我们感谢 NCFPB 综合核心设施的功能基因组学 (台北, 台湾) 和 NRPB 药物实验室 (台北, 台湾) 的技术支持。这项工作得到了台湾卫生基金会 (白介) 和科技部的研究补助金的支持, 台北, 台湾, 中华民国 [最 105-2320-b-002-047] 为伟瑶胡, [最 105-2628-b-002-051-MY3] 为康怡苏, 和 [MOST-105-2320-B-002-051-MY3] 为梁在林。

1. 底漆/模板准备
<ol><li>设计引物/模板与平衡的 G + C 内容之间的40% 和60% 之间的排序或 PCR 底漆设计。使用18到 21 nt 的引物进行适当的退火和更好的 MS 信号。</li>
	<li>设计模板, 设置50°c 为双工区域的最小熔化温度, 至少 7 nt 5 '-悬置, 以分离的信号之间的底漆和模板。 注: 例如, 对于表 1中的基板 P21/T28, 21-nt 底漆与 28 nt 模板配对。选项: 使用替代核酸 (如核酸酶抗硫代核苷酸键) 替换模板 3 ' 端上的最后4磷酸二酯键, 可以防止模板的非特定 3 ' 端退化可能造成的干扰 (例如,表 1中的 T28S4), 虽然这将增加模板准备的一些成本。</li>
	<li>采用标准淡化寡核苷酸无进一步纯化, 对本研究是满意的。使用寡核苷酸底漆/模板集中 100 pmol/µL 在 H2O 作为一个股票, 并存储在-20 摄氏度。检查底漆和模板的质量和纯度, 通过运行 MALDI 的寡核苷酸 (步骤 4-7), 以确保独特的峰值信号和良好的信噪比。</li>
	<li>确定校准和浓度规范化反应中相关序列的相等摩尔引物的相对 MALDI 的 MS 信号强度 (步骤 7) (图 2)。</li>
</ol>2. 校对反应
注: 相同的校对反应条件和协议适用于 DNA 修复 i、G i、C i 和 T i。
<ol><li>用 P21/T28 基板的 T G 错配作为表 1中校对的一个例子, 稀释底漆 P21 和模板 T28 到 12.5 pmol/µL 与 H2O;然后将稀释底漆的12µL 和12µL 稀释后的模板转移到1.5 毫升的灭菌离心管上。 注: 模板/底漆组合量足以应付2反应;相应地增加试剂的容量为多反应。</li>
	<li>紧紧地关上管子。在65°c 的水浴中孵化30分钟, 然后在37°c 的水浴中再加30分钟, 最后在冰上确保适当的退火。</li>
	<li>对1.5 毫升灭菌离心管, 添加8µL 的底漆和模板组合 (50 pmol), 2 µL 10x 校对反应缓冲器, 4 µL 的 4 ddNTPs 混合 (2 毫米的每 4 dNTPs), 和 H2O 高达18µL. 轻弹管子混合。 注: 1x 校对反应缓冲器包含50毫米氯化钠, 10 毫米氯化镁2, 1 毫米 dithiothreitol, 10 毫米三盐酸 (pH 7.9 在25°c)。参见材料表为商业来源的10x 校对反应缓冲器。反应中每个 ddNTP 的最终浓度为0.1 毫米。</li>
	<li>稀释的 DNA 聚合酶在冰冷的1x 校对反应缓冲到期望的浓度 [例如, 到一个1.5 毫升的离心管, 添加4µL 1x 校对反应缓冲器和1µL 5 U 克莱诺聚合酶从商业来源 (表材料)]。随时将稀释后的酶储存在冰上。 注: 稀释后的 DNA 聚合酶的用量足以应对2反应;相应地增加酵素的容量为多反应。体积为2.0 µL, 含2.0 克莱诺聚合酶, 可以校对超过 50 pmol 的 T G 终端不匹配 P21/T28 在5分钟。</li>
	<li>Prewarm 离心管与基质混合从步骤2.3 到预期的反应温度 (例如, 通常为37°c 为克莱诺聚合酶和25°c T4 DNA 聚合酶)。然后将稀释后的 DNA 聚合酶的2.0 µL 从步骤2.4 转移到 prewarmed 基质混合物中, 然后轻轻地将管子混合在一起。</li>
	<li>在室温下, 用酶和基片离心出几秒钟, 在 3200 x g 处旋转反应的成分。立即转移反应到加热块或水浴在期望的孵化温度和在步骤2.5。</li>
	<li>
终止反应
	<ol>
<li>添加等量的缓冲苯酚, 将其混合涡流几秒钟, 并将其离心在离心 3200 x g 的环境温度5分钟。<ol>
<li>将水相转移到干净的离心管, 加入等量的氯仿, 将其混合涡流几秒钟, 并将其离心在离心 3200 x g 处, 室温下3分钟. 将水相转移到干净的离心管中, 并在cubate 它在95°c 为5分钟;然后把它放在冰上。 注意: 苯酚和氯仿是危险化学品。处理和废物处置应遵循制度规定。</li>
		</ol>
</li>
		<li>或者, 使用酸性淬火协议。为此, 添加2µL 1 米 HCl 停止在冰上的反应6分钟, 然后添加0.64 µL 1 M 二乙醇胺 (DEA), 以中和的 pH 值的反应混合物和孵化它在95°c 为5分钟;然后把它放在冰上。</li>
	</ol>
</li>
</ol>3. 树脂除盐污染
<ol><li>使用的树脂铲从商业脱盐套件 (见材料表), 采取足够的树脂覆盖384酒窝, 6 毫克树脂酒窝板的酒窝。</li>
	<li>通过刮过盘子的顶部, 均匀地 (6 毫克/酒窝) 将树脂分散在所有的酒窝上。回收任何多余的树脂, 并将其替换在瓶子中。</li>
	<li>将2.7 步的最终反应产物从离心管转移到384井板上。分配16µL 的 H2O 稀释最终反应产物在每个井, 然后密封板和离心机, 以消除任何泡沫。</li>
	<li>取下密封, 将384井板与反应产物倒置, 盖上树脂填充的酒窝板。</li>
	<li>翻转好的酒窝板三明治, 并小心地让树脂滴入384井板 (确保384井板是冲对金属钉在树脂填充的酒窝板)。</li>
	<li>除去顶部的酒窝板, 封住含有反应产物和树脂混合物的384井板, 简单地将其离心在 3200 x g 上, 以除去任何气泡, 并将其放在转子上, 使其在室温下进行脱盐, 以淡化其含量。</li>
	<li>树脂清理后, 离心384井板 3200 x 克5分钟, 将树脂压缩到井底。</li>
</ol>4. 将反应产物转移到矩阵芯片上
<ol><li>在 nanoliter 分配器 (nanodispenser, 见材料表) 中设置样品板。</li>
	<li>把矩阵芯片 (见材料表) 和384井板从3.7 节在相应的托盘。</li>
	<li>操作 nanodispenser, 以发现反应产物到芯片;按 nanodispenser 的触摸屏上的 "运行" 按钮开始。</li>
	<li>检查在屏幕上包含饱和信息的样本点的自动捕获图像, 以确保芯片上的总斑点体积约为 5-10 nL。如果音量不足, 请重复取样。</li>
</ol>5. 建立质谱检测信息
<ol><li>准备包含导入的预期信号信息的. xls 格式的文件。例如, 在步骤2、3和4中使用 "P21_T28" (表 2) 中的 P21/T28 校对反应的设置。</li>
	<li>通过在设计器格式中右键单击导入检测组, 并从步骤5.1 中选择. xls 文件 (例如, "P21_T28"), 在应用程序程序中创建并定义新的检测方法 (参见材料表)。</li>
	<li>通过右键单击树顶并给它一个文件名 (例如, "CTT20171201" 表示检测代码和日期),通过屏幕左侧的Customer:Project:Plate选项树建立目标检测板。</li>
	<li>选择384井板类型, 按OK;将出现一个空白的盘子。</li>
	<li>在屏幕左侧选择适当的化验 (如P21_T28)。</li>
	<li>通过突出显示井和右键单击以选择添加丛, 为芯片上的每个样本斑点样本位置分配选定的检测 (例如, P21_T28)。</li>
	<li>在 xlsx 格式的芯片上编写所有测试的工作列表, 没有页眉 (例如表 3的 1201.xlsx)。通过"添加新示例项目" 选项导入工作列表。</li>
	<li>在工作列表中分配测试 (例如, 从 1201.xlsx) 到 P21_T28 检测的每个位置, 然后通过右键单击选择。</li>
</ol>6. MALDI MS 手术
<ol><li>使用应用程序将质谱仪与芯片连接 (见材料表)。</li>
	<li>选择屏幕右侧的默认设置。</li>
	<li>从步骤 5.3 (例如, CTT20171201) 中填充检测名称, 在芯片的拐角处输入芯片 ID, 并保存设置。</li>
	<li>启动 MS 控制程序 (参见材料表)。</li>
	<li>按进/出按钮, 取出侦察盘。将斑点芯片从步骤4.4 放置到侦察板上, 然后按进/出按钮, 使斑点芯片进入质谱仪。</li>
	<li>单击应用程序的 "获取" 按钮 (参见材料表) 以启动质谱并获取数据。</li>
</ol>7. 数据分析
<ol><li>打开数据分析程序 (见材料表)。</li>
	<li>浏览左上角数据库的树, 然后从步骤6.3 中选择芯片 ID。打开屏幕右侧的数据文件。</li>
	<li>单击频谱图标以显示质量频谱。若要裁剪特定范围的 m/z, 请右键单击以选择"自定义" 对话框, 然后在 "新建" 窗口中单击X 轴并设置所需的上限和下限, 然后按"确定" 以显示指定范围的频谱。</li>
	<li>通过右键单击导出, 导出用于记录保留的频谱。 注: 使用 1600 width/1,200 单位的刻度是计算机屏幕上数据检查的合理尺寸。<ol>
<li>选择 JPEG 文件类型, 单击目标, 选择浏览光盘(例如, 闪存盘 e:), 键入文件名 (例如, 1201-1. jpg), 然后单击 "导出"。</li>
	</ol>
</li>
	<li>对于数据定量, 请使用光标并单击峰值以显示左上角的峰值高度。<ol>
<li>或者, 打印导出的 JPEG 文件, 并手动使用标尺测量峰值高度。</li>
	</ol>
</li>
</ol>

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+single+nucleotide+extension+and+MALDI-TOF+mass+spectrometry+in+proofreading+and+DNA+repair+assay.">Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay.</a> DNA Repair. 61, 63-75 (2018).</li><li>Carver, T. E., Hochstrasser, R. A., Millar, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proofreading+DNA:+recognition+of+aberrant+DNA+termini+by+the+Klenow+fragment+of+DNA+polymerase+I.">Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (22), 10670-10674 (1994).</li><li>Santa Lucia, J., Hicks, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thermodynamics+of+DNA+structural+motifs.">The thermodynamics of DNA structural motifs.</a> Annual Review of Biophysics and Biomolecular Structure. 33, 415-440 (2004).</li><li>Su, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+I+proofreading+exonuclease+activity+is+required+for+endonuclease+V+repair+pathway+both+in+vitro+and+in+vivo.">DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo.</a> DNA Repair. 64, 59-67 (2018).</li></ol>

作者没有什么可透露的。

本研究描述了由选择的商用仪器 (见材料表) 分析的一步一步校对活动, 使用 MALDI MS。主要优点包括: 底漆和模板是免费的标签和易于执行, 允许更大的灵活性设计实验。30校对测试的流石灰完整处理需要4小时, 包括3小时手动执行校对反应和清理, 而 MALDI 的分析使用上述协议需要1小时完成。这种方法的主要缺点是化验所需的基底量。为了获得强烈的信号和一致的结果, 我们使用了 50 pmol ?...

dna 聚合酶在 dna 复制过程中的校对功能对于确保需要转移到后代1、2、3、4的遗传信息的高保真度至关重要, 5,6,7。能够评估聚合酶校对 exonucleases 的贡献将澄清保护遗传稳定性的机制。
放射性同位素标记和凝胶检测结合密度分析的 autoradiograms 或荧光粉成像8,9,10历来被用来检测 DNA 校对活动聚合 酶。虽然功能, 这些化验是费力的, 昂贵的, 不适合高通量格式。此外, 放射性同位素还遭受安全问题, 包括废物处理。另外, 通过荧光技术对校对活动进行了分析。例如, 2-aminopurine (2-AP) 可纳入延伸产品在体外聚合酶校对化验, 以产生荧光信号11,12。不幸的是, 这些方法受到低特异性, 因为 2 AP 可以对嘧啶和胞嘧啶。最近的方法包括一个敏感的基于 G 四的荧光开关探针, 用于聚合酶 3 '-5 ' exonuclease 检测13以及一个单一标记的荧光探针, 用于聚合酶校对试验, 克服了一些上述缺点14。由于需要对 DNA 基质进行特异标记, 因此对这些荧光方法的热情减弱。
与此相反, MALDI 的 DNA 分析的 MS 已经被应用于精确检测中, 在那里, 用未标记的 4 ddNTPs 的底漆延伸反应可以用来识别给定轨迹15、16、17和已广泛应用于临床应用的突变检测和癌症诊断18。利用这些基本原理, 我们建立了一个无标签的检测 DNA 聚合酶校对活动利用高分辨率, 高特异性和高通量潜力的 MALDI. 使用E。大肠杆菌DNA 聚合酶 I 克莱诺片段作为一种模型酶, dideoxyribonucleotide triphosphates (ddNTPs) 作为基质可以采取 "快照" 的校对产品后, 单核苷酸延长通过MALDI-飞行 MS (图 1)。
同样, 这种方法也被开发用于 DNA 修复化验, 其中含有 3 ' 倒数二 dI 病变的底漆受到一项模拟中 V 型修复中间体的一项检测。虽然不完全理解, 内 V 修复通路是唯一的修复系统, 已知使用的 exonuclease 的病灶切除19,20。使用 MALDI 的 MS, 我们显示一个明确定义的修复补丁, 其中 dI 可以被切除的第一个 2 nt 的底漆, 然后添加正确的补充核苷酸。
对于校对和 DNA 修复的研究, 该方法比以往的方法更快、更费力, 为机制和功能提供了补充信息。

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1125">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:331px;">Oligonucleotides</td>
			<td style="width:283px;">Mission Biotech (Taiwan)&#160;</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">phosphorothioate modified oligonucleotides</td>
			<td>&#160;Integrated DNA Technologies (Taiwan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNA polymerase I, Large (Klenow) Fragment</td>
			<td>New England Biolabs, MA</td>
			<td>M0210L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Klenow fragment (3&#39;&#8594;5&#39; exo-)</td>
			<td>New England Biolabs, MA</td>
			<td>M0212L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NEBuffer 2.1 (10X)</td>
			<td>New England Biolabs, MA</td>
			<td>B7202S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddATP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddGTP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddTTP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;, 3&#39; ddCTP</td>
			<td>Trilink Biotechnologies, CA</td>
			<td>N4005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dATP, dGTP, dCTP, dTTP set</td>
			<td>Clubio, Taiwan</td>
			<td>CB-R0315</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SpectroCHIP array</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#01509</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MassARRAY&#160;</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Typer 4.0 software</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#10145</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Clean Resin Tool Kit&#160;</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#08040</td>
			<td></td>
		</tr>
	</tbody>
</table>

proofreading and dna repair assay using single nucleotide extension and maldi-tof mass spectrometry analysis

维持基因组及其忠实的复制对保存基因信息至关重要。为了评估高保真度复制, 我们开发了一种简单的无标记和非无线电同位素方法, 利用矩阵辅助激光解吸电离与飞行时间 (MALDI) 质谱 (MS) 分析进行校对研究。在这里, 一个 DNA 聚合酶 [例如,克莱诺片段 (KF) 大肠杆菌 DNA 聚合酶 i () 在本研究中] 在所有四 dideoxyribonucleotide triphosphates 的存在, 用于处理不匹配的底漆模板双工。不匹配的底漆, 然后校对/延长和受 MALDI 的 MS。产品由底漆的质量变化区分到单核苷酸变异。重要的是, 校对也可以确定内部单一的不匹配, 虽然在不同的效率。2-4-核苷酸 (nt) 从 3 ' 年底的不匹配是有效地校对了由我, 和不匹配的 5 nt 从底漆总站只显示了部分更正。在 6-9 nt 从底漆 3 ' 末端没有校对发生内部不匹配。这种方法也可应用于 DNA 修复化验 (例如,评估内 V 修复通路基底损伤修复)。含 3 ' 倒数 deoxyinosine (dI) 病灶的底漆可由 i. 型矫正。事实上, 倒数第二 T i、G i 和 i 基板在添加正确的 ddN 5 '-单磷酸 (ddNMP) 之前, 有他们最后的 2 dI 含核苷酸, 而倒数第二个 C i 的不匹配是由我来容忍的, 允许引伸不修复, 显示的敏感性和分辨率的 MS 化验, 以测量 DNA 修复。

模板和底漆:
使用此处介绍的程序, 检查了从商业来源获得的等摩尔合成寡核苷酸模板和相关序列的引物, 以确定其纯度和质量 (图 3A; 注意匹配指定质量的信号和低背景) 以及峰值强度与分析物质量之间的关系 (图 2A)。测量和计算了峰值高度 (图 2B),...

Watch this Scientific Journal Video about Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis at JoVE.com

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

采用高分辨率 MALDI 质谱和单核苷酸扩展策略, 建立了 dna 聚合酶校对和 dna 修复实验的非标记非射电同位素方法。试验证明是非常具体, 简单, 快速, 并易于执行校对和修复补丁短于 9-核苷酸。

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

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Unit 1

DNA Replication

DNA Replication and Repair

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9.5K 次观看.  National Taiwan University. 采用高分辨率 MALDI 质谱和单核苷酸扩展策略, 建立了 dna 聚合酶校对和 dna 修复实验的非标记非射电同位素方法。试验证明是非常具体, 简单, 快速, 并易于执行校对和修复补丁短于 9-核苷酸。

视频: Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

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 ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

双DNA标尺研究单核苷酸分辨率的核糖体易位机制

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...