraction</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Life technologies</td>
			<td>AM9738</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Luciferase Control RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C606-4</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023-1L</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>BioRad</td>
			<td>170-8891</td>
			<td>Reverse transcription</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>175-5122</td>
			<td>Quantative PCR</td>
		</tr>
		<tr>
			<td>miRNeasy Micro Kit (50)</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Kit for total RNA isolation</td>
		</tr>
		<tr>
			<td>miScript II RT Kit (50)</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Kit for miRNA reverse transcription</td>
		</tr>
		<tr>
			<td>miScript Sybr Green PCR Kit (200)</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Kit for real-time PCR expression analysis of miRNAs</td>
		</tr>
		<tr>
			<td>Centrifuge 5424R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For centrifugation of 1.5ml or 2.0ml tubes at different temperatures. Max speed - 21130g</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For real-time PCR plate centrifugation at different temperatures. Max speed - 2039g</td>
		</tr>
		<tr>
			<td>My Cycler Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For reverse transcription</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time System/C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td>For quality control of RNA isolation</td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear</td>
			<td>Bio-Rad</td>
			<td>HSP9641</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>For real-time PCR plate sealing</td>
		</tr>
		<tr>
			<td>Research plus Single-Channel Pipette, Gray; 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>UX-24505-02</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P20</td>
			<td>Gilson</td>
			<td>F123600G</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P200</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 &#181;L</td>
			<td>Gilson</td>
			<td>17011782</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>Improves total RNA isolation efficiency</td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 mL Tubes, natural color</td>
			<td>Denville</td>
			<td>C2170</td>
			<td>RNA isolation and storage; reagent mix</td>
		</tr>
		<tr>
			<td>Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically</td>
			<td>Denville</td>
			<td>C18098-4 (1000910)</td>
			<td>Reverse transcription reaction</td>
		</tr>
		<tr>
			<td>Sharp 10 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1096-FR</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 20 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 200 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1122</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Tips LTS 1 mL Filter</td>
			<td>Rainin</td>
			<td>RT-L1000F</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (200)</td>
			<td>Qiagen</td>
			<td>(it changes according to the miRNA)</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Gradient Master ver 5.3 Model 108</td>
			<td>BioComp Instruments</td>
			<td></td>
			<td>For preparation of sucrose gradients</td>
		</tr>
		<tr>
			<td>trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma Aldrich</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Amresco</td>
			<td>M108</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>iodoacetamide</td>
			<td>Gbiosciences</td>
			<td>RC-150</td>
			<td></td>
		</tr>
		<tr>
			<td>sequencing grade modified trypsine, porcine</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium bicarbonate</td>
			<td>BDH</td>
			<td>BDH9206</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A117</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A454</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A996</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 0.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 1.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>HLB &#181;Elution plate 30 &#181;m</td>
			<td>Oasis</td>
			<td>186001828BA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac concentrator</td>
			<td>Thermo Scientific</td>
			<td>Savant SPD2010</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>Qsonica</td>
			<td>Oasis180</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Sorvall Legend micro 21R</td>
		</tr>
		<tr>
			<td>LC trap column PepMap 100 C18</td>
			<td>Thermo Scientific</td>
			<td>160454</td>
			<td></td>
		</tr>
		<tr>
			<td>LC separation column PepMap RSLC C18</td>
			<td>Thermo Scientific</td>
			<td>164536</td>
			<td></td>
		</tr>
		<tr>
			<td>mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Orbitrap Elite ion trap mass spectrometer</td>
		</tr>
		<tr>
			<td>MSConvert software</td>
			<td>ProteoWizard Toolkit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorcerer-SEQUEST software</td>
			<td>Sage-N Research, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion

蛋白质翻译中动态变化的研究需要专门的方法。在这里, 我们研究了新合成的蛋白质对缺血和再灌注反应的变化, 采用离体灌注小鼠心脏结合 polysome 分析。为了进一步了解蛋白质转化过程中的动态变化, 我们以胞内核糖体 (核糖体或 polysomes) 为基因, 并恢复了线粒体 polysomes, 并对其 mRNA 和蛋白质分布进行了比较。高效率的分数 (许多核糖体附着于 mRNA), 效率低下 (附着的核糖体较少), 也包括线粒体 polysomes 和非翻译分数。miRNAs 还可以与正在翻译的基因关联, 从而降低了翻译的效率, 我们研究了跨分数的 miRNAs 分布。对基因、miRNAs 和蛋白质在基础灌注条件下的分布进行了考察, 在30分钟的全球无流缺血和30分钟后再灌注。在这里, 我们介绍了用于完成这一分析的方法, 特别是从蔗糖梯度提取蛋白质的方法, 因为这一点以前没有描述过, 并提供了一些具有代表性的结果。

mRNA 分析 mrna 的结果可以表达为一个特定的 mRNA 在每一个分数的分布 (图 3A);为量化, 结合 polyribosomal 翻译分数和比较非翻译分数 (图 3B), 提出了 mRNA 丰度在翻译到 nontranslating 分数的比率。额外的信息是通过检查高效率的 polysome 分数分别从低效率 polysome 分数 (并与 nontranslating 分数分开) 获得。这在分析 miRNAs 时尤为重要, 在低效率分数 (它们干扰其目标基因的蛋白质翻译) 中丰富。
如图 4所示, 在小鼠心脏受缺血和缺血/再灌注后, 一个特定 mRNA 改变其在翻译和 nontranslating 分数上的分布的典型结果。
miRNA 阵列分析 miRNA 数组分析的代表性结果如图 5所示。在这里, 分析了 miRNAs (定制设计的阵列板) 的子集, 表明 22 miRNAs 在缺血、9 miRNAs 缺血和再灌注过程中与 polysomes 相关, 7 是两种条件共同存在的。这表明, miRNAs 的相关性是动态的, 以应对缺血和再灌注的压力。
蛋白质组分析 虚假 (控制) 样品的重、轻、非翻译分数分析: 以质谱法鉴定总蛋白的 881;3, 46 和208蛋白的总和是独特的重, 轻和非反式分数, 分别 (图 6A)。大多数 (88%) 线粒体核糖体蛋白 (28S 和 39S) 被确定为光分数 (36 出 41) 和大多数 (88%) 胞浆核糖体蛋白 (40S 和 60S) 通常被发现在重和轻的分数 (53 出 60)确认有效的分离和蛋白质组能力识别重胞浆与较轻的线粒体核糖体蛋白。所鉴定的线粒体和胞浆核糖体蛋白的子集如图 6B所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58079/58079fig1.jpg"> 图 1: 蔗糖梯度上 polysome 馏分的典型紫外吸光度剖面.前三个分数代表空的容量在管材。在分数4-7 中收集了高效率的 (高分子量、蛋白大分子) 分数, 低效率 (低分子量, LMW) 分数在 8-11, 而非翻译 (和剩余的胞浆材料) 收集在分数 12-15 (nontranslating, NTR)。红线表示导电性, 蓝色的痕迹表明紫外线吸收率为 254 nm。右边是一个带有蔗糖梯度的试管的卡通片, 显示沉淀后 polysomes 的分布。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58079/58079fig2.jpg"> 图 2: Bioanalyzer 的 RNA 完整性控制分析.从心脏全裂解液中分离出的总 rna 的 rna 完整性数 (玲)。根据18S 和28S 峰值计算。的范围: 1-10, 和玲 = 10 代表一个非常好的完整性。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58079/58079fig3.jpg"> 图 3: 蔗糖梯度中 mRNA 分布的代表性特征.心脏受基底 Langendorff 灌注或缺血再灌注 (I/R)。(A)对 GAPDH (左) 和 COX4 (右) 的蔗糖梯度和 mRNA 含量进行了裂解物, 确定了各分数。图为基底灌注 (假、红线和钻石) 和缺血/再灌注 (I/R, 蓝线和正方形) 中每个分数 (x 轴上的分数数) 的相对 mRNA 丰度。叠加在情节是紫外吸收指示总 mRNA 含量 (浅灰色曲线) 和电导 (直灰色倾斜线)。框表示分数是如何汇集的。(B)条形图图形显示 GAPDH (左) 和 COX4 (右) 基因的翻译 (polysomes) vs nontranslating (NT) 分数的 mRNA 丰度比值。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58079/58079fig4.jpg"> 图 4: 代表性 mRNA 结果.根据实验组的结果, 对聚集分数 (高分子量、LMW、NTR) 中的一个 mRNA 进行定量分析。Langendorff 灌注后对小鼠心脏进行 Polysome 分馏。结果被绘制为% 的总 mRNA (上部面板) 和作为比例的 polysomes (高分子量谷蛋白 + LMW) nontranslating 分数 (NTR)。误差线表示每组5颗心脏的结果 (假、缺血或 I/R)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58079/58079fig5.jpg"> 图 5: 代表 miRNA 结果.downregulated miRNAs 在大鼠心脏缺血、缺血再灌注后的重度池中的 miRNA 分析显示和表达的路径。截止值: 1.5 倍。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58079/58079fig6.jpg"> 图 6: 质谱鉴定的蛋白质。(A)对重、轻和非反式分数常见和独特的蛋白质的维恩图。(B)确定的线粒体和胞内核糖体蛋白的子集。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58079/58079fig7.jpg"> 图 7: 测试蛋白质提取方法的示意图工作流.采用假 (对照) 样和重 (高效 polysomes) 分数和最高蔗糖含量对蛋白质提取方法进行了测试。红色数字表明由质谱法鉴定的蛋白质数量;如果显示两个数字, 则来自两个独立的实验。文中给出了详细的描述。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>分数</td>
			<td>基因 Ct</td>
			<td>荧光素酶 Ct</td>
			<td>ΔCt</td>
			<td>2ΔCt</td>
			<td>所有分数的总和</td>
			<td>每个分数的%</td>
			<td>Polysome/NTR 比</td>
		</tr>
<tr>
<td>高效 (高分子量)</td>
			<td>28.75</td>
			<td>22.79</td>
			<td>5.96</td>
			<td>0.016</td>
			<td rowspan="3">0.046</td>
			<td>34.64</td>
			<td rowspan="3">2.77</td>
		</tr>
<tr>
<td>低效率 (LMW)</td>
			<td>28.43</td>
			<td>22.63</td>
			<td>5.80</td>
			<td>0.018</td>
			<td>38.81</td>
		</tr>
<tr>
<td>非翻译 (NTR)</td>
			<td>29.12</td>
			<td>22.77</td>
			<td>6.35</td>
			<td>0.012</td>
			<td>26.55</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Polysome/NTR 比值 = (谷蛋白 + LMW)/NTR</td>
		</tr>
</tbody></table>表 1:mRNA 的样本分析.在定量 PCR 后, 对不同的聚集分数 (高分子量、LMW、NTR) 进行了一 mRNA 分析的方法。ΔCt = 基因 ct-荧光素酶 ct, 2ΔCt 为 2ΔCt。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">查找 SCM 最小值: 22.77</td>
		</tr>
<tr>
<td></td>
			<td>样品1</td>
			<td>样品2</td>
			<td>样品3</td>
		</tr>
<tr>
<td>Ct 教学</td>
			<td>22.92</td>
			<td>22.36</td>
			<td>22.58</td>
		</tr>
<tr>
<td>Ct 单片机</td>
			<td>22.81</td>
			<td>22.77</td>
			<td>22。9</td>
		</tr>
<tr>
<td>Ct 参考</td>
			<td>23.27</td>
			<td>23.55</td>
			<td>23.19</td>
		</tr>
<tr>
<td colspan="4">将所有 SCM 值规范化到最低限度</td>
		</tr>
<tr>
<td></td>
			<td>样品1</td>
			<td>样品2</td>
			<td>样品3</td>
		</tr>
<tr>
<td>Ct 单片机</td>
			<td>0.05</td>
			<td>0</td>
			<td>0.13</td>
		</tr>
<tr>
<td colspan="4">从教学语言和裁判的 Ct 值中减去这些值</td>
		</tr>
<tr>
<td></td>
			<td>样品1</td>
			<td>样品2</td>
			<td>样品3</td>
		</tr>
<tr>
<td>Ct 教学</td>
			<td>22.87</td>
			<td>22.36</td>
			<td>22.45</td>
		</tr>
<tr>
<td>Ct 参考</td>
			<td>23.22</td>
			<td>23.55</td>
			<td>23.06</td>
		</tr>
</tbody></table>表 2: polysome 和 nontranslating 馏分中 miRNA 表达比较的样本实时 PCR 计算.对缺血或缺血/再灌注后小鼠的聚集重组分进行了 miRNAs 和参考基因 (REF) 的分析。首先将 ct 值归一化至峰值控制 (SCM) ct 值, 然后用 2ΔCt公式对 miRNA 表达进行比较。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>方法</td>
			<td>样品容积 [mL]</td>
			<td>确定的蛋白质</td>
			<td>评论</td>
		</tr>
<tr>
<td>FASP</td>
			<td>1</td>
			<td>227</td>
			<td>过滤器中的大量蔗糖沉淀</td>
		</tr>
<tr>
<td>丙酮沉淀</td>
			<td>0。6</td>
			<td>372</td>
			<td>蔗糖在管底的粘性污泥;4:1 (试剂: 样品) 卷;无可见蛋白颗粒</td>
		</tr>
<tr>
<td>氯仿/甲醇沉淀</td>
			<td>0.32</td>
			<td>389</td>
			<td>蔗糖在管底的粘性污泥;8:1 (试剂: 样品) 卷;无可见蛋白颗粒</td>
		</tr>
<tr>
<td>TCA 沉淀 (在4°c)</td>
			<td>0。5</td>
			<td>405, 415</td>
			<td>无蔗糖沉淀;0.12:1 (试剂: 样品) 卷;管底可见颗粒</td>
		</tr>
<tr>
<td>再沉淀</td>
			<td></td>
			<td>85, 60</td>
			<td>收集了第一粒颗粒后上清的额外的 TCA 沉淀</td>
		</tr>
<tr>
<td>TCA 沉淀 (在-20 °c)</td>
			<td>0。5</td>
			<td>440, 430</td>
			<td>无蔗糖沉淀;0.12:1 (试剂: 样品) 卷;管底可见颗粒</td>
		</tr>
<tr>
<td>再沉淀</td>
			<td></td>
			<td>81, 55</td>
			<td>收集了第一粒颗粒后上清的额外的 TCA 沉淀</td>
		</tr>
</tbody></table>表 3:蛋白质提取方法的比较.FASP, 丙酮沉淀, 氯仿/甲醇沉淀, 和 TCA 沉淀在4°c 和-20 °c 评估。目标是最大限度地确定蛋白质的数量。在第二批材料上对 TCA 降水进行了评估, 以确认重现性。

Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

在这里, 我们提出了一个协议, 执行 polysome 分析的孤立灌注小鼠心脏。我们描述的方法, 心脏灌注, polysome 分析, 并分析 polysome 分数的基因, miRNAs, polysome 蛋白质组。

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

dynamic-proteomic-mirna-analysis-polysomes-from-isolated-mouse-heart

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.5K 次观看.  Cedars-Sinai Medical Center. 在这里, 我们提出了一个协议, 执行 polysome 分析的孤立灌注小鼠心脏。我们描述的方法, 心脏灌注, polysome 分析, 并分析 polysome 分数的基因, miRNAs, polysome 蛋白质组。

视频: Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

科研

生物化学

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

人视网膜解剖与 RPE-脉络膜的蛋白质组分析

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. 
In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.

Here, we present an optimized on-filter digestion protocol with detailed information about the following: protein digestion, peptide purification and data independent acquisition analysis. This strategy is applied to the analysis of expressed prostatic secretions-urine samples and allows high proteome coverage and low missing value label-free profiling of the urinary proteome.

通过 FASP 消化和数据独立分析，EPS-尿液的蛋白质配置文件

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

活植物细胞中的Förster共振能量转移测量

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...