Dr. Rawi Ramautar would like to acknowledge the financial support of the Veni grant scheme of the Netherlands Organization of Scientific Research (NWO Veni 722.013.008).

注意:这里描述了使用鞘CE-MS的代谢谱研究该协议仅用于实验室使用。下面列出的程序是根据最近发表的作品4,5。进一步的实验细节，可以在这些文件中找到。此前使用此协议，请所有相关的材料安全数据表（MSDS）。请使用所有适当的实验室安全程序，包括安全眼镜，白大褂和手套，在开展该协议所列的实验时。 1.试剂溶液和样品的制备<ol><li> 背景电解质的制备（BGE） <ol><li>准备新的BGE溶液（10％（V / V）乙酸，pH值2.2）的每一天。 </li><li>加9.0毫升水到10ml玻璃小瓶中，并在通风橱中加入1.0毫升乙酸的水。调匀使用涡旋该溶液中。 </li></ol></li><li> 代谢STANDAR的制备ð混合<ol><li>溶解50微升含有60阳离子代谢成50微升水中的50μM的阳离子标准混合物中，将溶液充分混合。在-80°C储存在不使用时。 </li><li>溶解50微升含有30阴离子代谢成50微升水中的50μM的阴离子标准混合物中，将溶液充分混合。存储此溶液，以等分试样，以防止相同的标准混合物的冷冻/解冻循环，在-80℃下，不使用时。 注:当代谢产物在-80℃妥善保存标准混合物连续3个月保持稳定。 </li></ol></li><li> 提取物对胶质母细胞瘤细胞系的制备 <ol><li>洗粘附人的U-87 MG成胶质细胞瘤细胞三次用1ml冰冷的0.9％氯化钠溶液21中 。 </li><li>加入2毫升冰冷的甲醇/水的溶液（8/2，体积/体积），以贴壁细胞并用橡胶放倒细胞刮刀21刮除。 </l我&gt; <li>收集2分钟在管的甲醇/水的溶液和ultrasonicate。 </li><li>在16100 xg离心4℃10分钟加氯仿至甲醇/水级分（最终比8/8/2，体积/体积/体积）并离心样本。 </li><li>收集甲醇/水层，并用真空浓缩器蒸发该馏分。通过鞘CE-MS重组在50μl水中分析干燥的材料。在不使用时存放在-80℃的样品。 </li></ol></li></ol> 2.设置鞘CE-MS系统<ol><li> 裸石英墨盒安装与多孔尖端发射极 <ol><li>将新的裸石英墨盒在CE仪器的多孔尖端发射极（共30微米内径×90厘米长）。 </li><li>在50psi施加向前漂洗用100％甲醇的软件15分钟控制CE仪器并进行目视检查液体是否从capil流出在此冲洗步骤毛细管出口。此外，在相反的方向上在50psi进行漂洗使用BGE溶液可视地检查液体是否从导电毛细管流出5分钟。 </li><li>重复步骤2.1.2在万一没有液滴形成已在毛细管出口被观察到100磅的压力。如果在该步骤中没有观察到液滴形成安装一个新的裸石英盒。 </li><li>在50psi 10分钟用水冲洗分离毛细管在50psi进行10分钟，随后用0.1 M氢氧化钠在50psi进行10分钟，然后用水，最后在50与BGE psi的10分钟。 注意:步骤2.1.1至2.1.4只需要安装新的毛细管墨盒。 </li></ol></li><li> 毛细多孔尖端发射极到ESI-MS耦合 注:以耦合CE毛细管MS，确保MS仪器被校准并连接到CE系统之前。设置ESI电压0适合用纳米喷雾源的MS仪器。气体1，天然气2接口加热器温度在非常低的流量并没有用作ESI只把它运用离子喷雾电压设置为1500 V.设置窗帘在5 PSI发生。 <ol><li>从水管除去石英盒的喷雾器尖端和在纳米喷雾源适配器安装它用于耦合到MS仪器。确保在CE仪器的BGE小瓶的高度匹配喷雾器尖端的高度。 </li><li>通过在50psi与BGE漂洗5分钟检查通过导电毛细管液流。在此冲洗步骤在ESI喷雾器针的基部的液滴形成应观察。 </li><li>冲洗用BGE分离毛细管在50psi在向前方向10分钟。液滴形成应在多孔尖端发射极（喷雾器尖端）的前端在该步骤中被观察到。 </li><li>在C的距离的多孔尖端发射极到MS入口的入口位置IRCA 2〜3毫米。 </li><li>用1分钟的升温时间申请30千伏的电压，并开始在m / z范围获取MS数据从65至1000 米/ z计算使用0第一电喷雾电压代谢谱的研究。 注:质谱应该是无效的信号，因为应该没有电。 </li><li>设置ESI电压为1,000V，而测量数据的携带。加大与200伏增量ESI电压直到恒定背景信号观察至少15分钟。 </li><li>通过在x，y或z方向，以查看哪个位置提供的最大的和最稳定的MS信号（总离子电泳）移动它优化相对于该MS入口中心的多孔尖端发射极的位置。 </li><li>优化多孔尖端发射极的位置和确定最佳ESI电压后，设置ESI电压为0V，并使用了5分钟的升温时间为30千伏降低CE的电压为1千伏。 </li><li>创建一个MS法全光照克最佳ESI电压在CE仪器的代谢物标准和生物样品分析的方法CE。 </li></ol></li></ol> 3.代谢物标准和生物样品分析<ol><li> 在鞘CE-MS系统的性能评价 <ol><li>转让20微升的阴离子代谢物的标准混合物成适合在CE小瓶中，并把这个小瓶在进口样品盘空100微升microvial（PCR瓶）。 注:在microvial一个可靠的注射所需的最小体积为2微升。 <ol><li>启动STEP 2.2.9期间创建的MS采集负离子模式的方法，并随后开始使用软件控制CE仪器的CE序列。 </li><li>在1.0磅10冲洗BGE分离毛细管在50psi 3分钟，然后注入在2.0磅60秒（对应于毛细管体积的3％20 NL），然后通过BGE注射秒。 注意:此后，MS数据采集被触发。 </li><li>适用-30千伏的电压为1.0分钟，0.5磅的用于在入口30分钟的压力的斜坡时间。 30分钟电泳分离后，停止MS数据采集和利用5分钟的升温时间（CE电压的逐渐减少电泳分离提高了多孔尖毛细管发射器的耐用性之后）降低CE电压为-1千伏。 </li></ol></li><li>样品进样之间，用清水冲洗毛细管，0.1M氢氧化钠，水和BGE各在30psi 3分钟。 </li><li>通过确定迁移时间和所分析的阴离子代谢物的混合物的信号强度分析所记录的数据。 </li><li>评估阴离子代谢物标准是否10和28分钟之间出现在该地区。 </li><li>检查是否三种结构相关的异构体， 即 ，D-葡萄糖-1-磷酸，D-葡萄糖-6-磷酸和D-果糖-6-磷酸是部分分离， 即 ，前两个峰之间的分辨率为大约0.75和最后两个峰的是大约0.50（ 见图1）。 注:1.5的决议表明了两个相邻峰的基线分离。 </li><li>重复步骤3.1.1和3.1.2阳离子代谢的混合物。确保MS检测现在处于正离子模式和CE电压为+30千伏。 </li><li>通过确定迁移时间和所分析的阳离子代谢物的混合物的信号强度分析所记录的数据。 </li><li>评估阳离子代谢物标准是否出现在8和22分钟之间的区域。检查异亮氨酸和亮氨酸是否迁移15和15.5分钟之间，并确定如果分辨率大约0.5。 </li></ol></li><li> 生物样品的分析 <ol><li>重复步骤3.1.1和3.1.2的胶质母细胞瘤细胞系的提取物的阴离子代谢轮廓。 注:代谢CON样品预处理之后获得10吨对应于大约20个细胞/ NL的细胞密度，因此，一个20 NL注射是每次分析400个细胞的等价物。 。 </li><li>创建用于使用5 MDA质量精度的代谢产物乳酸（M / Z 89.0243）提取离子电泳，并检查该信号强度是否高于100,000计数。 </li><li>重复步骤3.1.1和3.1.2的胶质母细胞瘤细胞系的提取物的阳离子代谢轮廓。 注:一个高度信息丰富的总离子电泳应该在此模式下20 NL注射液被观察到。 </li><li>的分析或在不使用时后，用水冲洗所述毛细管在50psi进行15分钟，并在毛细管的入口部分存储在一个含有小瓶水和多孔质部（出口部）中的管也含有水。 </li></ol></li></ol>

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We have nothing to disclose.

采用多孔尖端发光体A鞘CE-MS法已经提出了高极性和电荷的代谢物的分析。这种方法的独特之处在于阴离子或阳离子代谢产物只能由切换MS检测和CE电压极性进行概要。广泛的生物样品中的高极性和电荷的代谢物能够以高的分离效率，这是结构上相似的代谢物是至关重要的，并与在（低）纳摩尔范围检测限进行分析。所提出的协议集中在使用鞘CE-MS的对于细胞提取物的代谢谱，以举例说明用于生物样品的代谢谱的方法的效用。此处所描述的方法也可以用于其它类型的生物样品的代谢谱，如人尿5，考虑到一个适当的样品预处理过程被使用。 该鞘CE-MS方法具d被基于一个多孔尖端发射极，它允许CE的固有的低流动性的用法。在这种情况下，稳定的ESI信号是一个先决条件可再现代谢谱的研究。因此，重要的是，喷雾器尖端被正确地定位在MS入口的前面。在此设置中，在ESI过程主要依赖于BGE的性质，因此，BGE优化是非常关键的。所述鞘结构相比，可加入各种鞘液体组合物以提高离子化效率鞘液的CE-MS系统少的通用性。所述鞘ESI喷雾器针需要完全填充导电液体（ 即 ，BGE溶液）。不稳定ESI信号可能导致从部分或完全堵塞毛细管。在与BGE高压冲洗就可以解决这个问题。否则分离毛细管需要更换。在此之前的分析性能考核，稳定的ESI背景信号应产生的第一是从一天到另一相一致。 需要使用标准的代谢产物为混合物，每天检查鞘CE-MS方法进行代谢谱研究的分析性能。在相同的实验条件下，一致的迁移时间， 即低于3％的变化对于内天（N = 10）和日间（N = 5），使用的代谢物标准混合物（12.5μM）的20 NL注射，峰高度/地区（低于15％的变化）和车牌号码（60,000至400,000范围）应获得。检测限应该在纳摩尔范围对大多数代谢物标准。只有当这些条件都满足是准备用于生物样品的代谢谱的方法。如果不是，则MS仪器需要调整和重新校准或需要更改多孔尖端毛细管发射极。 CE-MS之间的一种有效的漂洗步骤的分析是非常重要，不仅是为了防止潜在的交叉污染也能保持分离性能。潜在交叉污染可通过污染与样品，因此通过置换新BGE小瓶解决BGE小瓶引起。当鞘CE-MS法是不使用时，它断开分离毛细管并存储在水中的毛细管，并与在含有水以延长毛细管寿命的管保护套筒浸没外侧的入口侧是重要的。 总之，所提出的无鞘CE-MS法示出了根据在此协议报告的过程中使用时，生物样品的代谢谱的巨大潜力。在这个阶段，实验室间比数据肯定，以评估这种做法对代谢的（长期）的重现性和稳健性需要鞘CE-MS。该协议可能会刺激这样的研究。各种分析挑战仍需要考虑。为了获得最佳的PERFormance，在CE电流应该优选低于5μA和在此阶段仅在91厘米的长度，有可能妨碍高通量测定法的发展提供的毛细多孔尖端发射器被保持。另外，用于阴离子代谢轮廓这可能不是最优化的用于实现结构相关的糖磷酸酯的基线分离低pH分离缓冲液。同样重要的是，只有阴离子代谢产物可以分析这些（部分）带负用过的分离条件下充电。下一步骤是为目前评估鞘CE-MS法用于临床代谢谱研究的效用，单一多孔尖端的毛细管发射器只能用于多达100生物样品的分析。 总体来看，在鞘CE-MS方法的进一步发展将在代谢组学， 即领域打开一个新的方向，朝中的生物功能有更深的了解充裕的限制情况。

在当代代谢，高端分析分离技术被用来进行分析，以获得一个代表广泛代谢物类的读出的生物体1的生理状态。代谢组学研究的最终目标是获得一个答案，一个给定的生物/临床问题。目前，人类代谢组数据库包括代表内源性和外源性化合物（从营养物，微生物群，药物和其它来源后者始发）4万多代谢物的条目2。定在物理 - 化学性质和这些代谢物的浓度范围的巨大多样性，不同的分离机制的多个分析技术应结合以轮廓尽可能多的代谢物尽可能给定生物样品中的使用。例如，Psychogios 等人使用五个分析分离技术的组合为代谢教授导致检测4000多个不同的化学代谢物3的人血清iling。 在本文中，将注意力投放到最近开发的CE-MS策略生物样品4,5的代谢谱。在CE中，更具体地毛细管区带电泳（CZE;通常称为CE）化合物是分离的它们的电荷多尺寸比率的基础上，因此，此分析技术是非常适合于极性及带电代谢物的分析。 CE的分离机理是从基于色谱的技术根本不同的，由此提供对生物样品6-8的代谢组合物的互补视图。曾我和同事是第一个显示的CE-MS的效用为代谢物的生物样品9,10在全局分析。到现在为止，CE-MS的代谢组的可行性和效用已得到广泛证实11-15。CE经由鞘液接口技术16,17通常耦合到MS;然而，由于由鞘液的毛细管流出的稀释，检测灵敏度固有损害。 最近，证明的是，使用一个无鞘接口相比，CE-MS利用经典鞘-液界面5,18,19显著改善本多种生物样品中的代谢物的检测范围。例如，被人体尿液中鞘CE-MS检测到大约900分子特征，而与鞘液CE-MS 5中观察到约300分子特征。所使用的无鞘接口是基于多孔尖端发射极，其通过Moini 20发明，使得在组合的有效使用的CE的固有的低流动性的纳米ESI-MS。 为了刺激代谢组学领域的使用鞘CE-MS的，提出的协议描述该方法如何可用于生物样品中的高极性代谢物的分析，作为例示用于从成胶质细胞瘤细胞系提取物的分析。它表明，也可用于使用完全相同的毛细管和分离条​​件，从而减少分析时间和的全局分析提供一个单一的分析平台的阴离子代谢物的分析阳离子代谢物的分析的无鞘CE-MS法充电代谢物。该协议还介绍了与MS仪器鞘多孔尖端发射极的有效调整的策略。

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CESI 8000 instrument</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">A98089</td>
			<td style="width:395px;">OptiMS adapter required to couple CESI to MS</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">OptiMS Fused-Silica Cartridge, 30 &#956;m ID x 90 cm total length</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">B07367</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">OptiMS Adapter for Sciex Nanospray III source</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">B07363</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CESI vials</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">B11648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro vials</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">144709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glacial acetic acid</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:181px;">A6283</td>
			<td>Use in fume hood</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Cationic metabolite mixture</td>
			<td style="width:167px;">Human Metabolome Technologies</td>
			<td style="width:181px;">H3304-3034</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Anionic metabolite mixture</td>
			<td style="width:167px;">Human Metabolome Technologies</td>
			<td style="width:181px;">H3304-1031</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Methanol (LC-MS Ultra Chromasolv)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:181px;">14262</td>
			<td>Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hydroxide solution</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:181px;">72079</td>
			<td>0.1 M</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">U-87 MG Glioblastoma cell line</td>
			<td style="width:167px;">Sigma</td>
			<td>89081402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:167px;">Sigma</td>
			<td align="right" style="width:181px;">650498</td>
			<td>Toxic; use in fume hood</td>
		</tr>
	</tbody>
</table>

sheathless capillary electrophoresis&#8211;mass spectrometry for metabolic profiling of biological samples

在代谢组学，广泛的分析技术被用于复杂样品中（内生）的代谢产物全球分析。在本文中，一个协议被提出用于通过毛细管电泳 - 质谱（CE-MS）的生物样品中的阴离子和阳离子的代谢物的分析。 CE是非常适合高极性带电代谢物的分析化合物的电荷 - 大小比率的基础上分离。最近开发的鞘接口设计， 即，多孔尖接口，用于连接CE电喷雾（ESI）MS。这种接口方法允许组合的有效使用的CE的固有的低流动性的与MS，导致对大范围的极性代谢物类纳摩尔检测限。这里介绍的协议是基于在低pH的分离条件采用裸熔融石英毛细管与多孔尖端发射极对的一个分析生物样品中的代谢物类宽阵列。它证明了相同的无鞘CE-MS法可以仅切换MS检测和可用于阳离子代谢物，包括氨基酸，核苷和小肽，或阴离子的代谢产物，包括糖磷酸盐，核苷酸和有机酸，的分析分离电压极性。各种生物样品中的高度信息丰富的代谢概况，如尿，脑脊髓液和胶质母细胞瘤细胞系的提取物，可通过该协议中的CE-MS分析的不到1小时来获得。

所提出的无鞘CE-MS法是能够提供高效率的，即，塔板数范围从60,000至400,000，在用10％乙酸（pH为2.2）作为BGE纳摩尔检测限阴离子和阳离子的代谢物分布。对于高极性阴离子代谢物的分析方法的分离性能是展示三个结构相关的糖磷酸异构体（ 图1）。虽然这三个分析物不能获得基线分离，部分分离足以允许其被MS选择性检测这些分析物具有相同的精确质量。所述鞘CE-MS法的对数细胞的限制，代谢谱的电位即 20 NL喷射对应于400个细胞（细胞密度为大约20个细胞/ NL）中，展示了阳离子代谢物中的提取物的分析在胶质母细胞瘤细胞系（图2），其中上述一个的S / N比≥5检测到超过300个分子的功能。 <img alt="图1" src="/files/ftp_upload/54535/54535fig1.jpg" /> 图1.糖磷酸异构体的分析鞘CE-MS提取离子为负离子模式与鞘CE-MS得到的三种糖磷酸异构体（25μM）电泳。实验条件:BGE，10％乙酸（pH为2.2）;分离电压，-30千伏（+0.5磅施加在CE毛细管入口）;样品注射，2.0磅60秒。经许可后转载4。 <a href="https://www.jove.com/files/ftp_upload/54535/54535fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54535/54535fig2.jpg" /> 图2.异亮氨酸和L的分析通过鞘CE-MS提取离子的正离子模式与鞘CE-MS获得的两个氨基酸的异构体（25μM）电泳eucine。实验条件:BGE，10％乙酸（pH为2.2）;分离电压，+ 30千伏;进样，2.0磅，持续60秒。 <a href="https://www.jove.com/files/ftp_upload/54535/54535fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54535/54535fig3.jpg" /> 图3. 鞘CE-MS的潜力在细胞系提取物分析阳离子的代谢物。代谢轮廓（总离子电泳）在正离子模式下与鞘CE-MS成胶质细胞瘤细胞系的提取物观察到的。实验条件:BGE，10％乙酸（pH为2.2）;分离电压，+ 30千伏;样品注射，2.0磅60秒。经许可后转载4 </s了&gt;。 <a href="https://www.jove.com/files/ftp_upload/54535/54535fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Sheathless Capillary ElectrophoresisÃ¢â‚¬â€œMass Spectrometry for Metabolic Profiling of Biological Samples at JoVE.com

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

使用鞘多孔提示界面设计的毛细管电泳 - 质谱联用生物样品的代谢谱的协议提出。

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

sheathless-capillary-electrophoresismass-spectrometry-for-metabolic

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.6K 次观看.  Leiden University. 使用鞘多孔提示界面设计的毛细管电泳 - 质谱联用生物样品的代谢谱的协议提出。 

视频: Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

使用中性毛细管单克隆抗体亚型的毛细管电泳分离

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

科研

生物化学

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

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 ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

电子顺磁共振在生物样品环境温度和 77 k 的应用

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

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 ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

极快速和特殊的代谢标签的RNA在Vivo与4-Thiouracil （Ers4tU）

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...