SW和烟草花叶病毒是在美国国立卫生研究院支持的NCRR和NHLBI。 BSK和HS是由美国国立卫生研究院的NINDS和NIGMS支持。

实验步骤实验步骤（图2）包括四个部分，下面是一个分步进行的方式描述。 第1部分：亚克隆和表达P2X2受体的C -末端。 我们已经表达P2X2受体在细菌的C -末端全长识别脑蛋白与之结合。 <ol><li> C -末端（残基353-472）的P2X2受体（图1）经PCR扩增，克隆到PGEX 4NT1（GE生命科学）和测序验证。 </li><li>重组质粒转化E.表达重组蛋白的大肠杆菌（BL21） 。 </li><li>甘油包含P2X2 CT - GST重组质粒的细菌（大肠杆菌BL21）股票被刮掉，用无菌枪头，接种到5毫升卢里亚Bertani（LB）媒体与合适的选择标记“（50克/ mL氨苄青霉素）和在37C轨道摇床孵育过夜。 </li><li>用适当的抗生素（50μg/ mL氨苄青霉素），分别接种到250毫升LB培养基过夜的文化37C在轨道摇床孵育2-3小时在250 RPM，直到在600 nm的光密度达到~~ 0.6-0.7。 </li><li>文化是1毫米的IPTG诱导蛋白表达3小时，并进一步培养在37C。 </li><li>当时的文化纺5000克4C（文化1毫升是保存的表达水平进行分析，并标示为“诱导”）为15分钟。弃上清液，沉淀重悬于20毫升生理盐水（STE）的Tris - EDTA缓冲液（10毫米的Tris - HCl，150 mM氯化钠，1 mM EDTA的pH值8.0）。 </li><li>暂停15分钟在50毫升猎鹰管纺5000克。弃上清液和半干燥的颗粒被冻结在- 70C过夜。 </li><li> - 70C颗粒，悬浮在冰冷的裂解液（2％（W ​​/ V）sarkosyl 15毫克，溶菌酶，氯化钠150毫米，50毫米的Tris PH7.5，5毫米DTT和完整的蛋白酶抑制剂片）40毫升冰浴30分钟，和悬浮培养。在此孵化，3毫升谷胱甘肽琼脂糖4B珠20毫升磷酸缓冲液（PBS）和20毫升的T - PBS（0.1％（V / V）的PBS tritonX - 100），然后用PBS洗涤。珠子悬浮在1 mL PBS。 </li><li>菌悬液冷冻在液氮中解冻3次，超声（1S开/关，冰3分钟30微米），并进一步培养30分钟，用4％（V / V）Triton X - 100，10 mM的硫酸镁4和2毫米ATP在冰上。 </li><li> 20000克，4C和沉淀15分钟，被丢弃（沉淀和SDS - PAGE保存到测试在细胞质和细胞膜蛋白质的量的1毫升上清样本）裂解液中剥离出来。上清孵育1小时（或过夜），在4C的珠子。 </li><li>纺500克珠5分钟，弃上清液（样品保存的未绑定的分数）。珠洗的T - PBS五倍（涤洗控制保存）。 </li><li>珠子，然后悬浮在PBS给予50％（W / V）浆，存放在冰箱，直到使用4C。估计蛋白浓度由Bradford法（每制造商的指示）。 </li></ol>第2部分：全脑裂解液的制备 P2X2受体是已知的要丰富大脑中的8的表达。在目前的分析，我们试图找出与P2X2受体从大鼠全脑细胞裂解液（图3）相互作用的蛋白质。 <ol><li>成年大鼠脑收获（处死动物，根据加州大学洛杉矶分校IAACUC批准的协议，并根据美国国立卫生研究院实验动物的照顾和使用指南），并立即冲洗冰冷的PBS（3X）。 </li><li>裂解细胞（10毫升〜5倍的收获脑湿重）冰冷的裂解缓冲液含有150毫米的NaCl，50毫米的Tris - HCl，pH值7.4，10 mM的EDTA，EGTA 1毫米，1毫米的氟化钠，1毫米娜3 VO 4 PMSF，1毫米，5微克/毫升的亮肽素，1％（V / V）NP - 40，和一种蛋白酶抑制剂的鸡尾酒平板添加到收获的大脑。 </li><li>脑组织均质玻璃Dounce匀浆冰，直到混合均匀的一致性。 </li><li>在2000克5分钟取出完整的细胞，细胞裂解液中剥离出来。上清收集和纺60分钟再次删除完整的细胞器和膜聚集在29000克。 </li><li>离心后的上清液转移到一个干净的管。全脑裂解液的蛋白浓度测定Bradford法（通常〜15毫克/毫升）。 </li><li>裂解液分装和存储在- 70C。 </li></ol>第3部分：P2X2的C -尾部相关蛋白的分离为了确定P2X2 CT - GST的具有约束力的合作伙伴，从整个大脑细胞裂解液，一拉下来检测，通过使用CT - GST固定在谷胱甘肽琼脂糖4B珠为诱饵。对于控制，消费税单独绑定珠使用。 <ol><li>脑裂解precleared在4C 1小时的消费税珠。颗粒被丢弃上清液用于进一步分析。 </li><li>同等数量的商品及服务税和P2X2的CT - GST（10微克）绑定谷胱甘肽琼脂糖4个B珠孵育过夜，100克precleared可溶性蛋白大鼠全脑裂解液，裂解液4C的转动。 </li><li>珠纺在1000克5分钟，弃上清液。 </li><li>珠裂解液洗一次，并在修改Laemli缓冲液煮沸4％（W / V）SDS，10％（V / V）2 - 巯基乙醇，甘油2％（V / V），0.004％（W / V）溴酚蓝和0.125中号TrisCl pH值6.8] 5分钟。 </li><li>对样品进行了5分钟和10％的SDS - PAGE（图4）分离的上清纺5000克。凝胶染色SYPRO Ruby的蛋白凝胶染色（按制造商的指示）和在紫外线照射下的可视化。 P2X2的CT相关蛋白进行了比较与回收为诱饵，利用GST空。 4个生物复制被执行（即4个大脑和4个独立的下拉）。 </li></ol>第4部分：蛋白质的鉴定。 凝胶电泳分离的蛋白质从凝胶上切下 ，注意通过质谱12鉴定：在整个过程中没有灰尘，以减少角质污染的关键。 实验者应戴上口罩，发网和手套，在任何时候都永远无法触摸凝胶地区利益，没有使用手套。 <ol><li> P2X2下拉收回所有可见的蛋白质乐队用干净的刀片切和泡（图4）。 GST空相应的凝胶地区下拉巷也以同样的方式切除，而不带染色（GST空诱饵特定的频段也被切除，并确定）以下的水平，以确保蛋白质的存在染色的灵敏度是不容错过。 </li><li>凝胶片孵育50毫米碳铵200μL50％（V / V）乙腈（ACN），在室温下15分钟振荡管。重复此清洗步骤。 </li><li>凝胶片加入200μL乙腈（凝胶块收缩，成为不透明的颜色）脱水。 </li><li>乙腈被删除，干凝胶块〜10分钟speedvac。 </li><li> 30μL新鲜配制的10 mM的DTT/10 mM的三（2 - 羧乙基）膦盐酸盐（TCEP），足量的凝胶块，然后孵育沉浸件。这是留在56C水浴30分钟。 </li><li>下一步的数码地面电视服务的解决办法是更换100μL500毫米，50％的乙腈溶液碳铵和凝胶，ammounium碳酸氢钠洗净切片。 </li><li>洗涤液的吸气和200μL乙腈脱水凝胶。 </li><li>乙腈然后取出100μL新鲜配制的100毫米碘乙酰胺的解决方案添加到烷基免费sulfhydryls。 25分钟室温在黑暗中进行这一步。 </li><li>碘乙酰胺的解决办法是删除和凝胶块50毫米，碳铵50％乙腈（pH值8.0）2分钟与200μL洗涤，而震荡。重复此清洗步骤。 </li><li>取出后洗涤液，凝胶块孵育200μL乙腈，这是当时被speedvac删除〜10分钟。 </li><li>在这一步的凝胶块，胰蛋白酶消化的准备。凝胶片孵育30μL胰蛋白酶溶液（20毫微克/μL工作浓度），并置于冰上10分钟要肿凝胶。删除多余的胰蛋白酶是水合凝胶颗粒碳酸氢铵50毫米与30μL覆盖整个消化被允许在37C进行12至16小时，使他们沉浸。 </li><li>五μL5％（V / V）甲酸加入到停用的胰蛋白酶和逮捕消化。 </li><li>管振荡〜15分钟，离心，简单地把液体的试管底部，然后转移到一个干净的标记0.5毫升的小瓶。 </li><li>下30μL0.1％（V / V）50％乙腈甲酸加入到凝胶块，所以他们只是受两次振荡10-15分钟，短暂离心分离。此步骤重复一次，更换步骤之间的甲酸乙腈。 </li><li>最后的液体被删除，所有萃取液合并在一个单一的小瓶。 〜30 UL的speedvac体积减小。该样本是现在准备反相纳米液相色谱和质谱的蛋白质鉴定。在这个特定的例子中，这一步是开展orbi - 罗马和世界阱串联质谱仪从ThermoFisher（质量准确度，快速蛋白检测的占空比和一般健壮的操作系统功能选择），虽然其他型号和制造商的仪器可以很容易地描述到这一点的方法相结合。 </li><li>现简要介绍了质谱分析的细节和标准。所有肽的分离反相纳米LC（Eksigent）和肽类加载到C18反相柱在3μL/ min的流速。流动相A为0.1％甲酸和2％的水乙腈，流动相B为0.1％甲酸的乙腈和20％的水。肽列在220 NL /分钟使用线性渐变从5％B级50％B级超过90分钟，然后到95％，超过5分钟乙的流速洗脱，最后5分钟保持恒定的95％乙。光谱获取数据依赖模式MS扫描和MS / MS扫描的LTQ使用Orbi陷阱。肽进行鉴定通过搜索对大鼠IPI数据库使用SEQUEST算法集成到Bioworks软件包v.3.53光谱。每个肽符合下列条件：XCorr≥2（1）≥3（2），≥4（3），和DeltaCN&gt; 0.1。用于蛋白质鉴定的所有光谱手动检查，并没有接受蛋白质时不到二肽。只有蛋白质目前每四个P2X2下拉，在GST空下拉缺席被认为是为进一步分析。 </li></ol><img src="/files/ftp_upload/1178/1178fig1.jpg" alt="图1" /> 图1。一个P2X2受体亚基的示意图 。 答 ：卡通说明一个P2X2受体亚基的拓扑结构。胞浆域由N和C末端。 P2X2受体（红色）的C -末端为诱饵拉下来检测 。P2X2受体在这项研究中所使用的C -末端氨基酸序列<img src="/files/ftp_upload/1178/1178fig2.jpg" alt="" /> 图2。流程图和时间表达所使用的协议，净化，下拉和蛋白质的鉴定，我们显示该协议与时间线的轮廓。成年大鼠脑裂解准备新鲜立即拉下来分析前。 <img src="/files/ftp_upload/1178/1178fig3.jpg" alt="" /> 图3。 P2X2 C -末端在大鼠脑组织的网络二进制的蛋白质组学分析示意图 。P2X2受体的融合与消费税珠势必GST蛋白C -末端使用下拉检测。脑裂解液和培养固定的重组蛋白的蛋白质。裂解液洗不作承诺的分数。蛋白质质谱鉴定。 <img src="/files/ftp_upload/1178/Fig%204%20Singh%20et%20al.jpg" alt="" /> 图4。鉴定P2X2受体的C -末端的具有约束力的合作伙伴。SYPRO染色凝胶，呈现出与C -末端的融合与GST（蓝色框）受体相互作用的假定蛋白质谱。商品及服务税单（黄色盒子）和谷胱甘肽琼脂糖珠单独控制车道也所示。箭头指示作进一步的分析质谱切除独特的乐队的例子。

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离子通道是一个不可分割的膜蛋白的主要类。它们包含装满水的毛孔，选择性地允许跨质膜离子的运动了他们的电化学梯度。离子通道门打开和关闭状态之间。 P2X配体门控离子通道的情况下发射器（如ATP），或触发门控步骤是，它可能是由与其他蛋白的相互作用调节。在过去的十年见证在我们了解如何P2X受体结合ATP的13增加，但仍了解较少的辅助蛋白，在调节P2X功能方面的作用。在本议?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetonitrile</td>
<td>Reagent</td>
<td>JT Baker</td>
<td>9829-02</td>
<td>&nbsp;</td>
<tr>
<td>Acrylamide</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0156</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1507-01</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
<tr>
<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotritol (DTT)</td>
<td>Reagent</td>
<td>EMD</td>
<td>3860</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1474-01</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene Glycol tetraacetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
<td>&nbsp;</td>
<tr>
<td>Formic acid</td>
<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione Sepharose 4B beads</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-5132-01</td>
<td>&nbsp;</td>
<tr>
<td>Hydrochloric acid (HCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1758</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>15502</td>
<td>&nbsp;</td>
<tr>
<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I1149</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani (LB) Media</td>
<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L8511</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>Reagent</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Sulphate (MgSO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride (NaCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Flouride (NaF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Orthovanadate (Na3VO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P40</td>
<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
<td>&nbsp;</td>
<tr>
<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
<tr>
<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>170-3125</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N',N'-Tetramethylethylenediamine (TEMED)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9281</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

配体门控离子通道1在中枢神经系统的突触联系的基础。在哺乳动物中，有三个家庭的配体门控通道：循环的半胱氨酸，谷氨酸门控和P2X受体通道 2 。在每一种情况下变送器具有约束力，导致毛孔开放，通过离子流及其电化学梯度。许多配体门控通道，也渗透到钙离子的3，4，有下游信号的角色（如基因调控），可能会超过通道的开放时间。因此，配体门控通道信号在广泛的时间尺度从几毫秒到数天不等。鉴于这些重要的角色，有必要了解如何配体门控离子通道本身是由蛋白质所规管，以及如何将这些蛋白质可能调整的信号。最近的研究表明许多，如果不是全部的渠道可能是部分蛋白信号复合物6。在这篇文章中，我们将解释如何识别的蛋白质结合P2X2受体胞浆域的C -末端的各个方面。 P2X受体是ATP门控的阳离子通道，由7个亚基（P2X1，P2X7）组成。 P2X受体广泛表达在大脑中，在那里他们介导兴奋性突触传递和神经递质的释放 7的突触前便利。 P2X受体在兴奋和非兴奋性细胞介导的神经信号，炎症和心血管功能 8的关键角色。 P2X2受体在神经系统的9的丰富，是本研究的重点。每个P2X亚基被认为是拥有两个膜跨越段（TM1和TM2）的，由胞外区 7和细胞内的N和C末端（图1A）7分开。 P2X亚基10（P2X1，P2X7）显示30-50％的序列同源性氨基酸水平的 11 。 P2X受体只包含三个亚基，这是最简单的离子型受体的化学计量。 P2X2 C -末端由120个氨基酸（图1b）组成，并含有几种蛋白质对接的共识网站，支持这一假设P2X2受体信号复合物的一部分。不过，虽然有几个功能已被归因于P2X2受体9 C -末端没有研究描述分子的合作伙伴，这种蛋白质通过细胞内C -末端全长夫妇。在此方法纸中，我们描述了蛋白质组学的方法来识别与P2X2受体的C -末端全长相互作用的蛋白质。

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

我们描述一个简单的协议，以确定大脑的蛋白质结合ATP门控P2X2受体的C末端全长。这种方法的延伸和系统应用到所有P2X受体预计将导致一个更好地了解P2X受体信号。

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

proteomics-to-identify-proteins-interacting-with-p2x2-ligand-gated

Research

JoVE Journal

Biology

14.6K 次观看.  David Geffen School of Medicine, University of California, Los Angeles. 我们描述一个简单的协议，以确定大脑的蛋白质结合ATP门控P2X2受体的C末端全长。这种方法的延伸和系统应用到所有P2X受体预计将导致一个更好地了解P2X受体信号。

视频: Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

科研

生物学

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

使用范围，以确定潜在的监管图案协同调控基因

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

自下而上和猎枪蛋白质组学鉴定一个全面的蛋白质组耳蜗

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

糖肽捕获的细胞表面蛋白组学

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.