MH从“德意志研究联合会”（DFG）的承认支持。作者 贡献：MH设计的研究，KT，JS和MT进行研究和分析的数据，KT和MH写文章。

1。 衣藻的培养<ol><li>以下三莱茵衣藻株在本研究中使用：WT cc124，WT CW15-arg7（细胞壁缺陷和精氨酸营养缺陷型），一个ΔPSI突变体菌株29和一个pgrl1敲除菌株4。 </li><li>所有菌株均生长在Tris-乙酸盐-磷酸盐（TAP）的介质30，在25℃用20-50的连续光强度μE/米2秒，振摇在120rpm。 ΔPSI的文化应该被包裹着一些纸巾&lt;5μE/米2秒的曝光。 </li><li>标记株（ΔPSI和WT cc124，分别）的培养物含有7.5 mM的15 N的NH 4 Cl等 ，而文化对于未标记株（WT CW15-arg7和pgrl1，分别）含有7.5 mM的14 N的NH 4 Cl等 。细胞具有生长为至少4 GENERAT离子全部实现标签和必须保持在指数生长期。 </li><li>于实验开始计数并稀释细胞至1×10 6个细胞/在至少为750毫升/应变的体积ml的密度的前一天。 </li></ol>请注意，两种类型的不连续蔗糖密度梯度在以下方案中描述。根据Takahashi 等人 9的光系统蔗糖密度梯度用来在过夜离心分离不同的光合蛋白质复合物从分离和溶解的类囊体，并且必须在一天前制备（参见方案2）和类囊体蔗糖密度梯度8顷应用的类囊体分离过程中（见协议3）。 2。光系统蔗糖密度梯度准备<ol><li>浇注梯度需要三种股票的解决方案： <ol><li> 2M的蔗糖</li><li> 10％的β-DM，在H 2 O的</li><li> 0.5M的甲基甘氨酸，pH 8.0的氢氧化钠（NaOH） </li></ol></li><li>使用这些个股，准备了以下解决方案： <table border="0" cellpadding="0" cellspacing="0" width="544"><tbody><tr height="20"><td height="20" style="height:20px;width:94px;">浓度： </td><td style="width:75px;"> 130万</td><td style="width:75px;"> 1.0M的</td><td style="width:75px;"> 0.85米</td><td style="width:75px;"> 0.7M的</td><td style="width:75px;"> 65万</td><td style="width:75px;"> 0.4M的</td></tr><tr height="20"><td height="20" style="height:20px;width:94px;"> 2M的蔗糖</td><td style="width:75px;"> 13毫升</td><td style="width:75px;"> 10毫升</td><td style="width:75px;"> 8.5毫升</td><td style="width:75px;"> 7毫升</td><td style="width:75px;"> 6.5毫升</td><td style="width:75px;"> 4毫升</td></tr><tr height="20"><td height="20" style="height:20px;width:94px;"> 10％46;-DM </td><td style="width:75px;"> 100微升</td><td style="width:75px;"> 100微升</td><td style="width:75px;"> 100微升</td><td style="width:75px;"> 100微升</td><td style="width:75px;"> 100微升</td><td style="width:75px;"> 100微升</td></tr><tr height="20"><td height="20" style="height:20px;width:94px;"> 0.5M的甲基甘氨酸</td><td style="width:75px;"> 200微升</td><td style="width:75px;"> 200微升</td><td style="width:75px;"> 200微升</td><td style="width:75px;"> 200微升</td><td style="width:75px;"> 200微升</td><td style="width:75px;"> 200微升</td></tr><tr height="20"><td height="21" style="height:20px;width:94px;"> H 2 O的</td><td style="width:75px;"> 6.7毫升</td><td style="width:75px;"> 9.7毫升</td><td style="width:75px;"> 11.2毫升</td><td style="width:75px;"> 12.7毫升</td><td style="width:75px;"> 13.2毫升</td><td style="width:75px;"> 15.7毫升</td></tr></tbody></table></li><li>使用14毫米x 89毫米离心管中，并倒入梯度非常缓慢，开始以较低的密度最高的解决方案蔗糖密度解决方案。 </li><li>倾每个只有1毫升的1.3和1.0M溶液和2ml的溶液的其余部分。 </li><li>离开梯度隔夜在寒冷的房间。 </li></ol> 3。厌氧诱导和类囊体膜8的隔离<ol><li>开始的类囊体膜的分离之前，通过用4小时氩气鼓泡引起的厌氧条件。发泡可以通过玻璃吸管与培养的恒定混合有磁力搅拌棒的培养烧瓶中进行。 </li><li>通过在2,500 xg离心制粒细胞5分钟，从类囊体膜的分离，重悬在H 1缓冲液（0.3M的蔗糖，25mM的HEPES（pH值7.5），5mM的MgCl 2的）。 （请注意，与类囊体膜样品SH隔离时开始乌尔德保持在冰上，以避免蛋白质降解，所有离心步骤都在4℃和手套工作，强烈建议避免角蛋白污染。） </li><li>颗粒细胞5分钟，2,500 xg离心，重悬在H1的缓冲区。 </li><li>通过与1500百帕氮气压力喷雾器打破细胞有两个通道（无细胞壁的菌株做只有一个通道）。 </li><li>降速细胞7分钟，2,500×g的。 （在此步骤之后将上清液应该浅绿，如果不是，则电池单电池破损没有成功。） </li><li>悬浮细胞在H2缓冲液（0.3M的蔗糖，5毫米的HEPES（pH值7.5），10 mM的EDTA（pH值8.0））和颗粒细胞在32,800×g的10分钟。 </li><li>悬浮细胞在H3的缓冲液（1.8米蔗糖，5毫米的HEPES（pH值7.5），10 mM的EDTA（pH值8.0））和均质颗粒与陶工（没有绿色的颗粒应保持在这一工作步骤结束）。 </li><li>在浴缸类囊体准备蔗糖密度梯度ES（25毫米x 89毫米）由从底部开始有一层12毫升H3缓冲液与细胞，倒入12毫升H4缓冲的中间层（1.3M蔗糖，5mM的HEPES（pH 7.5）中，10毫摩尔EDTA（ pH 8.0）中），和在顶部12毫升H5缓冲液（0.5M蔗糖，5mM的HEPES（pH 7.5）中，10毫摩尔EDTA（pH8.0）中）。浇注梯度时要小心，避免了三层的混合。 </li><li>平衡了H5缓冲和离心类囊体蔗糖密度梯度为1小时，在70,700×g的。 </li><li>从类囊体蔗糖密度梯度除去囊体条带，并与适当量H6缓冲液（5mM HEPES（pH 7.5）中的10mM EDTA（pH 8.0）中）稀释它们。 </li><li>降速细胞在37,900×g离心20分钟。如果颗粒不紧，更H6缓冲是需要这种离心步骤。 </li><li>类囊体悬浮在小体积H6缓冲区，并进行测定叶绿素浓度。 </li></ol> 4。叶绿素浓度31的测定</suP&gt; <ol><li>叶绿素量的确定是用80％的丙酮进行​​。 </li><li>混合995微升80％的丙酮和5μl，以H6缓冲液囊体（1:200稀释）。 </li><li>涡流几秒钟，直到没有绿色颗粒保持，然后停止旋转，在14.1×g离心5分钟。 </li><li>取上清液，测定灭绝在663.6和646.6 nm和750 nm处，分别。 </li><li>使用下列公式计算叶绿素金额： <ol><li> Ç 叶绿素a [毫克/毫升] =（0.01225 * E 663.6 - 0.00255 * E 646,6）*稀释倍数</li><li> Ç 叶绿素b [毫克/毫升] =（0.02031 * E 646.6 - 0.00491 * E 663.6）*稀释倍数</li><li> Ç 叶绿素 [毫克/毫升] = C 叶绿素a + C 叶绿素b </li></ol></li></ol> 5。光系统蔗糖密度梯度加载中<ol><li>计算类囊体，β-DM和H6缓冲区需要的金额每个梯度： <ol><li>每个梯度应装有700微升总体积为0.8毫克/毫升的叶绿素在0.9％的β-DM（溶解β-DM中H 2 O），填充H6缓冲液的剩余量。 </li></ol></li><li>对于增溶准备不同的等份与类囊体，β-DM和H6缓冲区每个梯度。 </li><li>离开样本20分钟，冰与定期混合（反相）每隔几分钟（溶解步骤）。 </li><li>以最大速度离心（14,000×g离心）为10分钟，在4°C。 （离心后，将沉淀物应该是小而发白，而上清液为暗绿色。） </li><li>加载上清对梯度。 </li><li>用H6缓冲液并在4℃下旋转利用超速离心机在134,470×g离心过夜（14小时）平衡 （请注意，使用如本协议提出了光系统蔗糖密度梯度光合复合物的分离成功是全光照时才能实现克新鲜分离的类囊体，由于预测为增溶和复杂的分离是困难的与已经之前冻结类囊体膜中工作时作出。） </li></ol> 6。光系统蔗糖密度梯度分离<ol><li>采取梯度的照片。 </li><li>使用针和分馏梯度中加入500μl管穿刺孔在管的底部。或者，所述分馏也可以通过使用96孔板（微孔板）上进行。 </li><li>当使用微板，确定不同的梯度级分的吸光度在675纳米。 （在这一步骤中的样品可以贮存于-80℃下几个星期。） </li></ol> 7。 SDS-PAGE和免疫检测 （请注意，只有对WT的对比ΔPSI比较的免疫印迹分析已选择分数为后续质谱分析。对于实验WT与pgrl1 FR行动者是由在不同部位的吸光度的方法。） <ol><li>独立的30微升的WT和ΔPSI在13％SDS-聚丙烯酰胺凝胶32梯度每个分数的。 </li><li> ANR1（TEF7）26，PGRL1 34，CAS号6，PSI的亚基PSAD 32和PSII核心亚基D1：使用下列抗体进行免疫印迹分析33。使用的所有抗体用稀释1:1,000（增强化学发光检测法），除了D1抗体，其中应用稀释为1:10,000被使用。 </li><li>基于免疫检测的结果，采取ANR1，PGRL1及PSAD的峰组分WT和ΔPSI（这里馏分6和13，分别），混合30微升部分6从WT和ΔPSI，并与部分13从做同样的无论筹备工作，并再次将它们分开在13％SDS聚丙烯酰胺凝胶。 </li><li>对于WT与pgrl1的定量比较采取了C通过测量在不同的梯度级分的吸光度测定，混合30μl的两个样品随后在13％的SDS聚丙烯酰胺凝胶上分离的EF-超复合馏分。 </li><li>染色的蛋白条带在4℃的考马斯溶液（85％亚磷酸，757 mM的硫酸铵，1.2％的考马斯亮兰，20％甲醇）处理2小时，在室温或过晚</li><li>要删除非特异性染色，洗几次与双蒸2 O。 </li><li>切车道成1mm的凝胶块，并继续进行胶内消化。 （可替换地，样本可以干燥几分钟，在真空离心机和贮存几周的凝胶内消化，然后再继续）。 </li></ol> 8。胶内消化（从舍甫琴科等人 35修正） 请注意，准备在即将使用前的所有缓冲区和解决方案，并采取玻璃瓶含有乙腈（ACN）的缓冲区。&lt;BR /&gt;注意！用ACN工作可能是有害的，更多的信息可以从以下网站下载：http://www.sciencelab.com/msds.php?msdsId=9927335（2013年）。 <ol><li> &gt; 10卷双蒸2 O（〜200微升）的30秒洗凝胶块。 </li><li>孵育凝胶块用300微升25毫米的NH 4 HCO 3为15分钟，振摇，除去液体。 </li><li>孵育凝胶片用300微升的25mM NH 4 HCO 3在50％ACN 15分钟，在摇动（通过混合乙腈和50mM NH 4 HCO 3以1:1的比例制备），除去液体。 </li><li>如果凝胶块还是蓝色的，重复的NH 4 HCO 3和NH 4 HCO 3 /乙腈洗，直到大部分的考马斯亮被删除。 （虽然大部分考马斯染色的应被删除，这是没有必要完全脱色的凝胶块。） </li><li>加入100μl乙腈脱水的凝胶片5分钟。凝胶块应该收缩ND看起来完全白色。 </li><li>删除尽可能多的ACN，尽量进行胰蛋白酶消化。或者，样品可以贮存于-20℃下几个星期。 </li><li>添加每个波段10-20微升20纳克/微升胰蛋白酶在10％乙腈/ 25 mM的NH 4 HCO 3（新鲜摊薄），继续冰样本。 </li><li> 30分钟后，检查是否所有溶液吸收，并在必要时添加更多的胰蛋白酶缓冲。凝胶块应完全覆盖胰蛋白酶缓冲。继续冰样本。 </li><li>离开凝胶块在冰上另外90分钟，以用胰蛋白酶饱和它们。 （关键步骤：胰蛋白酶肽的产率与在冰上增加孵育时间大大增加了酶进入聚丙烯酰胺基质慢速扩散的可能，因为它需要有一个稍微过量的溶液覆盖所述凝胶片，以避免碎片。隔夜孵化期间跌倒干。） </li><li>消化在37℃4-6小时。对于需要实验最大的肽恢复，消化过夜。 </li><li>消化后降速简明缓冲区。 </li><li>超声处理管5分钟，再洗脱肽和离心机。 </li><li>收集上清液，并将其转移至新管。 </li><li>在超声波水浴中15分钟80微升的30％乙腈/ 1％甲酸（FA）进行肽的提取。 </li><li>与80μL30％乙腈/ 1％FA在超声波水浴中15分钟进行提取。 </li><li>与80μL70％乙腈/ 1％FA在超声波水浴中15分钟进行提取。 </li><li>再次和池上清液离心与以往洗脱液。 （FA用于灭活胰蛋白酶和肽增加溶解度。） </li><li>完全在真空离心机干燥的肽溶液。 （此时样品可存放数周在-20℃下继续进行质谱分析之前。） </li><li>重悬肽6微升的MS缓冲液（5％乙腈，0.1 V / V FA，水）和超声为2-5分钟。 </li><li>离心样品在maximu米速度5分钟，以去除颗粒物质。 </li><li>使用4μL上清进行质谱分析。 </li></ol> 9。与“Proteomatic”MS-数据分析与开源软件“Proteomatic”（可在http://www.proteomatic.org/下载），一个平台，允许发电和MS / MS数据评估管道完成后，通过进行数据分析免费和商业软件36。简单地说，设置如下所述，更详细的信息，可以在6,26找到。 <ol><li>的质谱数据鉴定和量化 从实验WT与ΔPSI识别和蛋白质的定量进行了与OMSSA（版本2.1.4 37）和qTrace 26，分别描述6,26。从实验中野生型与pgrl1以下更新管线的MS数据分析方法：<ol><li>除了​​OMSSA 37，蛋白质也被确定了X！串联（版本2013年2月1日38）。两种算法通过检索对由衣 JGI的基因模型数据库4.3版结合奥古斯数据库版本10.2，以及对NCBI的加入数据库生成的数据库应用数据库BK000554.2和NC_001638.1。 </li><li>通过使用一个目标诱饵的方法39进行质谱鉴定2肽。一个诱饵每个蛋白通过随机洗牌的胰蛋白酶肽，同时保留nonproteotypic肽的冗余创建的。 </li><li>使用小于0.01和结果进行过滤，用5 ppm的前体质量耐受后验误差概率（PEP）的阈值与该软件qvality（0.3.3 40版）进行肽的统计验证。 </li><li>肽从OMSSA和X！串联运行被加入并与qTrace量化<sup&gt; 26采用以下过滤器的步骤：要求MS 2鉴定，补充蛋白质名/组信息，需要两个妹妹肽。 </li></ol></li></ol>为了研究蛋白质之比是否朝向彼此显著不同在来自WT和pgrl1混合CEF-超复合馏分，进行统计学分析应用软件SPSS（版本21）。该色素b 6 / f复合体的亚基测试对彼此以及蛋白质FNR，FTSH2和HCF136对PSI亚基。每蛋白质每扫描计数四个重复的肽比例进行了测试正态分布与夏皮罗 - 威尔克斯测试。由于肽种群不是正态分布（P &lt;0.001），非参数统计被执行。首先，Kruskal-Wallis检验施加评估组间显著发散。只有当这个测试预测显著差异团体s之间，进行进一步的分析，预测两个独立的组间显著差异与采用Mann-Whitney-U检验。

作者宣称没有竞争的经济利益。

利用稳定同位素标记不同的蛋白质组定量研究已经发表在最近几年。在这些实验中通常有两种不同的样品进行比较，其中一个样品都标有一个稳定的同位素。其后，两个样品的蛋白质或肽组合在相等的比率，并进一步处理一起48。这种研究常常想比较定义孤立的细胞区室（ 例如叶绿体，线粒体，或类囊体膜）暴露于不同应激条件26,34,49调查向上或向下调节特定蛋白质。所描?...

在植物和藻类类囊体膜的光合过程可以以线性和环状的方式发挥作用。在线性电子流（LEF）光系统I（PSI），光系统II（PSII）和细胞色素b 6 / f电子转移，最终从水到NADP + 1，导致NADPH和ATP 2的产生。与此相反，循环电子传递（CEF），这被称为像状态2 3和厌氧条件4，结果在由注入电子返回到电子传递链的再还原氧化的PSI不同的环境条件下被诱导。这个过程可以发生在任何的色素b 6 / f复合体1的基质侧或在质体醌库5并产生ATP，但没有NADPH 2。 所提出的协议的目的是展示一个质谱（MS）基于米法认列在莱茵衣藻的类囊体膜多蛋白复合物来获得不同的条件（通过比较基因不同的菌株例举）下洞察这些配合物的组合物的比较，定量分析。这种方法在出版物由寺岛等被使用。在2012年呈现持续进修基金C中的钙离子依赖的调节衣藻由多蛋白复合物包括蛋白CAS号，ANR1和PGRL1 6介导的。该过程将通过比较分析CEF-超复合两种基因不同菌株的组成，从而采取贴标两个菌株与重氮（15 N）的一个优势来解释。简要地，该协议包括类囊体膜的制备，随后的洗涤剂溶解，并在蔗糖密度梯度光合配合物分馏。梯度分离后，选定压裂两株蒸发散是基于等体积混合，通过SDS-PAGE随后在凝胶内消化和随后的定量质谱分析分离。 如上所述，CEF是不同的环境条件下诱导，并从2010年出版演示了CEF功能，超复合的隔离状态下的2锁定单元格衣藻 7，这是由于超速离心过程中蔗糖密度梯度分离溶解的类囊体膜进行。从岩井等 7种不同的，所提出的协议说明了CEF-超复合的厌氧生长C.隔离莱茵衣藻培养物下面的替代程序。这包括在类囊体隔离协议的变化，以及关于在溶解步骤的差异和蛋白质复合物通过超速离心分离。在本协议中，类囊体膜通过施加由蔡氏和Bennoun 8公布的程序，而用于类囊体膜制剂通过岩井等的缓冲液中分离人含有25mM的MES，0.33M的蔗糖，5毫摩尔MgCl 2，1.5 mM氯化钠（pH6.5）中所描述的9。用0.7-0.8％的洗涤剂（N-十三烷基-β-D-麦芽糖苷）进行增溶，在冰上在岩井和同事的情况下30分钟，而这里所描述的增溶方法依赖于使用0.9％的去污剂（正十二烷基-β-D-麦芽糖苷（β-DM））和仅20分钟在冰上进行。两组用每毫升0.8毫克叶绿素为与各自的洗涤剂的溶解。对于光合作用复合体从溶解的类囊体膜的分离岩井等人采用蔗糖浓度0.1-1.3 M之间，而该协议的作者使用浓度为0.4-1.3米的最后一个区别是离心速度，相比是较低的在EArlier出版。 类囊体膜与非离子洗涤剂然后再进行蔗糖密度梯度分级分离的增溶作用已经在大量的研究，从20世纪80年代，直至今日7，9-14和还蛋白的代谢标记的应用是在蛋白质组学领域中的广泛的方法中得到应用。所描述的方法通过将其在重氮如在15 N的NH 4 Cl等 ，其掺入到所有氨基酸导致大规模的形式唯一氮源的存在下培养适用于比较两种菌株之一的15 N代谢标记移取决于肽的氨基酸序列。当分析的内一个MS运行14 N和15 N的混合物，该质量转移可以被用来确定样品原点为每种肽的丰度和可以计算表示为对应的相对丰度相肽ING蛋白质15。 C.上众多的定量蛋白质组学研究莱茵衣藻是可用的，这比较确定蛋白质的量来分析实验条件（ 例如 ，由于营养16-19或光胁迫20,21改变蛋白质组）之间变化，蛋白质组。相比，这些研究中，在目前提出的方法的样品的等体积相结合，并进行分析。这种设置允许以研究蛋白质的迁移行为的梯度内，此外，分析不同的复合物相对于所研究的菌株的组合物。 此方法将被解释主要集中在三种蛋白质：第一候选是叶绿体本地化钙传感器蛋白CAS，这被证明是涉及光驯化C中藻 22。钙被认为是一个重要的信号ING离子为激活由于不同的生物和非生物胁迫最终导致基因表达的变化和细胞生理学和23有人提出，叶绿体可能有助于细胞内Ca 2 +通过CAS蛋白22,24,25的信号通路。第二蛋白是ANR1（厌氧反应1 6），其被证明是缺氧的生长条件下在C诱导的蛋白藻 26。值得注意的是，中科院以及ANR1被认定为CEF-超复合，而且，通过使用反向遗传学的方法，它表明，这两种蛋白质有助于在功能上的CEF 在体内 6，支持他们为这种蛋白质复合体的功能性亚基作用亚基。第三蛋白是类囊体蛋白PGR5样1（PGRL1），将其显示参与在CEF中衣藻 4,27以及在拟南芥 5,28和也标识entified在岩井等人的工作。7 野生型（WT）相对于（对比）一个ΔPSI29株表现出缺失的PSAB基因，编码为一个重要的光系统I亚单位，这也是该组成部分：该方法将通过示出的两个不同的实验的结果呈现CEF-超复合和WT与一个pgrl1敲除株4。为每个实验的CEF-超复合的15 N和14 N-标记的菌株进行了比较之间的定量组成。

<table border="0" cellpadding="0" cellspacing="0" width="1135">
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a new approach for the comparative analysis of multiprotein complexes based on 15n metabolic labeling and quantitative mass spectrometry

所引入的协议提供了在类囊体膜多蛋白复合物的分析工具，通过在不同条件下揭示见解复杂的组合物。在这个协议中的方法被证明通过比较蛋白复合物负责在莱茵衣藻的循环电子流（CEF），分离自遗传上不同的菌株的组合物。该方法包括类囊体膜的分离，随后通过基于差分代谢标记（14 N / 15 N）分离成多蛋白复合物通过蔗糖密度梯度离心法，SDS-PAGE，免疫检测和比较，定量质谱（MS）分析菌株。洗涤剂溶解的类囊体膜对蔗糖密度梯度装在平等的叶绿素浓度。超速离心后，将梯度分离成各种馏分，这是由大量spectromet分析RY基于等量。这种方法允许梯度组分中的成分进行调查，此外，分析不同蛋白质的迁移行为，尤其是专注于ANR1，CAS和PGRL1。此外，这种方法被证明了通过从以往的研究支持的结果（即先前描述的是CEF-超复合如PGRL1，FNR的一部分的蛋白质的识别和PSI-依赖的迁移，并在确认的结果与免疫印迹和另外细胞色素F）。值得注意的是，这种方法适用于解决范围广泛的有本协议可以通过， 例如用于多蛋白复合物成分从不同的环境条件下分离出的比较分析问题。

所引入的定量蛋白质组学方法的目的是多蛋白复合物的组成表征由基因不同C. CEF-超复合成分的比较分析表明类囊体膜衣藻株。所描述的方法已经被成功地由寺岛等人 6施加，并且包括从厌氧生长的培养物类囊体膜，随后洗涤剂增溶的隔离。随后，样品装载到蔗糖密度梯度的基础上平等的叶绿素量和复合物通过超速离心分级。用于定量质谱分析从不同菌株2梯度组分等体?...

Watch this Scientific Journal Video about A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry at JoVE.com

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

所描述的比较，定量蛋白质组学方法的目的是获得见解多蛋白复合物的组成不同的条件下，并证明通过比较基因不同的菌株。为定量分析从蔗糖密度梯度不同的馏分等体积完全由质谱混合并进行分析。

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

12.1K 次观看.  University of Münster. 所描述的比较，定量蛋白质组学方法的目的是获得见解多蛋白复合物的组成不同的条件下，并证明通过比较基因不同的菌株。为定量分析从蔗糖密度梯度不同的馏分等体积完全由质谱混合并进行分析。 

视频: A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

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 ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-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 ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

新技术脱发的定量分析小鼠使用灰度分析

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...