Work in the Ait-Si-Ali lab was supported by the Association Fran&#231;aise contre les Myopathies T&#233;l&#233;thon (AFM-T&#233;l&#233;thon); Institut National du Cancer (INCa); Agence Nationale de la Recherche (ANR), Fondation Association pour la Recherche sur le Cancer (Fondation ARC); Groupement des Entreprises Fran&#231;aises pour la Lutte contre le Cancer (GEFLUC); CNRS; Universit&#233; Paris Diderot and the ''Who Am I?'' Laboratory of Excellence #ANR-11- LABX-0071 funded by the French Government through its ''Investments for the Future'' program operated by the ANR under grant #ANR-11-IDEX-0005-01. EB was supported by an INCa grant.

1.接种HeLa-S3核盐可提取与染色质结合组分的制备<ol><li>细胞的集合<ol><li>在Dulbecco氏改良Eagle培养基（DMEM）中补充有10％胎牛血清，100U / ml青霉素和100μg/ ml链霉素（生长生长的HeLa-S3标示-HA-MyoD的和HeLa-S3标示-HA（对照细胞系）培养基），在37℃和5％CO 2在潮湿的培养箱中。 注意:在使用中描述的协议有可能成为建立的HeLa-S3稳定表达的Flag-HA-的MyoD和HeLa-S3空载体（HeLa细胞-S3标志-HA）转染细胞:19,20，或由作者提供。 </li><li>细胞扩增到每个细胞系（HeLa细胞-S3标志-HA-的MyoD和HeLa-S3标志-HA细胞）的10完全融合150毫米菜肴。 </li><li>收集上清（含非贴壁细胞亚群）为5升微调。 </li><li>用每150毫米盘10mL的PBS洗涤附着的亚群，trypsinize细胞在室温统1分钟023（0.05％胰蛋白酶-EDTA溶液，加入2ml为15厘米的培养皿）。 </li><li>添加4毫升每150毫米盘介质和收集细胞到50ml管中。 </li><li>从步骤1.1.3（在5升旋转器），并从步骤1.1.5细胞结合上清，加入500毫升新鲜的预温热的生长培养基中的每个细胞系。 </li><li>转移细胞悬液至5升旋转器和在潮湿培养箱中于37℃和5％CO 2培养细胞为至少60小时。 </li><li>加入500毫升新鲜生长培养基和生长的细胞24小时。 </li><li>添加1升的新鲜生长培养基和生长的细胞24小时。 </li><li>取出将1ml细胞悬浮液计数细胞并检查细胞存活力。彩色细胞悬液30微升用血球0.4％台盼蓝的30微升和计数活（不是蓝色）细胞在显微镜下。 </li><li> ;可以通过每日加入新鲜生长培养基6×10 5个细胞/ ml -保持细胞密度在2不超过1×10 6个细胞/ ml。最大值量一5升微调为2.5 L. 注意:对于下列步骤，由第2批进行- 2.5升培养物的密度为1×10 6个细胞/ ml。重复步骤1.1.12 - 1.1.14收集约20L （≈20克干粒料）每个细胞系在进行下一步之前。 </li><li>沉淀细胞，在500×g离心5分钟，离心在4℃下，并丢弃上清液。 </li><li>悬浮细胞，通过移液和离心机在500×g离心5分钟100毫升冰冷的PBS中于4℃以洗涤沉淀。 </li><li>通过重悬细胞用25ml冰冷的PBS中，将悬浮液转移至50ml管中并在4℃下以500 xg离心离心5分钟重复洗涤。 注:细胞沉淀可立即使用或被在液氮中冷冻并储存在-80℃下数天。 </li></ol></li><li>细胞核的分离 注:执行步骤1.2.1 - 1.4.3在寒冷的房间。 <ol><li>预寒意buffeRS和均质。 </li><li>如果使用冷冻物质，迅速在37℃下解冻水浴细胞沉淀。 </li><li>悬浮20克在20毫升细胞（≈20ml）中通过，用10毫升缓冲液中在第一，然后加入5毫升，然后加入低渗缓冲液A（ 表3）的（等于细胞沉淀的尺寸的体积）另外5毫升用新鲜的缓冲液洗井吸液管以获得最大的细胞。 </li><li>转印40毫升细胞悬浮到预冷却的均化用紧身杵。 </li><li>均质在均化器20次（在进出动作20）细胞和转移细胞悬液至50ml管中。 </li><li>洗均化用7ml蔗糖缓冲液（初始小区体积的1/3， 表3）中，回收的最大单元。快速传送该悬浮进50毫升管从步骤1.2.5保存细胞核和限制渗漏。 </li><li>染色30微升等分0.4％TR的30微升ypan蓝色，在显微镜下分析来控制裂解效率（如果裂解成功，所有的核应蓝色）。如有必要，重复同质化的一步。 </li><li>离心机以10,000 xg离心7分钟，4℃以沉淀细胞核。保存上清液作为细胞质级分。 </li></ol></li><li>核盐提取（SE）级分的制备<ol><li>重悬细胞核沉淀在8ml蔗糖缓冲液（等于细胞核沉淀的尺寸的体积）。通过加入一滴一滴而在涡流8毫升高盐缓冲液（1细胞核沉淀体积， 表3）的充分混合以获得300mM NaCl的终浓度。孵育在冰上30分钟，每5分钟混合。 </li><li>通过加入蔗糖缓冲液8毫升（总体积的1/3）下降NaCl浓度至150mM的。离心机以13,000 xg离心10分钟，4℃。收集上清（核盐萃取物，SE）。 注:要么继续执行步骤1.3.3或离开SE制备染色质结合的级分，然后超离心两馏分同时期间在冰上级分。 </li><li>超离心机SE在4℃下以85000 xg离心30分钟。取上清（≈26ml）清洗。 </li><li>冷冻在液氮中100μl等分的。继续执行步骤2"蛋白质复合体净化"，使用提取的其余部分。 注意:等分试样被用作步骤5.1.1期间的输入控制。 </li></ol></li><li>染色质结合态的制备<ol><li>重悬沉淀（从步骤1.3.5）。精心在7ml蔗糖缓冲液（等于粒料的大小的体积）。 </li><li>添加氯化钙2至1mM的终浓度（28微升的0.5M 的 CaCl 2 14 ml悬液）来激活MNase（步骤1.4.4）并混合。 </li><li>预热悬浮液在37℃1分钟。 </li><li>加入70微升微球菌核酸（MNase）从0.5获得0.0025 U /μL（1/200稀释终浓度U /μL股票），混匀。 </li><li>孵育在37℃下正好12分钟。混合每4分钟。 </li><li>立即置于冰上反应。 </li><li>停止MNase活性，添加112微升的0.5M EDTA pH 8.0的（1/125稀释）的4 1mM的终浓度。孵育在冰上5分钟。 </li><li>超声处理5次在高振幅1分钟，两人之间的超声处理允许1分钟（10分钟计）休息。 </li><li>超速离心机在85000 xg离心30分钟，4℃。 </li><li>收集上清（富含核分数，NE，≈12ml）中。 </li><li>冷冻在液氮中50微升等分。继续执行步骤2"蛋白质复合体净化"，使用提取的其余部分。 注意:等分试样被用作步骤5.1.1期间的输入控制。 </li></ol></li></ol> 2.蛋白质复合物纯化注:进中从每个细胞系（HeLa细胞-S3标示-HA-MyoD的和控制，HEL平行提取A-S3标志-HA）。 <ol><li>基于升旗净化<ol><li>预洗旗树脂。使用标志树脂的600微升（从商用50％股票）每个实验点（SE和NE馏分对于每个细胞系）。 </li><li>转印树脂成15ml试管，重悬在13ml冷TEGN（ 表3），离心2分钟以1000 xg离心并去除上清。重复5次。在最后一次洗涤悬浮标示树脂分成相等的体积TEGN（300微升每个实验点）之后。 </li><li>每个实验点，混合（分别在15ml试管为12毫升NE和在50ml管中的26毫升SE馏分，）洗涤标示树脂的600微升和相应的蛋白质提取物。孵育在4℃下旋转轮过夜。 </li><li>离心机在4℃，1000 XG 2分钟，抛开上清液，并保持在4℃，直至净化效率试验（2.2）的结果。 </li><li>对于SE的样品，重悬旗树脂在4ml TEGN和转移到15毫升管。重复此步骤3次，每次转印到相同的15ml试管，以避免失去珠。在1000 xg离心和4℃并除去上清液离心2分钟。 </li><li>洗涤标示树脂中使用13毫升TEGN中，并在1000 xg离心，4℃离心2分钟15毫升管7倍。 </li><li>最后一次洗涤，悬浮在1毫升TEGN和转让至1.5 ml后低结合管。在1000 xg离心和4℃并除去上清液离心2分钟。 </li><li>重悬在4毫克/毫升200微升标记肽溶液在每个实验点pH 7.8。混合珠和肽。加入200μlTEGN缓冲液中并在4℃孵育至少4小时或过夜，以洗脱结合的蛋白质。 </li><li>在1000 xg离心离心2分钟，在4℃并转移在树脂和上清液（标示洗脱液），以放置在一个2毫升管的空旋转柱。 </li><li>离心柱，收集遗留在2 ml管上清。 </l我&gt; <li>通过加入TEGN缓冲器到列收集旗树脂并进行用200μl的标记肽溶液（4毫克/毫升）的第二标志洗脱在4℃过夜每个实验点。 </li><li>重复步骤2.1.9 - 2.1.10收集第二次洗脱。结合的第一和第二洗脱液。 </li></ol></li><li>净化效果测试 注:在步2.1.11 - 2.1.12，进行SDS-PAGE和银染色（2.2.1 - 2.2.2）。 <ol><li>取15微升洗脱液，加4倍样缓冲液的5微升和2μl10X还原剂，煮沸5分钟，在96℃下并运行SDS-聚丙烯酰胺4 - 根据制造商的说明12％的凝胶。 </li><li>染色使用银染色试剂盒凝胶（按照制造商的说明）。如果有标志-HA-的MyoD和标志-HA之间的蛋白质模式明显不同模拟洗脱液，特别是标志-HA的MyoD的带（约50 kDa的， 图1A），进入下一个STE页。 </li></ol></li><li>血凝素（HA）为基础的分离纯化<ol><li>洗涤的HA树脂通过转移到15ml试管中，在13ml TEGN的重悬，并在4℃下以1000 xg离心离心2分钟。重复洗涤步骤5次。后在相同体积TEGN缓冲器的最后洗涤悬浮珠。 注意:体积必须根据基于旗纯化效率进行调整。开始从商业50％的股票300微升每个实验点。 </li><li>转移HA树脂每个实验点至1.5 ml低结合管。离心2分钟，在4℃下1000 xg离心，消除了最大的洗涤缓冲液（ 表3）的和从基于旗纯化添加洗脱液至HA树脂。 </li><li>孵育在4℃下旋转轮过夜管。 </li><li>离心机在4℃，1000 XG 2分钟，抛开上清液，并保持在4℃，直至净化效率测试的结果（2.3.12）。 </li><li>回覆暂停在0.5ml TEGN的HA树脂，转移到一个新的低结合1.5mL管中。重复再悬浮一次加入到同为1.5毫升管，以避免在1000 xg离心和4℃失去珠离心2分钟。除去上清液。 </li><li>通过用1ml TEGN的每一次，离心以1000 xg离心2分钟，4℃，并除去上清液（避免丢失珠）洗涤珠8倍。最后一次洗涤后，转移到珠0.5毫升管。 </li><li>除去尽可能多的上清液越好。在pH 7.8珠粒以洗脱结合蛋白加入100微升的HA游离肽溶液（4毫克/毫升）。孵育在4℃下旋转轮过夜。 注意:需要的HA肽的量取决于所充满的复合物来进行调整。 </li><li>在1000 xg离心离心2分钟，在4℃和树脂和上清液（HA洗脱物）转移到放置在2毫升管的空旋转柱。 </li><li>离心柱收集遗留的上清液2 ml管。 </LI&gt; <li>执行用100μlHA肽（4毫克/毫升）的每个实验点的第二洗脱。孵育在4℃下旋转轮过夜。重复步骤2.3.8 - 2.3.9收集第二次洗脱。 </li><li>结合的第一和第二洗脱液。 </li><li>通过SDS-PAGE和银染色试验净化效率（见步骤2.2）（ 图1A）。 </li><li>集中的HA洗脱物，使用离心过滤器单元具有根据制造商的说明10 kDa截止的（标称分子量极限，NMWL）30微升。 </li><li>划分浓缩样品分为两部分:22.5（3/4）和7.5（1/4）微升。卡扣冷冻在液氮中并存储样本的1/4在-80℃。使用3/4部分用于步骤3。 注意:1/4冷冻部分是用于通过免疫印迹质谱结果的确认（参见步骤5.1）。 </li></ol></li></ol> 3.质谱分析注:以下步骤应与质谱设施，将进行分析讨论。 <ol><li>补充样品3/4（22.5微升），4倍样缓冲液7.5微升和3.3微升10X还原剂中，并在96℃煮沸5分钟。 </li><li> 12％的凝胶 - SDS-聚丙烯酰胺4上负荷的样品。在150V恒定5分钟运行凝胶，以允许所有的蛋白质不经分离进入凝胶。 </li><li>用水冲洗凝胶;定为20 - 30分钟用固定液（10％乙酸，50％甲醇，40％的超洁净的水），然后洗固定凝胶5次在超洁净的水。 </li><li>切含有蛋白质，贮存于1ml水的频带在1.5ml管中，发送到质谱设施。 </li></ol> 4.数据分析 注意:这是用于分析的一般指导。确切的步骤，将取决于被MS设施提供的数据的特定模式用于执行分析（ 例如，21,22）。 <ol><li>比较确定的蛋白在与对应阴性对照制剂获得的名单"的MyoD"洗脱液名单。从分析中排除两个列表中发现的蛋白质。 </li><li>删除具有剩余的列表中标识的肽≤2总数的蛋白质。 </li><li>访问功能注释和所获得的使用DAVID生物信息学资源蛋白质的列表的功能分类（http://david.abcc.ncifcrf.gov/home.jsp）和分类 ​​蛋白23,24的列表。 </li><li> （可选）从列表中选择"搭便车"，收取无核蛋白质具有很强的偶然结合DNA 25负电荷删除。这些含有细胞骨架，细胞质，线粒体，膜和受体蛋白质（蛋白质折叠，在表1骨架和杂项组蛋白和2）。&lt;/ LI&gt; <li>比较所得到的由SE和网元级分的MyoD交互件的列表，以确定特定于每个级分（ 图2）共同的蛋白质和蛋白质。 </li></ol> 5.确认通过Western blot鉴定交互件<ol><li>确认在HeLa-S3标志-HA-的MyoD和HeLa-S3标志-HA <ol><li>在冰上步骤2.3.14（7.5微升）获得解冻洗脱液样品。添加12微升TEGN缓冲液，10微升4倍装载缓冲液中，10×3微升还原剂。煮沸样品在96℃5分钟。 </li><li>准备输入样本的印迹。 <ol><li>从步骤1.3.9和1.4.11解冻等分试样并用BCA试剂盒（按照制造商的说明）测量蛋白浓度，例如。 </li><li>使用15微克每车道的蛋白质。测量提取物的合适量和调整样品的体积达到15微升与TEGN缓冲器。 </li><li>加入5微升4倍样缓冲液和1.5微升10X还原剂。煮沸样品在96℃5分钟。 </li></ol></li><li>执行与感兴趣抗体（特异于MS分析过程中确定伙伴候补）使用15微升洗脱液样品的免疫印迹分析。使用输入提取物作为内源性蛋白的水平（ 图3A）的控制。 </li></ol></li><li>确认在相关蜂窝系统<ol><li> C2C12成肌细胞的鼠标总核提取物的制备。 <ol><li>在潮湿的培养箱中生长C2C12小鼠成肌细胞在补充有15％胎牛血清的DMEM培养基，100U / ml青霉素和100μg/ ml链霉素，在37℃和5％的CO 2。不要超过80％的细胞汇合保持增殖状态的细胞。 </li><li>生长C2C12的至少两个150毫米菜肴进行免疫沉淀的一个点（一种抗体）（IP）。吸出介质，用10毫升的PBS洗涤细胞两次。 </li><li>刮细胞和收进15ml试管。铈ntrifuge在250 xg离心在4℃下5分钟。弃上清。 </li><li>估计细胞沉淀（= V 细胞 ）的容积。轻轻悬浮在低渗的缓冲液B 3体积V CELL粒料破坏细胞质膜（ 见表3）。 </li><li>添加10％0.44 x垂直细胞体积的NP-40，以达到1％的最终浓度（体积/体积）。轻轻颠倒管数次​​混匀。 </li><li>添加的SR（ 表3）的0.89 x垂直细胞体积，以保持核的完整性。轻轻颠倒试管几次以均匀的悬浮液，并离心5分钟，在2000 xg离心以沉淀细胞核。 </li><li>除去上清液（细胞质分数）。估计细胞核沉淀（= V NUC）的体积。重悬细胞核沉淀在蔗糖缓冲液的一个体积V NUC。 </li><li>通过加入一滴一滴而在高盐缓冲液的旋涡1体积V NUC充分混合以获得300mM的终浓度NaCl和孵育在冰上30分钟。混合每5分钟。 </li><li>添加蔗糖缓冲液中的一个体积V NUC（降低NaCl浓度至150毫摩尔）和氯化钙2（最终浓度为1mM）。混合。 </li><li>预热悬浮体在37℃下1分钟，加入微球菌核酸至终浓度0.0025 U /μL（1/200 0.5 U /μL的股票）的。混合。 </li><li>孵育在37℃下正好12分钟。混合每3分钟。 </li><li>立即置于冰上反应，并添加EDTA（终浓度为4毫米）停止MNase活动。孵育在冰上5分钟。 </li><li>超声处理5次在高振幅每次0.5分钟。 2超声处理之间，允许0.5分钟（5分钟计）休息。 </li><li>超离心机在85000 xg离心30分钟，4℃。收集上清（总核提取物）。 </li><li>用BCA试剂盒（按照制造商的说明）测量总核提取物的蛋白质浓度，例如并继续immediat伊利步骤5.2.2。 </li></ol></li><li>内源性的MyoD的免疫沉淀（IP）。 <ol><li>要预清提取物，洗10微升的G蛋白琼脂糖珠为每个IP点。 <ol><li>转移的G蛋白琼脂糖珠的所需量到1.5毫升管，悬浮1.4毫升冷TEGN，离心机中2分钟，在1800 xg离心并去除上清。重复3次。 </li></ol></li><li>混合预洗涤G蛋白琼脂糖珠与总核提取物的合适体积。使用500微克每IP的总点数核提取蛋白质。 </li><li>孵育在旋转轮在4℃下2小时进行预清除提取物。 </li><li>离心机在1800 xg离心在4℃下5分钟。保留上清。 </li><li>混合的MyoD抗体的5微克或5微克兔IgG与在1.5ml低结合管500微克预清总核提取物。添加TEGN最多缓冲1毫升在4℃孵育的旋转轮过夜。 注意:请在F5.2.2.3一步期间ollowing步骤。 </li><li>每个IP点预洗7.5微升蛋白质A / G股珠的解决方案。 <ol><li>在1800 xg离心转移的珠的所需量到1.5毫升管，悬浮珠在1ml TEGN和离心机在4℃下2分钟。去除上清。重复3次。 </li></ol></li><li>重悬蛋白质A / G珠在1.5ml于4℃过夜封闭溶液（ 表3），并培育上的旋转轮。 </li><li>与16,000 XG在4℃下10分钟，ABS（步5.2.2.5）培养离心机总核提取物。保留上清。 </li><li>离心机在1800 XG阻止蛋白A / G树脂（步骤5.2.2.7），2分钟，4℃。弃上清，悬浮在1毫升TEGN缓冲。重新离心在4℃下1800×g离心2分钟洗出封闭缓冲液。 </li><li>悬浮阻断蛋白质A / G的树脂在1ml TEGN缓冲和分装成新的低蛋白结合管，以获得7.5微升一个BLO每个IP点cked蛋白A / G树脂。离心机在1800 xg离心在4℃下进行2分钟，并除去尽可能多的上清液。 </li><li>添加用绝对温育总核提取物（或对照IgG）与A / G树脂。混合并孵育在旋转轮上室温（RT）2小时。 </li><li>离心悬浮液在RT 1800 xg离心2分钟。弃上清，悬浮在1毫升洗涤缓冲液和转让暂停到新的低结合管。在RT孵育5分钟的旋转轮。 </li><li>在在RT 1800 xg离心2分钟，再悬浮于1ml的洗涤缓冲液中离心悬浮液并温育在室温在旋转的轮5分钟。重复4次。对于最后一次洗涤转移到一个新的较低的结合管。 </li><li>除去最大上清液。加7微升的洗涤缓冲液，4倍样缓冲液7微升和3微升10X还原剂至珠，混合和在96℃煮沸5分钟。 </li><li>执行带有int抗体蛋白质印迹分析erest（具体为合作伙伴候选免疫沉淀的蛋白质）。使用输入提取物的1％（5微克），为内源蛋白的水平（ 图3B）的控制。 </li></ol></li></ol></li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatics+enrichment+tools:+paths+toward+the+comprehensive+functional+analysis+of+large+gene+lists.">Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.</a> Nucleic Acids Res. 37 (1), 1-13 (2009).</li><li>Ohta, S., 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=The+protein+composition+of+mitotic+chromosomes+determined+using+multiclassifier+combinatorial+proteomics.">The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.</a> Cell. 142 (5), 810-821 (2010).</li><li>Yahi, H., 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=Differential+cooperation+between+heterochromatin+protein+HP1+isoforms+and+MyoD+in+myoblasts.">Differential cooperation between heterochromatin protein HP1 isoforms and MyoD in myoblasts.</a> J Biol Chem. 283 (35), 23692-23700 (2008).</li><li>Philipot, O., 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=The+core+binding+factor+CBF+negatively+regulates+skeletal+muscle+terminal+differentiation.">The core binding factor CBF negatively regulates skeletal muscle terminal differentiation.</a> PloS one. 5 (2), (2010).</li><li>Joliot, 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=The+SWI/SNF+subunit/tumor+suppressor+BAF47/INI1+is+essential+in+cell+cycle+arrest+upon+skeletal+muscle+terminal+differentiation.">The SWI/SNF subunit/tumor suppressor BAF47/INI1 is essential in cell cycle arrest upon skeletal muscle terminal differentiation.</a> PloS one. 9 (10), e108858 (2014).</li><li>Tanese, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+density+gradient+sedimentation+to+separate+and+analyze+multiprotein+complexes.">Small-scale density gradient sedimentation to separate and analyze multiprotein complexes.</a> Methods. 12 (3), 224-234 (1997).</li><li>Singh, K., 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+KAP1+phosphorylation+switch+controls+MyoD+function+during+skeletal+muscle+differentiation.">A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation.</a> Genes Dev. 29 (5), 513-525 (2015).</li><li>Cong, L., 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Yahi, H., Philipot, O., Guasconi, V., Fritsch, L., Ait-Si-Ali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modification+and+muscle+differentiation.">Chromatin modification and muscle differentiation.</a> Expert Opin Ther Targets. 10 (6), 923-934 (2006).</li><li>Sun, L., Liu, L., Yang, X. J., Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt+binds+prohibitin+2+and+relieves+its+repression+of+MyoD+and+muscle+differentiation.">Akt binds prohibitin 2 and relieves its repression of MyoD and muscle differentiation.</a> J Cell Sci. 117. 117 (Pt 14), 3021-3029 (2004).</li><li>Caretti, G., 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=The+RNA+helicases+p68/p72+and+the+noncoding+RNA+SRA+are+coregulators+of+MyoD+and+skeletal+muscle+differentiation.">The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation.</a> Dev Cell. 11 (4), 547-560 (2006).</li><li>Knoepfler, P. S., 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+conserved+motif+N-terminal+to+the+DNA-binding+domains+of+myogenic+bHLH+transcription+factors+mediates+cooperative+DNA+binding+with+pbx-Meis1/Prep1.">A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.</a> Nucleic Acids Res. 27 (18), 3752-3761 (1999).</li><li>Albini, S., Puri, P. 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The authors have nothing to disclose.

该方法允许详尽的鉴定转录因子的MyoD的合作伙伴。它揭示了MyoD的来诱导分化，即接种HeLa-S3细胞性异源系统MyoD的合作伙伴。因此，根据定义，确定MyoD的合作伙伴普遍表达。这些包括一般和序列特异性转录因子，染色质修饰酶，RNA加工蛋白质激酶（ 表1和2）。自的HeLa-S3细胞对MyoD的诱导转分化抗性，MyoD的活动主要是压制性以及所识别的伙伴可能是MyoD的辅阻遏。这是通过Id?...

真核多细胞生物是由不同的器官和组织。各功能性组织具有特定的基因图案表达，这是在各个分化步骤中确定。细胞分化涉及特定基因的活化，维持它们的表达，并且通常沉默一组基因，例如那些参与细胞增殖的。骨骼肌分化，或成肌，因而是一个多步骤的过程，与中胚层干细胞的确定为成肌细胞开始，然后导致这些成肌成第一单核，然后多核，肌管的终端分化。因此，成肌细胞被"确定"的细胞，其仍然能够增殖，但他们致力于骨骼肌谱系，因此可以单独使用分化成期间胚胎发育或成年肌肉再生的骨骼肌细胞。骨骼肌终端的过程不同entiation由特定遗传程序，与来自成肌细胞前体细胞的细胞周期，导致的增殖相关的基因，如E2F靶基因1明确沉默永久退出开始协调。实际上，终端分化的过程中，成肌细胞增殖阻滞是之前的骨骼肌特异性基因表达和成肌细胞的融合成肌管2的关键步骤。这样的程序允许成年肌肉干细胞，也称为卫星细胞，在以下骨骼肌损伤再生过程来区分。 哺乳动物肌形成是严重依赖于一个家庭生肌碱性螺旋-环-螺旋（bHLH结构）转录因子的MyoD，Myf5的，MRF4和肌细胞生成素，经常被称为家庭的骨骼肌确定因素或回收设施（肌肉调节因子3）3。他们每个人都在扮演着规范和DIF至关重要的作用骨骼肌细胞的ferentiation和具有特定的表达图案4 5-7 Myf5的和MyoD的活化构成了承诺细胞生肌谱系的决定性步骤，和肌细胞生成素的后续表达触发肌形成与骨骼肌特异基因，如MCK（肌肉肌酸激酶）的活化。生肌的bHLH转录因子与从先前无声轨迹8在肌肉的基因的激活MEF2家族的成员进行合作。它们也刺激骨骼肌基因的转录与普遍存在的bHLH蛋白，E12和E47，已知为E蛋白，其在各个基因调控区8结合所谓的E-盒异二聚体。捻，ID（分化抑制剂）等因素负调节这一过程中，通过与MyoD的对于E蛋白结合8竞争。 MyoD的是引发肌肉终末分化9视为主要参与者</s向上&gt;因为它具有诱导在许多完全分化非肌肉系统10-13一个生肌判定/分化（反分化）方案的能力。事实上，MyoD的强制表达诱导不同的细胞类型，甚至没有从另一个胚胎起源12衍生的转分化。例如，MyoD的可肝细胞，成纤维细胞，黑素细胞，神经母细胞，以及脂肪细胞转化成肌样细胞。的MyoD的反式分化行动涉及的生肌遗传方案的在非肌肉环境中的异常活化（特别是它的靶基因），伴随的原始遗传程序（尤其是增殖基因）的沉默。 在增殖成肌细胞，MyoD的表达，但不能激活其靶基因甚至当它结合到他们的启动子14-16。因此，对于MyoD的在未分化的成肌细胞不断表达的需求仍然相当埃卢西伯。 MyoD的抑制可能因镇压染色质修饰酶的招聘其靶基因在激活染色质重塑酶14,17加载之前增殖成肌细胞。例如，在增殖成肌细胞，MyoD的与转录共阻遏KAP-1，组蛋白脱乙酰酶（HDACs）和压制性赖氨酸甲基转移（KMTs），包括组蛋白H3赖氨酸9或H3K9和H3K27 KMTs相关联，并且积极地抑制其靶基因的表达通过建立本地镇压染色质结构14,17。重要的是，最近的一份报告指出，MyoD的本身由H3K9 KMT G9A从而抑制其反式激活活性16的直接甲基化。 参与非肌肉细胞的MyoD的这种转分化的表观遗传机制在很大程度上是未知的。值得注意的是，一些细胞系是对MyoD的诱导转分化抗性。因此，在HeLa细胞中，MyoD的或者是不活动的，或甚至还不如阻遏，而不是转录激活功能，由于缺乏染色质重塑复杂SWI / SNF 18 BAF60C亚基的表达。因此，这种模式可以选择，以更好地表征的MyoD诱导基因抑制的机制。它也适合于测定的MyoD在其靶位点以诱导压制性染色质环境及其关联伙伴，因此揭开MyoD的依赖性压制性机制在增殖成肌细胞微调终末分化的能力。 在这里，我们通过使用耦合到质谱（MS）表征串联亲和纯化（TAP-标签）描述的MyoD伙伴的标识的协议。使用的HeLa-S3稳定表达的Flag-HA-的MyoD的许可来获得足够的材料来净化从分馏核提取物MyoD的。在异源系统的MyoD伙伴的识别随后validati在一个相关的系统。

<table><tbody><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HeLa-S3</td><td>ATCC</td><td>CCL-2.2</td><td></td></tr><tr><td>C2C12</td><td>ATCC</td><td>CRL-1772</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spinner</td><td>Corning</td><td>778531</td><td></td></tr><tr><td>Homogenizer:&amp;nbsp; Dounce homogenizer&amp;nbsp;</td><td>Wheaton</td><td>432-1273 and 432-1271</td><td></td></tr><tr><td>Agitating device for spinners</td><td>Bellco</td><td>778531</td><td></td></tr><tr><td>Sonicator</td><td>Diagenode</td><td>UCD 200</td><td></td></tr><tr><td>Hemocytometer</td><td>Marienfeld Superior&amp;nbsp;</td><td>640610</td><td></td></tr><tr><td>Low-binding tubes</td><td>Sorenson</td><td>27210</td><td></td></tr><tr><td>Empty spin column</td><td>Bio-Rad</td><td>7326204</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SDS-polyacrylamide 4-12%&amp;nbsp; gel</td><td>Life technologies</td><td>NP0336BOX</td><td></td></tr><tr><td>4x loading buffer&amp;nbsp;</td><td>Life technologies</td><td>NP0007</td><td></td></tr><tr><td>10x reducing agent</td><td>Life technologies</td><td>NP0004</td><td></td></tr><tr><td>Silver staining kit</td><td>Life technologies</td><td>A8592</td><td></td></tr><tr><td>Centrifugal filter units, 10K</td><td>Millipore</td><td>UFC501024</td><td></td></tr><tr><td>Protein G agarose beads</td><td>Sigma-Aldrich</td><td>P4691</td><td></td></tr><tr><td>Protein A/G&amp;nbsp; Resin</td><td>Thermo Scientific</td><td>53132</td><td></td></tr><tr><td>Flag resin (anti-Flag M2-agarose affinity gel)</td><td>Sigma-Aldrich</td><td>A2220</td><td></td></tr><tr><td>HA resin (monoclonal Anti-HA agarose)</td><td>Sigma-Aldrich</td><td>A2095</td><td></td></tr><tr><td>MNase</td><td>Sigma-Aldrich</td><td>N3755</td><td></td></tr><tr><td>Flag free peptide (DYKDDDDK)</td><td>Ansynth Service BV</td><td>Custom synthesis</td><td>Resuspend up to 4 mg/ml in 50 mM Tris-HCl, pH 7.8</td></tr><tr><td>HA free peptide (YPYDVPDYA)</td><td>Ansynth Service BV</td><td>Custom synthesis</td><td>Resuspend up to 4 mg/ml in 50 mM Tris-HCl, pH 7.8</td></tr><tr><td>Bicinchoninic acid based protein assay kit : BCA kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Protease inhibitors</td><td>Sigma-Aldrich</td><td>S8830</td><td></td></tr><tr><td>Luminol-based enhanced chemiluminescence (ECL) HRP substrate</td><td>Life technologies</td><td>34075</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>Sheared salmon sperm DNA (Deoxyribonucleic acid sodium salt from salmon testes)</td><td>Sigma-Aldrich</td><td>D1626</td><td></td></tr><tr><td>Spermine tetrahydrochloride</td><td>Sigma-Aldrich</td><td>S1141</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich</td><td>S0266</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>MOPS running buffer</td><td>Life technologies</td><td>NP0001</td><td></td></tr><tr><td>DMEM (high glucose)</td><td>Sigma-Aldrich</td><td>D0822</td><td>Pre-warm&amp;nbsp; at 37 &amp;deg;C before use</td></tr><tr><td>Trypsin-EDTA (0.05% phenol red</td><td>Life technologies</td><td>25300-054</td><td></td></tr><tr><td>Serum</td><td>GE healthcare life sciences&amp;nbsp;</td><td>PAA A15-102</td><td>Each lot of serum has to be first tested for your cells.</td></tr><tr><td>Penicillin and Streptomycin&amp;nbsp;</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Life technologies</td><td>15250-061</td><td></td></tr><tr><td>Water (sterile-filtered)</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HA from rat (12CA5)</td><td>Roche</td><td>11583816001</td><td></td></tr><tr><td>Flag</td><td>Sigma-Aldrich</td><td>A8592</td><td></td></tr><tr><td>MyoD</td><td>Santa Cruz</td><td>sc-32758</td><td>To use for western blotting</td></tr><tr><td>MyoD</td><td>Santa Cruz</td><td>SC-760</td><td>To use for immunoprecipitation and western blotting</td></tr><tr><td>CBFbeta</td><td>Santa Cruz</td><td>FL-182&amp;nbsp;</td><td></td></tr><tr><td>HP1alpha</td><td>Euromedex</td><td>2HP2G9</td><td></td></tr><tr><td>HP1beta</td><td>Euromedex</td><td>1MOD1A9AS</td><td></td></tr><tr><td>HP1gamma</td><td>Euromedex</td><td>2MOD1GC</td><td></td></tr><tr><td>Suv39h1</td><td>Cell Signaling Technology</td><td>8729</td><td></td></tr><tr><td>BAF47</td><td>BD Biosciences</td><td>612111</td><td></td></tr><tr><td>Myf5</td><td>Santa Cruz</td><td>SC-302</td><td></td></tr><tr><td>Tubulin</td><td>Sigma-Aldrich</td><td>T9026</td><td></td></tr><tr><td>Actin</td><td>Sigma-Aldrich</td><td>A5441</td><td></td></tr><tr><td>IgG Mouse</td><td>Santa Cruz</td><td>SC-2025</td><td></td></tr><tr><td>IgG Rabbit</td><td>Santa Cruz</td><td>SC-2027</td><td></td></tr><tr><td>Goat anti-rat -HRP</td><td>Sigma-Aldrich</td><td>A9037</td><td></td></tr><tr><td>Goat anti-rabbit -HRP</td><td>Sigma-Aldrich</td><td>A6154&amp;nbsp;</td><td></td></tr><tr><td>Goat anti-mouse -HRP</td><td>Sigma-Aldrich</td><td>A4416</td><td></td></tr></tbody></table>

identification of myod interactome using tandem affinity purification coupled to mass spectrometry

骨骼肌终末分化开始于多能中胚层前体细胞向成肌细胞的承诺。这些细胞具有仍然增殖的能力，或者它们可以分化和融合成多核肌管，其中，还maturate形成肌纤维。骨骼肌终末分化是由多种转录因子的协调动作协调，特别是肌肉调节因子或回收设施的成员（MyoD的，肌细胞生成素，Myf5的，和MRF4），也称为成肌的bHLH转录因子家族。这些因素精心转录调控网络中的染色质重塑复合物合作，以实现骨骼肌肉发生。在这方面，MyoD基因被认为是引发肌肉终末分化的主生肌转录因子。这个概念是由MyoD的与非肌肉细胞转化成骨骼肌细胞的能力增强。在这里，我们描述了用于识别MyoD的一种方法为了阐明参与骨骼肌终端分化的不同因素以详尽的方式蛋白伙伴。长期目的是了解参与骨骼肌基因的调控， 即 ，MyoD的目标的表观遗传机制。 MyoD的伙伴通过从耦合到质谱（MS）表征的异源系统中，随后有关伙伴的骨骼肌终末分化过程中的作用的验证使用串联亲和纯化（TAP-标记）标识。先天性肌无力，肌强直性营养不良，横纹肌肉瘤和缺陷在肌肉再生:生肌因子，或它们的异常调节异常的形式，与许多肌肉障碍相关联。因此，生肌因素提供潜在治疗靶点池肌肉疾病，既涉及导致疾病本身和再生机制，可以提高疾病的治疗机制。因此，详细understan由生肌因素控制分子间相互作用和基因计划的鼎是有效治疗的合理设计是必不可少的。

理解的MyoD活性的调节，我们进行使用生化纯化的MyoD复合物的详尽描述，根据的MyoD的双重标记的形式，随后通过质谱法（MS）的免疫纯化。表达旗HA-MyoD的标记和表达旗HA许可证控制细胞系使用的HeLa-S3细胞，以获得足够的材料通过执行旗HA-MyoD的双亲和纯化，净化MyoD的复杂。 我们分级细胞提取物到细胞质和细胞核分数，然后再分...

Watch this Scientific Journal Video about Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry at JoVE.com

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

identification-myod-interactome-using-tandem-affinity-purification

Research

JoVE Journal

Biology

9.8K 次观看.  Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité. MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

视频: Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

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

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

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

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

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

科研

生物化学

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) ...

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 ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

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 ...

生物学

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

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

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

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

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 ...

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 ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

量化和站点本地化两个翻译后修改的同步亲和力丰富

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

<meta charset="utf8"></head><body>通过 16 重串联质量标签标记结合二维色谱和质谱进行高通量和深度蛋白质组分析

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

通过 16 重串联质量标签标记结合二维色谱和质谱进行高通量和深度蛋白质组分析

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

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此视频中的章节

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

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

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

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

蛋白复合物的鉴定

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定

量化和站点本地化两个翻译后修改的同步亲和力丰富

通过 16 重串联质量标签标记结合二维色谱和质谱进行高通量和深度蛋白质组分析

通过 16 重串联质量标签标记结合二维色谱和质谱进行高通量和深度蛋白质组分析