这项工作是从加拿大安大略基因组研究所，加拿大基因组，创新，创新的安大略省，加拿大卫生研究院研究补助金JG和AE红表示E.基金会的资金支持coli菌株DY330从Donald L.法院（美国国家癌症研究所，冯检基，MD）是一种礼物。

1。 SPA标记基因特异性E.建设大肠杆菌 DY330应变<ol><li>包围的质粒pJL148 SPA-标签的DNA序列和卡那霉素的抗生素抗性标记盒（阚R）被用作聚合酶链反应（PCR）扩增7中的一个模板。一名45 nt的特定基因的正向引物，位于紧接在帧的目标基因的终止密码子的上游侧与一个27 bp的（5&#39;-AGCTGGAGGATCCATGGAAAAGAGAAG -3&#39;）标记特定的正向引物，和一个45 nt的基因特异的反向引物，位于紧接在帧与一个27 bp的（GGCCCCATATGAATATCCTCCTTAGTT 5&#39;-3&#39;），标记特定的反向引物，靶基因的终止密码子下游的是用于扩增的SPA-标签和Kanŕ盒从pJL148使用以下的PCR循环条件为：94 ℃下5分钟，30个循环的94℃1分钟，55℃下1分钟，68℃下2分10秒，然后通过68℃10分钟。这些扩增的序列服务s衬底为异位引入的λ-红同源重组机制（见下文）。 </li><li>将PCR产物用Qiaquick PCR纯化试剂盒进行纯化，以去除盐，可能会干扰电穿孔。纯化的PCR产物随后针对整合DY330菌株，其中的λ-Red重组机械表示10在一个特定的基因的3&#39;末端（紧邻上游侧的本机的终止密码子）。 </li><li> DY330λ-红表达株在2毫升的Luria-Bertani（LB）培养基中生长过夜，在32℃以180rpm摇动。 1毫升过夜培养随后接种到70毫升的新鲜LB培养基中，在500毫升的锥形烧瓶中。接种物在32℃下生长，通过以180rpm摇动，直到OD 600达到约0.8。 </li><li>将培养物转移到新鲜的250毫升锥形烧瓶中，烧瓶中，在水浴中在42℃下轻轻振荡孵育的细胞被诱导180 rpm离心15分钟。诱导后，立即在冰水浆浴保温烧瓶振摇至少30分钟。 </li><li>冰冷的文化立即转移到预冷的50ml聚丙烯管中，并进行离心6分钟，在4℃下3,993 xg离心将细胞沉淀重新悬浮在50毫升冰冷的无菌水，并离心，再次在4℃下以3993×g离心6分钟然后将细胞沉淀重新悬浮于1毫升冷水转移到1.5ml Effendorf管，在4℃下持续20秒，并以最大的速度离心两个或三个用冰冷的水洗涤步骤后，将细胞沉淀再悬浮终于在700微升冰冷无菌水，得到的电感受态细胞中。 </li><li>通过电穿孔，纯化的扩增子的1微升40微升电感受态细胞中导入。细胞在用Bio-Rad GenePulser II具有以下设置：2.5千伏，25μF用脉冲电穿孔的控制器200Ω。经过同源重组和整合，转化，成功地重组到染色体上的标记/盒的选择是基于抗菅直人（ 图1A）。选择多个转化子用于Western印迹以验证正确的代（ 即阳性信号）的SPA-标签的融合蛋白与抗FLAG M2抗体，该抗体是有选择性的对FLAG表位的SPA-标签。 </li><li>证实羧基末端SPA-标签融合菌株培养在一个大型隔离可溶性的蛋白质复合物，从收获的细胞使用的SPA纯化协议。亲和标记纯化过程中所涉及的步骤的概要的图1B中所示。 </li></ol> 2。培养和超声<ol><li>接种100微升的SPA-标签E.入50毫升优质肉汤（TB）的大肠杆菌甘油液体培养基用50μl的25微克/毫升邻f阚溶液在250毫升的锥形烧瓶中。过夜培养的速度增长，在32°C。 注：由于温度诱导λ红盒的控制下的一个温度敏感的阻遏物10在应变DY330，SPA-标签的大肠杆菌菌株在32℃下生长 </li><li>转移10毫升过夜培养的新鲜TB990毫升补充25微克/毫升的菅直人在4升的烧瓶。将培养生长在32℃下以恒定以250rpm振摇，5至6小时，直到OD 600达到〜2至3。 </li><li>转移1升E.大肠杆菌 SPA，的标记文化，清洗离心瓶和旋转的细胞在4°C贝克曼J6-HC离心 ​​机@ 3993 xg离心15分钟。 </li><li>弃去上清液，将离心瓶。重悬E.大肠杆菌细胞沉淀用25ml的超声缓冲液。确保在任何时候都保持在冰上的离心瓶。 </li><li>再悬浮颗粒转移至50毫升聚丙烯Falcon管中，并用液氮冷冻。冷冻的细胞储存在-80℃下长期使用。 </li><li>之前，超声处理，完全解冻的冷冻细胞保持在冰上粒料。解冻的样品被转移到一个无菌的不锈钢杯置于冰上超声处理。探头浸没到样品中，并超声处理3分钟。一个额外的2分钟允许过热冷却样品。 </li><li>超声处理的细胞裂解液转移到预冷的无菌离心管中，并在4℃下进行离心分离15分钟两次使用JA-17转子@ 35,267 xg离心（16000转）在Beckman离心机。 注意：在膜蛋白质的纯化的情况下，超声处理的细胞裂解物只有一次离心分离15分钟，因为我们不知道哪部分的膜蛋白，即，无论是在颗粒或上清液中的馏分。因此，为了避免过多的损失的mem跨膜蛋白，我们旋转在高速离心15分钟，且上清液进行随后进行纯化处理（参见下面的步骤），其中1％的洗涤剂用于溶解膜蛋白的细胞。 </li><li>从离心管的上清液小心转入到一个50毫升聚丙烯Falcon管中，并用液氮冷冻。超声处理的冷冻的细胞提取物在-80℃的6个月期间的最大存储以备将来使用。 </li></ol> 3。亲和纯化<ol><li>在使用之前，100微升抗旗M2珠洗涤的AFC缓冲液（10倍体积的30毫摩尔Tris-HCl，150mM的NaCl，0.1％的洗涤剂，和0.1-0.5毫TCEP [三（2 -羧乙基）膦-盐酸]）没有洗涤剂和还原剂的TCEP（三（2 - 羧乙基）膦）。 </li><li>冷冻，超声处理的细胞提取物是通过放置在冷水管解冻。解冻的细胞提取物保温用3μl的苯并激酶核酸酶在4℃下30分钟到该混合物中，添加非离子型洗涤剂的Triton X-100（洗涤剂的最终浓度应为0.1％）和200μl的悬浮液的抗旗M2琼脂糖珠。 注意：对于所有的可溶性蛋白质纯化，0.1％的Triton X-100的用来最大限度地减少非特异性吸附，其中作为一种膜蛋白，不同的温和的非离子型去污剂，如C12E8（Octaethylene乙二醇十二烷基醚），DDM净化（ n-十二烷基β-D-麦芽糖苷）和麦芽糖-新戊基乙二醇（MNG）可以在1％洗涤剂浓度使用，以提高增溶。由于不同的洗涤剂有不同的膜蛋白增溶效率，建议使用一个以上的洗涤剂来执行独立的蛋白质纯化物。 </li><li>的内容是通过旋转管为3小时，在4℃下使用LabQuake摇床轻轻混合。旋转3小时后，离心管在1,700×g离心6分钟。将上清液则removed小心，尽可能地不打扰宽松的珠丸。 </li><li>将沉淀重新悬浮于剩余的上清液，然后转移到0.8×4厘米Bio-Rad公司的聚丙烯制备柱。确保删除之列的底部插座插头，使洗脱液的重力流排水。洗柱5次与TEV裂解步骤。 </li><li>排出洗涤后的洗脱液后，在塔的底部出口被封闭。给相同的列，卵裂进行通过添加约5至10微升（50单位）在色谱柱上的TEV蛋白酶，1X的AFC缓冲液和400微升。确保关闭柱顶部的上限。列包含珠粒过夜，4℃过夜旋转轻轻使用LabQuake摇床。 </li><li>取下瓶盖的顶部和在塔底部的插座插头，和漏极TEV酶切后回收的洗脱液含有200μl的悬浮液中钙调蛋白 - 琼脂糖珠，洗涤10到一个新的列大量的钙调素结合缓冲。 </li><li>柱的顶部和底部的坚固地拧紧，并且在该列中的内容被通过旋转3小时，在4℃下使用LabQuake摇床轻轻混合。 </li><li>经过3小时的旋转，洗脱液被排出，通过删除的列的顶盖和底塞。该柱1X钙调蛋白由一个严格的洗涤用400μl的洗涤缓冲液中钙调素的结合缓冲液中，然后用200μl的洗涤四次。 </li><li>在四个级分50μl的一个新的Eppendorf管中，用1X钙调蛋白的洗脱缓冲液洗脱结合的蛋白质。分布在两个干净的Eppendorf管中，以等体积的洗脱馏分。这两个管，其后使用高速真空干燥。 </li><li>干燥后的洗脱液从一个管是用于运行的银染色凝胶，而其他被存储在-80℃下，使用质谱法之前。 </li></ol> 4。银染<ol><li>对于银staini纳克，3X SDS（十二烷基硫酸钠）样品缓冲液中的体积的一半加入到50微升的干燥的洗脱液。沸腾的混合物5分钟后，将样品加载到SDS-聚丙烯酰胺凝胶。 </li><li>后凝胶运行完毕后，它被小心地放入定影溶液（50％甲醇和10％乙酸）和20分钟的旋转振荡器上轻轻搅拌。该凝胶在20％乙醇中10分钟，然后漂洗。 </li><li>固定凝胶10分钟用500ml双蒸水彻底洗涤两次，以确保均匀的背景低。 </li><li>然后轻轻搅拌该凝胶在500毫升钠硫代硫酸钠1分钟，然后由两个用蒸馏水洗涤步骤，持续20秒。 </li><li>水被丢弃，并且该凝胶中温育30分钟，0.1％硝酸银的200毫升。经过该时间后，硝酸银被除去，持续20秒，凝胶，用蒸馏水洗涤，以除去过量的硝酸银。 </li><li>终于在75毫升的手激动凝胶显影溶液。当达到所需的强度，显影溶液被丢弃，并加入80毫升乙酸，使反应停止。确保孵育凝胶在乙酸，为最少20分钟为适当的可视化的凝胶带。 </li></ol> 5。蛋白质和样品制备的质谱分析<ol><li>向干燥的样品中，加入50微升的消化缓冲液，0.9微升的100mM的TCEP-HCl（三（2 -羧乙基）膦-盐酸），并在室温下孵育45分钟的混合物 用于还原步骤。接着，加入1微升的500mM碘乙酰胺温育另外40分钟，使样品烷基化在黑暗。 </li><li>孵化的第二轮后，添加固定化胰蛋白酶的1微克到混合物中，并在37℃下孵育5小时或在室温下过夜。确保使反应停止，加入1微升的乙酸。 </li><li>预湿的Millipo，重新Zip的提示通过吸液的润湿性和平衡溶液的10μl的移液管尖。分配浪费的解决方案。接着吸取10微升的洗涤溶液并和分配溶液的浪费。重复此步骤两次。 </li><li>对于给小费的肽混合物的有效结合，吸液混合肽混合物的20倍。附着到前端的肽混合物通过抽吸和分送洗涤溶液洗掉。重复此步骤两次有效结合的肽混合物的小费。 </li><li>含有结合的肽的前端，吸液的润湿性和平衡溶液10微升，和分配到一个干净的eppendorf管中。重复此步骤两次。洗脱的样品在高速真空干燥后，可以立即进行分析的样品，通过质谱法或贮存于-80℃，在使用前。 </li></ol> 6。 LTQ Orbitrap Velos的质谱仪蛋白质鉴定钍Ë孤立的复合物的多肽组分确定使用的LTQ Orbitrap Velos的混合串联质谱仪。的Orbitrap具有优异的分辨能力（&gt; 60,000全宽半高，或FWHM）和质量准确度（&lt;2 ppm），最大限度地减少MS / MS抽样的无关的非特异性背景控制净化污染物检测，同时高速Velos离子阱组件可以检测和使用两个电子转移解离和碰撞诱导解离模式的片段的低丰度肽。高置信度的匹配导致MS / MS谱之间的映射到参考E.大肠杆菌的蛋白质序列数据库搜索算法，如SEQUEST和STATQUEST 11的概率算法，像评估过程中的每一个匹配的序列。总的谱号，肽序列的唯一性和阴性对照（ 即模拟）净化污染物检测到共同的背景被认为是实现一个低的实证假显示covery率。下面的步骤进行蛋白质鉴定： <ol><li>微色谱柱填充有〜10厘米为3μm的Luna-C 18的树脂中，并且连接到一个Proxeon纳升电离子源被放置在符合文书的Orbitrap。 </li><li>一个Proxeon纳米流的二进制的HPLC泵是用于提供一个稳定的尖流量约300 NL分钟-1在多肽分离。 </li><li>为了达到肽洗脱，根据样品的复杂性有机缓冲液梯度设置。例如，下面的梯度通常设置的大肠杆菌大肠杆菌 SPA样品：在1分钟内，从2％至6％的溶剂B增加，在38分钟至24％，至100％，在接下来的4分钟，保持在100％，持续1分钟，然后在1分钟内减少到2％，最后15分钟保持在2％。流动相：溶剂A具有HPLC梯度95％水和5％乙​​腈，0.1％甲酸，而溶剂B有HPLC梯度5％的水和95％乙腈，含0.1％甲酸阿哲四。针尖处的流速设定为60分钟至300 NL分钟-1。 </li><li>运行在60000决议通过一个完整的质量扫描随着质谱仪周期的，随之而来的串联碎片的质量扫描所收集的最激烈的前体离子。动态排除，单一同位素质量的选择，依赖于数据的实时采集仪的功能被启用。 </li><li>光谱使用一个数据库搜索算法对数据库的大肠杆菌如SEQUEST搜索大肠杆菌的蛋白质序列，并将结果进行统计过滤使用一个的概率算法如STAQUEST 11，以确保低假发现率。只有高置信度（99％概率）考生选择，而比赛显示超过10 ppm的质量误差的理论肽质量进一步考虑被淘汰。 </li></ol>

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Cell Proteomics. 2, 96-106 (2003).</li><li>Vlasblom, J., 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=GenePro:+a+Cytoscape+plug-in+for+advanced+visualization+and+analysis+of+interaction+networks.">GenePro: a Cytoscape plug-in for advanced visualization and analysis of interaction networks.</a> Bioinformatics. 22, 2178-219 (2006).</li><li>de Berardinis, V., 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=A+complete+collection+of+single-gene+deletion+mutants+of+Acinetobacter+baylyi+ADP1.">A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1.</a> Mol. Syst. Biol. 4, 174 (2008).</li><li>Babu, M., 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=Sequential+peptide+affinity+purification+system+for+the+systematic+isolation+and+identification+of+protein+complexes+from+Escherichia+coli.">Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli.</a> Methods Mol. Biol. 564, 373-400 (2009).</li></ol>

没有利益冲突的声明。

这里所描述的一个关键方面SPA基于APMS方法的的标记内的天然染色体背景下进行，从而确保维持正常的基因调节（ 即原生饵子保存的，因此，表达水平不扰动）和本机稳定相关蛋白质复合物中回收在近内源性水平。正负极性问题，也避免包括选择标记朝外启动。这SPA-标记的方法是有效的，足以净化组件的低丰度的配合物不久的均匀性，包括膜相关的组件，即使亚基表达下降到只有几个分子?...

<table border="1">
	<tbody>
		<tr>
			<td>Materials</td>
			<td>Vendor</td>
			<td>and Catalog Numbers</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>I. Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Bioshop</td>
			<td>#KAN201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>#AMP201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Terrific-Broth medium</td>
		</tr>
		<tr>
			<td>Bio-Tryptone</td>
			<td>Bioshop</td>
			<td>#TRP 402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bioshop</td>
			<td>#YEX 555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Bioshop</td>
			<td>#GLY 002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Bioshop</td>
			<td>#PPM 302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Bioshop</td>
			<td>#PPM 303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. Bacterial Strain and Plasmid</td>
		</tr>
		<tr>
			<td>DY330</td>
			<td>&nbsp;</td>
			<td>Yu et al. (2000)10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pJL148</td>
			<td>&nbsp;</td>
			<td>Zeghouf et al. (2004)7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. PCR and Electrophoresis Reagents</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 X PCR buffer</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 mM dNTPs</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 mM MgCl2</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td># AGA002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Loading dye</td>
			<td>NEB</td>
			<td>#B7021S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Bioshop</td>
			<td># ETB444</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X TBE buffer</td>
			<td>Thermo Scientific</td>
			<td>#28355</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Bioshop</td>
			<td>#TRS001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Bioshop</td>
			<td>#BOR001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Sigma</td>
			<td># E6768</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNA ladder</td>
			<td>NEB</td>
			<td>#N3232L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5. Plasmid isolation and Clean-up Kits</td>
		</tr>
		<tr>
			<td>Plasmid Midi kit</td>
			<td>Qiagen</td>
			<td>#12143</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>#28104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6. PCR and Transformation Equipments</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>BioRad</td>
			<td>iCycler</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis</td>
			<td>BioRad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-Rad</td>
			<td>GenePulser II</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.2 cm electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>42 &deg;C water bath shaker</td>
			<td>&nbsp;</td>
			<td>Innova 3100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beckman Coulter TJ-25 centrifuge</td>
			<td>Beckman Coulter</td>
			<td>TS-5.1-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C Shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C large Shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C plate incubator</td>
			<td>Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>7. Electrophoresis and Western blotting</td>
		</tr>
		<tr>
			<td>Acrylamide monomer, N,N'- methylenebis-acrylamide</td>
			<td>Bio-Rad</td>
			<td>#161-0125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bioshop</td>
			<td># AMP001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>n-butanol</td>
			<td>Sigma</td>
			<td># B7906</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bioshop</td>
			<td>#TEM001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Whatman No. 1 filter paper</td>
			<td>Fischer Scientific</td>
			<td>#09-806A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mini protean 3 cell</td>
			<td>Bio-Rad</td>
			<td>#165-3301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>iBlot gel transfer device</td>
			<td>Invitrogen</td>
			<td>#IB1001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>Bio-Rad</td>
			<td>#162-0115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Flag M2 antibody</td>
			<td>Sigma</td>
			<td>#F3165</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Amersham</td>
			<td>#NA931V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pre-stained protein molecular weight standards</td>
			<td>Bio-Rad</td>
			<td>#161-0363</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chemiluminescence reagent</td>
			<td>PIERCE</td>
			<td>#1856136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>Clonex Corp</td>
			<td>#CLEC810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Quick Draw blotting paper</td>
			<td>Sigma</td>
			<td>#P7796</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C2 platform rocking shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>8. Sonication Equipment and Reagents</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson Ultrasonic</td>
			<td>#23395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>#SOD001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche</td>
			<td>#800-363-5887</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.5 mM TCEP-HCl</td>
			<td>Thermo Scientific</td>
			<td>#20490</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>9. Affinity Purification Reagents and Equipment</td>
		</tr>
		<tr>
			<td>0.8 x 4 cm Bio-Rad polypropylene column</td>
			<td>Bio-Rad</td>
			<td>#732-6008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Novagen</td>
			<td>#70746</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-FLAG M2 agarose beads</td>
			<td>Sigma</td>
			<td>#A2220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calmodulin-sepharose beads</td>
			<td>GE Healthcare</td>
			<td>#17-0529-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEV protease</td>
			<td>Invitrogen</td>
			<td>#12575-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>#T9284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>#C2661</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>#E3889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LabQuake Shaker</td>
			<td>Thermolyne</td>
			<td>#59558</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>10. Silver Staining Reagents</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Bioshop</td>
			<td>#MET302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Bioshop</td>
			<td>t#ACE222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium-thiosulfate</td>
			<td>Sigma</td>
			<td>#S-7143</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Fischer Scientific</td>
			<td>#S181-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Bioshop</td>
			<td>#FOR201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Bioshop</td>
			<td>#SOC512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>11. Reagents and Equipment for Protein Identification </td>
		</tr>
		<tr>
			<td>Trypsin Gold, Mass Spectrometry Grade</td>
			<td>Promega</td>
			<td># V5280</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 mM NH4HCO3</td>
			<td>Bioshop</td>
			<td>#AMC555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 mM CaCl2</td>
			<td>Bioshop</td>
			<td>#CCL302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>#A998-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma</td>
			<td>#F0507</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Sigma</td>
			<td>#95304</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>#16125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Millipore Zip-Tip</td>
			<td>Millipore</td>
			<td># ZTC18M960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>~10 cm of 3 &mu;m Luna-C18 resin</td>
			<td>Phenomenex</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Proxeon nano HPLC pump</td>
			<td>Thermo Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos mass spectrometer</td>
			<td>&nbsp;</td>
			<td>Thermo Fisher Scientific</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>12. Labware</td>
		</tr>
		<tr>
			<td>4 liter conical flasks</td>
			<td>VWR</td>
			<td>#89000-372</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml polypropylene falcon tubes</td>
			<td>Any Vendor</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.5 ml micro-centrifuge tubes</td>
			<td>Any Vendor</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>250 ml conical flaks</td>
			<td>VWR</td>
			<td>#29140-045</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml sterile culture tubes</td>
			<td>Thermo Scientific</td>
			<td>#366052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>VWR</td>
			<td>#479-3221</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>-80 &deg;C freezer</td>
			<td>Fisher Scientific</td>
			<td>#13-990-14</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed vacuum system</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			Buffers and Solutions
			1. 1 liter Terrific Broth (TB) media
			11 g Bio-Tryptone 
			22 g Yeast Extract 
			2% Glycerol 
			50 ml potassium salt stock solution
			2. Potassium Salt Stock Solution
			1.5 M K2HPO4 
			0.35 M KH2PO4
			3. Sonication Buffer
			20 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.2 mM EDTA 
			10% Glycerol 
			Before use add protease inhibitor (PI) and 0.1-0.5 mM TCEP
			4. AFC buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			5. TEV cleavage buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.2 mM EDTA 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			6. Calmodulin binding buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			2 mM CaCl2 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			7. Calmodulin wash buffer
			30 mM Tris-HCl pH 7.9 
			150 mM NaCl 
			2 mM CaCl2 
			0.1-0.5 mM TCEP
			8. Calmodulin elution buffer
			30 mM Tris-HCl (pH 7.9) 
			100 mM NaCl 
			10 mM EGTA 
			0.1-0.5 mM TCEP
			9. Developing solution (1L)
			37% Formaldehyde 
			30 g sodium carbonate 
			1000 ml distilled water
			10. Digestion buffer
			50 mM NH4HCO3 
			1 mM CaCl2
			11. Wetting and Equilibration solution
			70% acetonitrile (ACN) in 0.1% formic acid
			12. Washing solution
			100% H2O in 0.1% formic acid</td>
		</tr>
	</tbody>
</table>

identification of protein complexes in escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry

由于大多数大分子组件，系统识别的蛋白-蛋白相互作用（PPI）和确定的亚基组成的多蛋白复合物，可以帮助了解基因的功能和增进了解的生物系统1，2介导的细胞过程。可以被映射的物理相互作用，与高的信心vialarge规模接近生理条件下，基于与质谱（APMS）亲和纯化的染色体标记的蛋白质组合的内源性蛋白复合物的分离和鉴定的。这种方法已成功地应用在进化上不同的生物，包括酵母，果蝇，蠕虫病毒，哺乳动物细胞和细菌1-6。特别是，我们已经产生了一个羧基末端的顺序肽亲和（SPA）亲和纯化的天然蛋白复合物从培养革兰阴性大肠杆菌双标记系统，使用GEnetically听话的主机实验室菌株全基因组调查的基本生物学和保守的过程中原核生物1，2，7，非常适合。我们的SPA标记系统是类似于酵母8，9最初开发的串联亲和纯化方法，由钙调蛋白结合肽（CBP）的后跟的高度特异性的烟草蚀刻病毒 （TEV）蛋白酶的切割位点和三个副本FLAG表位（3X FLAG），允许两个连续两轮亲和富集。盒扩增后，序列特异性的线性编码的SPA-tag和可选择标记的PCR产物是集成的，表示帧作为羧基末端融合在DY330背景下，瞬时表达被诱导高效率的异源λ噬菌体重组系统10。随后的双步纯化使用调钙蛋白和抗FLAG亲和珠使高度选择性甚至大型文化低丰度蛋白复合物和有效的恢复。串联质谱法，然后使用，以确定稳定地共同净化蛋白具有高灵敏度（低纳克检出限）。 在这里，我们将详细介绍一步一步的程序，我们通常使用的系统蛋白标记，分离纯化和质谱为基础的分析可溶性蛋白复合物E.大肠杆菌 ，可以按比例增加和潜在的针对其他细菌物种，包括某些服从重组工程的机会病原体。由此产生的物理相互作用通常可以发现有趣的组件和连接提出新的机械链接。 PPI数据整合与的备用分子协会提供的数据，如遗传（基因 - 基因）的相互作用和基因组上下文（GC）的预测可以方便的多蛋白复合物的的全球分子组织的澄清在BI目的论的途径。 E.产生的网络大肠杆菌可以使用的功能结构，其他微生物的同源基因产品的功能注释目前缺乏深入了解。

Once tagged bait proteins, which are expressed at endogenous levels are affinity-purified from logarithmic phase cultures the samples were run on a silver-stain gel to visualize the individual polypeptide components of the isolated stable complexes. We also subjected a second portion of the affinity-purified protein samples to gel-free tandem mass spectrometry (LCMS) to identify the corresponding polypeptide sequences. The effectiveness of this APMS procedure is shown with a representative SDS-PAGE analysis of the compon...

Watch this Scientific Journal Video about Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry at JoVE.co

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

亲和纯化标签蛋白结合质谱（APMS）是一个功能强大的蛋白质相互作用网络系统的映射方法和调查的生物过程的机理的基础。在这里，我们描述了一个优化的顺序肽亲和力（SPA）APMS程序开发的细菌<em&gt;大肠杆菌</em&gt;，可用于分离和表征到接近均匀性，甚至从每个细胞的拷贝数低的稳定的多蛋白复合物。

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

identification-protein-complexes-escherichia-coli-using-sequential

Research

JoVE Journal

Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

48.2K 次观看.  University of Toronto. 亲和纯化标签蛋白结合质谱（APMS）是一个功能强大的蛋白质相互作用网络系统的映射方法和调查的生物过程的机理的基础。在这里，我们描述了一个优化的顺序肽亲和力（SPA）APMS程序开发的细菌大肠杆菌，可用于分离和表征到接近均匀性，甚至从每个细胞的拷贝数低的稳定的多蛋白复合物。

视频: Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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 ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...