Name: profiling thiol redox proteome using isotope tagging mass spectrometry
Uploaded: 2012-03-24T14:00:00
Duration: 12 min 7 s
Description: Watch this Scientific Journal Video about Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry at JoVE.com

笔者想感谢格雷格·马丁博士（康奈尔大学）和他的研究小组提供的DC3000应变，番茄种子，和建议。他们也想感谢督博士牟民建联协议蛋白质组学研究部和用友生物技术研究的跨学科方法开发援助中心的帮助。蛋白提取协议修改从Hurkman和田中16。适应cysTMT标签，步骤4至6条的协议，根据原来的热皮尔斯尔科技产品手册17。这项工作是由国家科学基金会（MCB的0818051小陈）。

1。育苗和准备细菌<ol><li>幼苗发芽在MetroMix 500混合土中生长室（160μmol光子与光照8小时的光，在22米-2 S -1（布雷顿森林公司）°C和16小时黑暗在20°C的1个星期。相对湿度为70％。用自来水浇灌幼苗，需要保持土壤变得干燥。 </li><li>两个同样大小的幼苗，然后移植到直径4“盆载的MetroMix 500土壤和成长为一个额外的3个星期，在相同的条件下为1.1。 </li><li> 野火（PST）是最初的T-挑染到王乙中等（KBM）板（每公升20克Proteose蛋白胨，4 0.1975Ğ硫酸镁，1％甘油，1.5 G K 2 HPO 4，琼脂18克），并成长为两天在28°C。一个单一的殖民地收集，混合成300μL液体国王乙中等（20克Proteose蛋白胨， 硫酸镁 0.1975Ğ </suB&gt;，甘油10毫升，10毫升（10毫升H 2 O K 2 HPO 4 1.5克）100倍的K 2 HPO 4股票，并蔓延到王乙中厚板。这是在28°C培养过夜</li><li>细菌接种准备从KBM板刮成H 2 O 20毫升细菌培养0.03 OD 600（〜10 6 CFU /毫升）准备在1 L接种缓冲液（10 mM的MgCl 2的 +400微升Silwett-70）。接种缓冲减去细菌的控制解决方案的准备。 </li></ol> 2。接种番茄PST和H 2 O 2的组织化学染色<ol><li>四个星期的老厂，接种到上述接种溶液浸泡30秒。在植物清除塑料袋后立即接种。 </li><li> 3&#39;-3&#39;二氨基联苯胺（DAB）染色11（1毫克/毫升在H 2 O的民建联，pH值3.8（HCL））三Days使用前达到完全溶解。该解决方案是震撼，在室温下混合。 </li><li>二十四小时治疗后，完全展开的叶片被拆除，放置在DAB染色表皮朝上，真空渗透15分钟。然后把叶在室温染色晚上在黑暗中。 </li><li>叶片在95％乙醇10分钟，煮沸，然后保持在75％的乙醇。 </li></ol> 3。蛋白质的提取<ol><li>收获后，番茄叶片磨成细粉使用迫击炮和杵与液体氮。 </li><li>为0.5 g组织，加入1.25毫升的Tris pH值8.8缓冲苯酚和0.5提取液1.25毫升/克（0.1米的Tris-HCl pH值8.8，10 mM的EDTA，0.9 M蔗糖），然后继续磨数多分钟在通风柜。 </li><li>转让提取橡树岭离心管（赛默飞世尔科技NALGENE公司），并在室温下2小时搅拌。 </li><li>在5000 XG离心和15°C为10分钟。顶端苯酚相转移到一个新的清洁管。 </li><li>回到提取等量的水相萃取苯酚缓冲区;在30分钟筛搅拌。离心和提取物转移到一个新的Falcon管。 </li><li>重复上述步骤，有更多的时间和提取转移到新的橡树岭管。 </li><li>苯酚提取蛋白质沉淀，加入0.1 M的醋酸铵在100％的甲醇（储存在-20°C）5卷。涡在-20°C孵育过夜。 </li><li>在20,000 XG，4℃收集的蛋白质沉淀20分钟离心。 </li><li>洗冷0.1M醋酸铵甲醇和2倍，80％冷丙酮沉淀2倍。加入1.5毫升70％冷乙醇沉淀和暂停。转移到2毫升离心管（美国科学）和14,000 RPM的离心机，4°C间为20分钟。除去乙醇。 </li><li>在Speed​​Vac浓缩干燥颗粒的短暂溶解的蛋白质EXTR行动缓冲区，如ReadyPrep蛋白抽提试剂盒试剂3（8 M尿素，4％CHAPS，40毫米Tris碱，2M硫脲）（Bio-Rad公司）。 14,000 rpm和20℃离心30分钟，以形成沉淀。收集上清。 </li><li>测量使用的CB-X蛋白检测，根据制造商的手册（基因型科技）的蛋白质浓度。 </li></ol> 4。样品制备和肽的标记与cysTMTs <ol><li>准备在2-5微克/μL浓度的蛋白质样品。在这里，我们每个质量标签使用100微克的蛋白质，20μL50μL样品。在这个实验中，所有6个标签。 </li><li>烷基化液中加入等量（100毫米的Tris-盐酸pH7.5的200毫米碘乙酰胺）的蛋白质样品，以阻止自由巯基组。缓冲区应准备新鲜。烷基化在37°C，持续1小时。 </li><li>加入1毫升80％冷丙酮在-20°C过夜沉淀蛋白质。沉淀蛋白离心14,000转，4°C下20分钟。洗沉淀3次，用80％的涡旋每次丙酮。去除上清，并允许上空气干燥而冰。 </li><li>在50μL裂解液（Tris碱，50毫米，6 M尿素1毫米EDTA）重悬沉淀。然后加入0.5μL100毫米三（2 - 羧乙基）磷（乙基）在室温下孵育一小时，以减少二硫键。 </li><li>准备加入20μL乙腈试剂管溶解标签的标签。涡和自旋向下的底部。 </li><li>洗出额外的TCEP使用1单片机3KD的离心过滤装置（Millipore公司）。加入50μL样品到单片机3KD柱，和50μL裂解缓冲列。 10,000 XG和4°C离心15分钟重复此步骤共三次。删除列和反转成一个干净的管。 1000 XG和4°C离心5分钟</li><li>检查pH值。如果需要，1 M盐酸调整pH值至7.0-8.0。 </li><li>加入5μL的cysTM每个样品（cysTMT的试剂盒提供高达500微克蛋白20μL的标记。此方法使用100微克的蛋白质，因此我们使用标记的五分之一。）ţ试剂。在室温下为2小时，使反应继续进行。 </li><li>样品结合Laemmli样品缓冲液以1:1的比例（Bio-Rad公司）。 </li></ol> 5。拆除非反应标签样品分馏<ol><li>样品煮沸5分钟和一个预制的12％的聚丙烯酰胺凝胶（Bio-Rad公司）分离。凝胶冲洗过滤H 2 O 5分钟一班，3倍和1小时与考马斯蓝染色（Bio-Rad公司）。凝胶的染色过夜与H 2 O </li><li>收集12个组分，从每个凝胶泳道。组分测定蛋白条带的浓度。分数被切成1毫米件，收集到1.5 mL管。 </li><li>凝胶块去染色，在H 2 50％乙腈和0.1 M碳酸氢铵</SUB&gt; O。必须重复此步骤，直到蓝色凝胶块（约3-4次，每次15分钟）中删除。 </li><li>德染色的凝胶块，干燥，用15分钟，上清液中删除，并使用1 Speed​​Vac离心干燥件100％的乙腈（包括凝胶件）。 </li><li>溶解胰蛋白酶（Promega公司）碳铵25毫米（作为合并的标记样品）在相同的体积比1:10（胰蛋白）。例如，600微克，体积为500μL蛋白质样品需要60微克的胰蛋白酶溶解在500μL25毫米碳酸氢铵。 </li><li>加入胰蛋白酶溶液的凝胶块上设置冰补充水分。如果不覆盖件增加50毫米碳酸氢铵缓冲。补液后，在37℃孵育12-16小时。 </li><li>从孵化样品中取出的液体蛋白提取物。 </li><li>停止反应，并删除任何剩余的蛋白质消化，甲酸5％，50％乙腈覆盖凝胶件......摇20分钟，在室温TE凝胶件mperature。将上清转移至提取的蛋白质样品管从5.7。重复两次提取过程。 Speed​​Vac离心使用提取肽擦干。 </li></ol> 6。样品富集cysT​​MT标记肽<ol><li>添加反TMT树脂的梯度0.5毫升50％泥浆管。 （梯度从带分馏凝胶浓度确定。如果馏分收集的暗带组成，需要更多的树脂。带弱的组分需要较少的树脂有较少的蛋白质。） </li><li>树脂，然后洗净，用1倍柱体积的三倍Tris缓冲液（TBS）（25毫米三，0.15 M氯化钠，pH值7.2）（Thermo Scientific的皮尔斯蛋白质研究产品）。 </li><li>每个样品中加入200μL1xTBS。样本，然后加入到反TMT树脂（Thermo Scientific的皮尔斯蛋白质研究产品），并在室温下搅拌2小时后，在4°C过夜摇摆</li><li>将样品列（RMO科学皮尔斯蛋白质研究产品）。 </li><li>每列洗净1X 200μL，TBS电视台的三倍。随后是0.05％CHAPS（1X TBS的溶解）洗涤三次。 </li><li>列，然后1X TBS的4米尿素洗涤三次。两百年的H 2 O的微升，然后用洗柱三倍。 </li><li>每个样品200微升50％洗脱缓冲液（0.4％TFA的50％乙腈）洗脱三次。 </li><li>样本，然后在真空浓缩干燥。 </li></ol> 7。质谱分析<ol><li>悬浮在3％乙腈12μL的样品与0.1％甲酸和5μL，直接注入到Eksigent NanoLC-1D高压液体层析柱（AB Sciex公司，美国）。 </li><li>将分离肽1 Proteopep II C18柱75微米的ID×20厘米（美国）新的目标，用4-60％的梯度（答：3％的乙腈，0.1％甲酸，B：97％乙腈，0.1％甲酸酸）在3微升/分钟超过60分钟。 </li><li>使用了Thermo Scientific的LTQ Orbitrap加大质谱仪检测肽使用前2×3实验单级MS组成的收购与高能量的C陷阱解离（HCD）碎片，其次是3毫秒，3 MS / MS谱图其次/质谱碰撞诱导解离（CID）的蛋白质鉴定。在这种模式下的参数为：隔离宽度：3.0米/ Z;碰撞能量：50％（10％）两个步骤。被选定为双和三重收取肽碎片。动态排斥参数设置为：重复计数= 1;重复时间= 60;排除列表的大小= 500;排除时间= 28。目标值如下：MS = 5 E 5的MS / MS（HCD）= 1，E 5。 FTMS的和300的MS / MS（HCD），离子传输时间设置为500。有两个microscans凯康光谱的需要。 </li></ol> 8。数据库搜索和定量<ol><li>所收购的CID和HCD数据是nalyzed使用Thermo Scientific的蛋白质组学发现者软件1.2（Thermo Scientific的的皮尔斯蛋白研究产品），通过一个分支的工作流程，实施记者离子量化（20 ppm的碎片离子的质量容忍）量化的比例。一个单独的段通过选择的频谱，频谱正规化，谱斑节点处理的MS谱。 </li><li>数据，然后对SEQUEST搜索引擎搜索。用20 ppm的质量公差。静态修改设置的cysTMT试剂（304.18大）和动态修改，包括磷酸化和蛋氨酸氧化（15.99大）。使用RNA数据（约35万来自哈佛大学的条目）13日在这两种情况下使用自定义的一个马铃薯lycopersicum的数据库组成。 </li></ol> 9。代表结果控制番茄叶和1 假单胞菌接种叶的一个形象代表如图1所示。观察治疗控制和假单胞菌处理叶片之间的差异。后叶被删除，染色使用民建联，组织化学染色的染色过程中允许迹象表明在叶组织活性氧（ 图2）。图2A是一个没有染色的控制叶的代表。 图2B是代表叶假单胞菌和积极的H 2 O 2的生产染色处理。蛋白质组差异的氧化还原调节蛋白的发现数据输出的一个例子是如图3所示。这种蛋白质是已知的氧化还原调节蛋白铁氧1月14日 ，已被证明是打在防守中的作用，针对假单胞菌丁香光伏番茄 15。峰值强度之间的控制和接种样品用于获取相对定量，这表明在ferredox的显着变化1氧化还原调节（P &lt;0.05）。高强度峰表明，这种蛋白质被氧化病原治疗。 图4是有相似的一种蛋白质之间的控制和接种样品氧化还原调控的一种蛋白质，蛋白质组发现数据输出的一个例子。类似强度的峰值，表明二硫键改变治疗未规定的存在。该方法将彻底改变科学家发现氧化还原反应半胱氨酸和二硫化物10。 <img alt="图1" src="/files/ftp_upload/3766/3766fig1.jpg" /> 图1。接种番茄的形象代表与控制解决方案（A）和假单胞菌 （二）离开。 &lt;跨度类=“的pdflinebreak”&gt; <img alt="图2" src="/files/ftp_upload/3766/3766fig2.jpg" /> 图2。一个形象代表民建联接种番茄染色与控制解决方案（A）和假单胞菌 （二）离开。叶使用3&#39;-3&#39;二氨基联苯胺染色。从叶片叶绿素含量在95％的乙醇煮沸。深色染色表示的H 2 O 2的存在。只留下接种细菌培养呈深色染色。 <img alt="图3" src="/files/ftp_upload/3766/3766fig3.jpg" /> 图3。蛋白质组发现数据输出铁氧还蛋白-1，不同的氧化还原调节蛋白14的例子。用于上述各峰的峰强度绝对定量。用于控制和接种样品之间的峰值强度进行相对定量。 <img alt="图4" src="/files/ftp_upload/3766/3766fig4.jpg" /> 图4。蛋白质组发现的一种蛋白质，具有相似的峰值强度之间的控制和接种样品数据输出的一个例子。类似强度的峰值，表明二硫键改变治疗未规定的存在。

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Commun. 211, 198-204 (1995).</li><li>Huang, H. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+resistance+to+bacterial+pathogens+affected+by+the+amount+of+ferredoxin-I+proteins+in+plants.">Disease resistance to bacterial pathogens affected by the amount of ferredoxin-I proteins in plants.</a> Mol. Plant Pathol. 8, 129-137 (2007).</li><li>Hurkman, W., Tanaka, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+of+plant+membrane+proteins+for+analysis+by+two-dimensional+gel+electrophoresis.">Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis.</a> Plant Physiol. 81, 802-806 (1986).</li></ol>

没有利益冲突的声明。

该协议提供的信息以及执行DAB染色标记的cysTMT氧化半胱氨酸量化。有利于在研究生产的活性氧以及蛋白质调控的效果接种野火马铃薯lycopersicum时，这些程序。在这个协议中提出的方法提供了一种方法来研究在叶组织的损害最少的方式，导致活性氧在整个叶样品。标签程序提供了一种利用半胱氨酸标记法检查潜在的氧化还原调节蛋白。审查时应激反应的早期阶段，这是有益的。 如同位素编码亲和标签（ICAT）和cysTMT方法可用于研究生物样品中潜在的氧化还原调节蛋白。 ICAT的允许标签和比较两个样本12。两种方法标记的半胱氨酸可以用于蛋白quantificATION 10,12。然而，cysTMT方法允许在实验变异的减少以及复10。标签的数量，使研究人员，包括复制或多个样品，在他们的实验设计。有更多的样本，提供了一个确定蛋白质较多的潜力。一个的cysTMT技术的主要缺点是，它破坏蛋白质鉴定为cysTMT标记肽（6.5-6.6）的选择性富集步骤的整体素质。在很大程度上取决于半胱氨酸残基在蛋白质序列的多肽蛋白质鉴定。富集质谱蛋白质鉴定前提交的胰蛋白酶样品的一部分，这个问题是可以克服的。 由于实验设计以及标签机制，cysTMT方法利用的性质，某些步骤是至关重​​要的。在执行蛋白质精度pitation和沉淀洗涤（3.9），它是重要保持冰上样品，以减少蛋白质的降解。在cysTMT标签，去除还原剂（4.6）是重要的，因为样品可以进行反向的标签。反向标签是可能的，如果减少试剂样品中仍然。如果样品标签后减少，cysTMT的标签可以被删除。一旦标签被添加到样品，pH值必须检查（4.7）为了有最佳的标记效率。此外，数据分析是依赖于需要的研究员，在使用该协议的最终目标是什么。它也被用来作为每个软件都有不同的算法软件的依赖。 本实验利用作为提高生产番茄活性氧物种的激发子病原体;然而，其他调节氧化还原反应，可测得相应。这个实验设计是适应其他植物和动物系统。 </P&gt;

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MetroMix 500 </td><td>布雷顿森林公司</td><td> TX-500 </td></tr><tr><td> 3,3&#39; - 二氨基</td><td> Sigma-Aldrich公司</td><td> D8001 </td></tr><tr><td> ReadyPrep顺序抽提试剂盒试剂3 </td><td> BIO-RAD </td><td> 163-2104 </td></tr><tr><td> CB-X蛋白检测</td><td>格诺科技</td><td> 786-12X </td></tr><tr><td> cysTMT试剂</td><td> Thermo Scientific的皮尔斯蛋白质研究产品</td><td> 90071 </td></tr><tr><td> Laemmli样品缓冲液</td><td> BIO-RAD </td><td> 161-0737 </td></tr><tr><td>生物安全Comassie的（的G-250染色） </td><td> BIO-RAD </td><td> 161-0786 </td></tr><tr><td>单片机3KD柱</td><td> Millipore公司</td><td> 42403 </td></tr><tr><td>固定化反TMT的树脂</td><td> Thermo Scientific的皮尔斯蛋白质研究产品</td><td> 90076 </td></tr><tr><td>离心机列</td><td> Thermo Scientific的皮尔斯蛋白质研究产品</td><td> 89896 </td></tr><tr><td> proteopep二C18柱</td><td>新目标</td><td> PFC7515-PP2安理会-10 </td></tr><tr><td> NanoLC-1D高效液相色谱法</td><td> AB SCIEX </td><td> 90389 </td></tr><tr><td>的LTQ Orbitrap XL </td><td> Thermo Scientific的</td><td> 0020137580 </td></tr><tr><td> Speed​​Vac离心</td><td> labconco </td><td> 7812013 </td></tr><tr><td>蛋白质的发现者1.2软件</td><td> Thermo Scientific的皮尔斯蛋白质研究产品</td><td></td></tr><tr><td>胰蛋白酶</td><td> Promega公司</td><td> V5111 </td></tr><tr><td>橡树岭离心管</td><td>热科学NalgeNE公司</td><td> 3139-0050 </td></tr><tr><td>离心管（2毫升） </td><td>美国科学</td><td> 1620年至2700年</td></tr><tr><td> 12％的Mini-PROTEAN TGX预制凝胶</td><td> BIO-RAD </td><td> 456-1043 </td></tr><tr><td>返回表格 &gt;生物安全的考马斯StainBottom表格</td><td> BIO-RAD </td><td> 161-0786 </td></tr><tr><td> TMT的富集试剂盒</td><td> Thermo Scientific的皮尔斯蛋白质研究产品</td><td> 90077 </td></tr></tbody></table>

profiling thiol redox proteome using isotope tagging mass spectrometry

野火光伏番茄应变DC3000不仅会导致细菌性斑点病在马铃薯lycopersicum，但也对甘蓝型油菜品种，以及对拟南芥 ，一种基因听话的寄主植物1,2。番茄细菌性斑点病过程中积累的活性氧（ROS），在与DC3000接种子叶表明了ROS在调节细胞坏死的作用。过氧化氢 ​​，活性氧的一个组成部分，是假单胞菌番茄植株接种后产生的。可以检测过氧化氢 ​​，使用1组织化学染色3&#39;-3&#39;二氨基联苯胺（DAB）4。 DAB染色与过氧化氢 ​​反应生成棕色染色的叶组织4。 ROS有一个细胞的氧化还原环境，它可以改变某些蛋白质的氧化还原状态的调节作用。半胱氨酸是一种重要的氨基酸，敏感氧化还原的变化。轻度氧化下，可逆的氧化半胱氨酸的巯基氧化还原传感器和信号转导，调节各种生理过程6,7。串联质谱标记（TMT）的试剂能够同时识别和定量蛋白质在不同的样品采用串联质谱法8,9复。半胱氨酸反应TMT（cysTMT）试剂可以选择性的标签和相对定量半胱氨酸的肽含有多达6个生物样品。每个的恒压cysTMT标签具有相同标称父的质量和组成的巯基反应组，一个MS-中性间隔臂和一个MS / MS记者10。样品标签后，蛋白酶消化。使用含有反TMT抗体树脂富集半胱氨酸标记的肽。在MS / MS分析，出现一系列的记者离子（即达126-131），在低质量的区域，提供相对定量的信息。工作流是有效降低样品的复杂性，提高动态范围和研究半胱氨酸修改。在这里，我们提出的Pst DC3000处理番茄的氧化还原蛋白质组分析（格兰德河）离开使用cysTMT技术。这种高通量的方法，有可能被应用到研究其他的氧化还原调节生理过程。

Watch this Scientific Journal Video about Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry at JoVE.com

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

活性氧水平升高，当细胞遇到压力条件。在这里，我们表明3&#39;-3&#39;二氨基联苯胺染色的例子，以及cysTMT标签和质谱分析的氧化还原蛋白质在<em&gt;野火</em&gt;处理番茄叶片。

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

profiling-thiol-redox-proteome-using-isotope-tagging-mass-spectrometry

Research

JoVE Journal

Biology

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 ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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 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 ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

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

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

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 ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...