程序在提取协议中常用的试剂和缓冲液: <ol><li>喷气推进实验室的培养基拟南芥悬浮细胞培养<ul><li> 3μM的H 3 BO 3 </li><li> 3微米硫酸锰4×H 2 O的</li><li> 1.1微米的ZnSO 4×7 H 2 O </li><li> 0.15微米KJ </li><li> 0.03微米的Na 2的MoO 4×2 H 2 O的</li><li> 3纳米氯化钴2×6 H 2 O </li><li> 3纳米硫酸铜4×5 H 2 O </li><li> 0.9毫米氯化钙2×2 H 2 O的</li><li> 0.5毫米硫酸镁4×7 H 2 O </li><li> 0.5微米的FeSO 4×7 H 2 O </li><li> 0.5微米的Na 2 EDTA×2 H 2 O的</li><li> 0.12微米盐酸硫胺素</li><li> 0.16微米烟酸</li><li> 0.097微米盐酸吡哆醇</li><li> 0.107μMNAA </li><li> 0.375 mM的KH 2 PO 4 </li><li> 0.061毫米的NaH2 PO 4×2 H 2 O的</li><li> 0.039毫米的Na 2 HPO 4 </li><li> 0.027 mM的甘氨酸</li><li> 0.56毫肌醇</li><li> 1.5％的蔗糖</li><li> 10毫米的K 14 NO 3或10毫米的K 15 NO 3 </li></ul></li></ol>注:pH值在JPL介质应调整到5.7，用KOH。该介质必须通过过滤或在使用前高压灭菌进行灭菌。 <ol start="2"><li>缓冲液H <ul><li> 100mM的HEPES-KOH（pH 7.5）中</li><li> 250mM的蔗糖</li><li> 10％（重量/体积）甘油</li><li> 5毫米EDTA </li><li> 5mM的抗坏血酸</li><li> 0.6％（重量/体积），PVP K-25或K-30 </li><li> 5 mM的DTT（补充新鲜） </li><li> 1 mM的PMSF（补充新鲜） </li><li>蛋白酶和磷酸酶抑制剂（补充新鲜） <ul><li> 50毫米氟化钠</li><li> 1毫米的Na 3 VO 4 </li><li> 1毫米苄脒</li><li> 0.3微米mikrocystin </li><li> 4微米亮肽素</li><li>蛋白酶抑制剂混合物</li></ul></li></ul></li><li>缓冲液R <ul><li> 5mM磷酸钾</li><li> 0.33M的蔗糖</li><li> 3毫米氯化钾</li><li> 0.1毫米EDTA </li><li> 1 mM的DTT（补充新鲜） </li></ul></li><li> TNE缓冲液<ul><li>的25mM的Tris-HCl，pH值7.5 </li><li> 150 mM氯化钠</li><li> 5毫米EDTA </li></ul></li><li>两相系统（6克 - 系统适用于高达15克样品鲜重）的制备方法 在下面列出和15毫升猎鹰管组合成分调匀: <ul><li> 20％（W / W）的葡聚糖​​T500 - 2.6克</li><li> 40％（W / W），聚乙二醇（PEG 3350） - 1.3克</li><li>蔗糖 - 0.678克</li><li>的0.2M磷酸钾，pH 7.8 - 0.15毫升</li><li> 2米氯化钾 - 0.014毫升</li><li>加水，直到两相体系的总质量为6克</li></ul></li><li>蔗糖溶液在TNE缓冲液<ul><li> 2.4 M，1.6 M，1.4 M，0.15M的</li></ul></li><li>试剂胰蛋白酶消化<ul><li> UTU:6M尿素，2M硫脲（pH值为8.0的10mM的Tris-HCl） </li&gt; <li>还原缓冲液（1微克/微升DTT水; 6.5毫米） </li><li>烷基化反应缓冲液（5微克/微升碘乙酰胺在水中，27毫摩尔） </li><li> LYSC内肽酶（0.5微克/微升） </li><li>胰蛋白酶，修改测序级（0.4微克/微升） </li></ul></li><li>试剂肽脱盐超过C 18的<ul><li>悬浮液:2％三氟乙酸（TFA），在水中的5％乙腈</li><li>溶液A:0.5％醋酸</li><li>溶液B:80％乙腈，0.5％乙酸</li></ul></li><li>试剂磷酸化肽富集<ul><li>溶液A:0.1％TFA，5％乙腈</li><li>溶液B:含0.1％TFA，80％乙腈</li><li> TiO 2的10微米</li><li>氨（股票25％溶液） </li><li>哌啶（股100％） </li></ul></li></ol> PROTOCOLS 1。 拟南芥悬浮培养细胞代谢标记<ol><li>种植拟南芥 Col-0中的细胞在整整14 N-JPL媒体来自17叶悬浮培养（18;请参阅"常用试剂和缓冲液"部分）的恒定光照条件下，在80〜100微摩尔/平方米的无菌瓶中15 N-喷气推进实验室中，一组文化2秒，23℃，在不断摇动在120rpm。 </li><li>维持细胞培养物，接种400毫升新鲜JPL培养基用40ml七日龄的细胞培养物在1升烧瓶中。 </li><li> 通过真空抽吸，通过用不锈钢丝网广泛的玻璃漏斗细胞培养收获发生。细胞积聚在网状板和在漏斗和可以容易地从那里收集。细胞被推荐在-80℃或在液氮中研磨之前将冻结。 </li></ol>注:对于15 N代谢标记细胞培养用K 15 NO 3为氮的唯一来源，至少有两个通道，两个多星期19。在忠武riments的比较蛋白质组学，用15 N标记的细胞培养，同时在普通培养基上保持未标记的文化。生物处理将被应用到任何标记的或未标记的培养，而其他用作对照（ 图1）。对于蛋白质制剂，两种文化将被合并20。在规划的实验装置，我们建议考虑相互的标签设置4中相同的处理，一旦应用到15 N-标记的细胞，一旦到了未标记细胞和未标记的分别或15 N标记的细胞作为对照组。在这种情况下，需要细胞培养物的双值。 2。质膜的纯化注:所有其他步骤都进行了冷室和/或冰面上，除非它被注明。 <ol><li>从大约1升的液态氮7天前细胞悬浮培养细胞均质化。该材料可以储存在-80℃直至进一步使用。 </li><li>基于鲜重混合标记的和未标记的冷冻的细胞培养物的相同量的烧杯中，并立即添加2体积的缓冲液H的。若膜微区的准备，建议至少使用7克两个标记和未标记细胞的鲜重。 </li><li>将烧杯在振荡器上振摇直到材料被熔化，并将溶液是液体和没有冰晶。 </li><li>通过一层的Miracloth的过滤匀浆入50ml离心管中。 </li><li>平衡的样品用缓冲液H和以10,000 xg离心8分钟。 </li><li>加载上清液进入预冷超离心管（ 如转子Beckman Coulter公司SW31Ti），与约32毫升的量</li><li>用缓冲液H平衡的样品和通过以100,000×g离心离心在4℃下用贝克曼SW32Ti转子30分钟沉淀的微粒。 </li><li>离心后去除上清。 </l我&gt; </ol>注:上清液含有可溶性蛋白质。少量的这部分可被保存以供进一步的蛋白质沉淀也可溶性蛋白和随后的分析。 <ol start="9"><li>重新悬浮在缓冲液中的R各自量每个样品的膜沉淀并加载到U1的两相体系的顶部（ 图2）。如果6克两相系统时，加载刚好是2克重悬膜沉淀。 </li></ol>注意:缓冲器的R的终体积取决于将要使用的（〜1.6缓冲液的R毫升使用6克系统时，需要的话）的两相系统的大小。它建议使用以下的缓冲液重悬如此纯净缓冲器可以被添加到两相体系中，以获得所要求的重量，而不是使用太多的缓冲液溶解，并不能在整个样品加载到两相体系。 <ol start="10"><li>加载纯缓冲r作为用于日相同体积Ë样品再悬浮到U2。 </li><li>慢慢地倒置他们30倍（约1反转/秒）混合U1和U2管。 </li></ol>注意:不要大力摇晃两相体系。它可以引起缺乏的阶段，在超速离心步骤分离。 <ol start="12"><li>离心样品在1500×g离心4℃10分钟。 </li><li>离心后去除上清。 </li><li>从U1的转移上相到U2和在1500×g离心重复离心在4℃下10分钟。 </li><li>将上阶段从U2到SW41Ti超速离心管和五卷缓冲的R稀释调匀。 </li></ol>注:最终上相可能不得不被分成独立的超速离心管之前，它可稀释5倍。 <ol start="16"><li>离心样品以200,000×g离心在4℃下用SW41Ti转子一小时。 </li><li>重悬膜沉淀在SMaL公司的TNE缓冲液升量（通常为200μL缓冲液使用）耐洗涤剂膜隔离。 </li></ol>注:少量这一部分可以被保存为普通肝素质膜的分析。 注:该质膜组分可贮存于4℃过夜分馏成耐洗涤剂膜和洗涤剂可溶部分之前。 3。耐洗涤剂膜的制备<ol><li>检查通过Bradford测定细胞膜级分的浓度。 </li><li>加的Triton X-100至质膜组分。洗涤剂的最终浓度应保持之间0.5-1％。洗衣粉蛋白质重量的重量比应保持13-15之间。 </li><li>摇样品在约100rpm转速，在4℃下进行30分钟。 </li><li>添加三卷2.4米蔗糖的处理质膜的一个量，获得1.8米决赛中蔗糖的浓度。&lt;/ LI&gt; <li>通过加载为图3中所示的1.8M的级分的顶部的各自的蔗糖溶液制备在SW41Ti超速离心管的蔗糖梯度。 </li><li>离心样品以250,000×g离心在4℃下18小时使用贝克曼SW41Ti转子。 </li><li>小心地从转子拆下超速离心管，以避免这可能是因为在0.15 M/1.4 1M蔗糖界面乳状环可见低密度带的妨害。有时什么也看不到，但部分仍包含耐洗涤剂的蛋白质部分。 </li><li>除去为0.75毫升的量从渐变的顶部馏分。馏分2和3，其覆盖大约150毫升相间环和下面0.5毫升以上体积表示的耐洗涤剂的膜部分。收集这些部分分为15毫升猎鹰管。级分9和10中包含的洗涤剂溶解的膜级分，并且还可以收集用于比较。作进一步的分析，池FRACT离子2和3以及馏分9和10。 </li></ol> 4。提取蛋白，从耐洗涤剂组分由甲醇/氯仿注:所有步骤均在室温下进行。 <ol><li>加入4倍体积的甲醇收集到的分数和涡彻底。 </li><li>加入1体积的氯仿，涡旋彻底。 </li><li>加入3体积的水，旋涡彻底。 </li><li>离心样品在2000×g下使用台式离心机（ 如的Eppendorf 5417R）5分钟。 </li><li>除去蛋白层中的相间上面的水相。 </li><li>加入3倍体积的甲醇，涡旋彻底。 </li><li>离心样品在4000×g离心10分钟。 </li><li>除去上清液，并干燥沉淀。将干燥的沉淀物是准备在溶液消化。 </li></ol> 5。在溶液中的胰蛋白酶消化注意:在此过程中的所有步骤都完成时室温下用变性剂，以减少氨基酸侧链的不必要的衍生化。 <ol><li>在体积6M尿素，2M thiurea，pH值8（UTU）小溶解样品。如与样品（通常大约40微升）兼容使用尽可能低的音量。 </li><li> 10分钟，以沉淀任何不溶性物质溶解在UTU在5000×g离心在台式离心机（ 如 Eppendorf公司5417R）自旋样品。 </li><li>最终溶液的pH值应接近8.0的最佳胰酶消化。请与pH值条。 </li><li>加入1μl的还原缓冲液每50微克样品蛋白质孵育30分钟，在室温下进行。 </li></ol>注:蛋白质含量只有一个粗略的估计是必要的。当样品量是有限的，最好是牺牲精度，而不是在蛋白质测定浪费样品。 <ol start="5"><li>加入1μl的烷基化反应缓冲液，每50微克样品蛋白质孵育20分钟，在室温下在黑暗中。 </li><li>加入0.5微升的lysC的每50微克样品蛋白质和在室温下3小时孵育恒定振摇在700rpm。如果有必要，摘要也可过夜进行。但是，不建议在温暖的温度延长时间，因为它可以增加肽损失，由于水pH值为8的条件下吸附到塑料管材料。 </li><li>稀释样品用4倍体积的10mM Tris-盐酸，pH为8。 </li></ol>注意:此步骤是绝对必要的，以稀释的尿素浓度为胰蛋白酶，是高盐非常敏感。 <ol start="8"><li>加入1μl每50微克样品蛋白质的胰蛋白酶孵育过夜，在室温下用恒定振摇在700rpm。 </li><li>酸化样品，以0.2％TFA的终浓度，以达到pH为2（添加约1/10体积的2％三氟乙酸）。 </li></ol>注:样品可以储存在 - 20°C，直到进一步的使用，但它是更好地存储他们StageTips在4℃，如果它是很短的时间（一周），或将其StageTips脱盐后存储。 6。的C 18 - StageTips制造<ol><li>在平坦，干净的表面上放置一个Empore磁盘C18如一次性塑料培养皿中。 </li><li>用钝头皮下注射针头直径为1.5mm的切出一个小圆盘。磁盘坚持在针和可转移到枪头。 </li><li>推盘出针和由一片熔凝硅石或管道中的针的内配合的固定在移液管尖的锥形。 </li></ol>注:StageTips可在室温下21贮存干燥。 7。使用C 18-StageTips肽脱盐和浓缩<ol><li>通过将50微升溶液B到准备StageTip条件为C 18-StageTip。旋转的离心分离机的前端在2,000 rpm持续2分钟在台式Çentrifuge（ 如 Eppendorf公司5417R）。 </li></ol>注意:使用的适配器旋转StageTips在Eppendorf离心机，液体将被收集在2 ml反应管中。对于较大规模的制剂，舞台技巧也可以放置成一个尖端机架的200μl的提示96和液体可收集在微量滴定板。 注意:切勿使 ​​用更高的速度超过3000 XG在台式离心机（ 如 Eppendorf公司5417R）由于从尖端纺出了C 18盘的风险。 <ol start="2"><li>用100微升溶液A旋转尖端的离心机在5000 XG在台式离心机（ 如 Eppendorf公司5417R）平衡的StageTip。 </li><li>重复步骤2。 </li><li>由样品仔细吹打成枪头与椎间盘内将样品到磁盘上。 </li></ol>注:一个光盘可以结合约。 100微克蛋白质。 <ol start="5"><li>旋在CE的提示ntrifuge直到样品的总容积通过了C 18盘。 </li><li>用100微升溶液A的附带的秘诀在离心洗StageTips两次。 </li></ol>注意:洗涤并加载StageTips可以储存在4℃下进行长达一周。 <ol start="7"><li>洗脱的样品用40微升溶液B的收集在一个新的1.5毫升的反应管中的洗脱液。 </li><li>浓缩样品中的快速真空。在理想条件下，停止脱水过程，因为这里仅有大约1或2个微升的液体。 </li></ol>注:干样品可以储存在-80℃多年。 <ol start="9"><li>加的9微升悬浮液对样品的最终体积，并将其转移到微量滴定板进行质谱分析。 </li></ol>注意:所使用的重悬浮缓冲液的最终体积是依赖于实验者和质谱仪的灵敏度的需要使用。 8。用于磷酸化肽富集的替代协议注:对于磷酸化肽富集，蛋白质提取步骤2中，必须在磷酸酶抑制剂的存在下进行。 注意:实际蛋白质浓度不需要被用于下面的步骤确定。它足以具有的蛋白质含量的粗略估计，以避免不必要的样品损失<ol><li>编写的C 8-StageTips（后续步骤6相同的协议，用于制备C 18 - StageTips）。 </li><li>转移1毫克的TiO 2-珠准备C 8 - StageTips的顶部（使用1毫克的TiO 2，每100微克蛋白质）。 </li><li>平衡在TiO 2的提示用100微升溶液C，自旋在2000×g离心5分钟，在台式离心机（ 如 Eppendorf公司5417R）。 </li><li>混合100从步骤7.7消化和脱盐肽微克（应该是在100微升的解决方案B）用100μl溶液A的</li><li>加载混合样品到平衡的TiO 2列;旋在1000×g离心5分钟。 </li><li>收集流过，再重新放入到同一个提示（通过第二遍后，保持流量）。 </li><li>用100微升溶液A洗尖;旋在2000×g离心5分钟在台式离心机（ 如 Eppendorf公司5417R）。 </li><li>洗脱样品与50微升5％的氨水中。 </li><li>洗脱的样品用50μl5％哌啶的（洗脱液都可以组合）。 </li><li>立即用50微升20％磷酸酸化的样品。 pH应在约2。 </li></ol>注:除在TiO 2的提示和检索粉末。可以用溶液B，并再次用于一个或两个以上的回合。 <ol start="11"><li>再次脱盐酸化过的样品C 18的提示（见第8步）。 </li></ol> 9。肽混合物的LC-MS/MS分析<ol><li>悬浮脱盐phosphopeptides在悬浮缓冲液。 </li><li>负载重悬样品到所选择的LC-MS/MS系统并获取光谱数据依赖模式在60000全宽的半峰建议的解决方案。 </li></ol>注:为了获得最佳的碎片，无论是中性丢失扫描，应适用22，如果可用多级激活23。如果扣除非经常性可用它将使肽骨架碎片而不磷酸24的损失。 注意:需要从原始数据峰值列表要被提取，并提交给数据库的识别以及定量。在这里，我们描述了使用吉祥物和MSQuant，它适用于从LTQ，LTQ-的Orbitrap和LTQ-FT工具（Thermo Scientific的）原始数据文件的设置。 <ol start="3"><li>使用DTAsupercharge在MSQuant的"助手"菜单中提取峰列表。使用默认设置。 </li><li>提交产生的​​峰值列表文件，以吉祥物的搜索引擎。铬itical设置:母离子的质量误差为10ppm，MS / MS质量公差0.5大。固定修饰:carbamidomethylation半胱氨酸，可变修饰:甲硫氨酸的氧化，磷酸化的丝氨酸，苏氨酸，酪氨酸。定量方法:15 N代谢标记。 </li><li>除吉祥物结果作为网页文件。 </li><li>加载吉祥物结果文件和原始文件到MSQuant。选择感兴趣的所有蛋白质并运行"自动定量"。该程序将读取全扫描谱图从原始文件，并执行峰积分为每个肽的标记和未标记的合作伙伴。 </li><li>导出定量结果以电子表格程序或制表符分隔的文本文件。这些数据可以被提交到Excel或再使用软件如Statquant 25或饼干26进一步的统计分析。 </li></ol>

<ol>
	<li>Alexandersson, E., Saalbach, G., Larsson, C., Kjellbom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+plasma+membrane+proteomics+identifies+components+of+transport,+signal+transduction+and+membrane+trafficking.">Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking.</a> Plant Cell Physiol. 45, 1543-1556 (2004).</li><li>Brown, D. A., Rose, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+of+GPI-anchored+proteins+to+glycolipid-enriched+membrane+subdomains+during+transport+to+the+apical+cell+surface.">Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.</a> Cell. 68, 533-544 (1992).</li><li>Lanquar, 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=15N-metabolic+labeling+for+comparative+plasma+membrane+proteomics+in+Arabidopsis+cells.">15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.</a> Proteomics. 7, 750-754 (2007).</li><li>Kierszniowska, S., Walther, D., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratio-dependent+significance+thresholds+in+reciprocal+15N-labeling+experiments+as+a+robust+tool+in+detection+candidate+proteins+responding+to+biological+treatment.">Ratio-dependent significance thresholds in reciprocal 15N-labeling experiments as a robust tool in detection candidate proteins responding to biological treatment.</a> Proteomics. 9, 1916-1924 (2009).</li><li>Singer, S. J., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluid+mosaic+model+of+the+structure+of+cell+membranes.">The fluid mosaic model of the structure of cell membranes.</a> Science. 175, 720-731 (1972).</li><li>Simons, K., Ikonen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+rafts+in+cell+membranes.">Functional rafts in cell membranes.</a> Nature. 387, 569-5672 (1997).</li><li>Baumgart, T., 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=Large-scale+fluid/fluid+phase+separation+of+proteins+and+lipids+in+giant+plasma+membrane+vesicles.">Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.</a> Proc. Natl. Acad. Sci. USA. 104, 3165-3170 (2007).</li><li>Silvius, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+membrane+molecules+between+raft-and+non+raft-domains:+insights+from+model+membrane+studies.">Partitioning of membrane molecules between raft-and non raft-domains: insights from model membrane studies.</a> Biochim. Biophys. Acta. 1746, 193-202 (2005).</li><li>Tanner, W., Malinsky, J., Opekarova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+plant+and+animal+cells,+detergent-resistant+membranes+do+not+define+functional+membrane+rafts.">In plant and animal cells, detergent-resistant membranes do not define functional membrane rafts.</a> Plant Cell. , (2011).</li><li>Lauwers, E., Andre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+yeast+transporters+with+detergent-resistant+membranes+correlates+with+their+cell-surface+location.">Association of yeast transporters with detergent-resistant membranes correlates with their cell-surface location.</a> Traffic. 7, 1-15 (2006).</li><li>Kierszniowska, S., Seiwert, B., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+Arabidopsis+sterol-rich+membrane+microdomains+by+differential+treatment+with+methyl-&#223;-cyclodextrin+and+quantitative+proteomics.">Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-&#223;-cyclodextrin and quantitative proteomics.</a> Mol. Cell. Proteomics. 8, 612-623 (2009).</li><li>Simons, K., Toomre, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+rafts+and+signal+transduction.">Lipid rafts and signal transduction.</a> Nat. Rev. Mol. Cell Biol. 1, 31-39 (2000).</li><li>Maeda, Y., Kinoshita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+remodeling,+trafficking+and+functions+of+glycosylphosphatidylinositol-anchored+proteins.">Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins.</a> Prog. Lipid Res. 50, (2011).</li><li>Keinath, N. F., 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=PAMP-induced+changes+in+plasma+membrane+compartmentalization+reveal+novel+components+of+plant+immunity.">PAMP-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity.</a> J. Biol. Chem. 285, 39140-39149 (2010).</li><li>Stanislas, T., 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=Quantitative+proteomics+reveals+a+dynamic+association+of+proteins+to+detergent+resistant+membranes+upon+elicitor+signaling+in+tobacco.">Quantitative proteomics reveals a dynamic association of proteins to detergent resistant membranes upon elicitor signaling in tobacco.</a> Mol. Cell. Proteomics. 8, 2186-2198 (2009).</li><li>Minami, A., 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=Alterations+in+detergent-resistant+plasma+membrane+microdomains+in+Arabidopsis+thaliana+during+cold+acclimation.">Alterations in detergent-resistant plasma membrane microdomains in Arabidopsis thaliana during cold acclimation.</a> Plant Cell Physiol. 50, 341-359 (2008).</li><li>Pauly, M., Eberhard, S., Albersheim, P., Darvill, A., York, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+mur1+mutation+on+xyloglucans+produced+by+suspension-cultured+Arabidopsis+thaliana+cells.">Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells.</a> Planta. 214, 67-74 (2001).</li><li>Jouanneau, J. P., Peaud-Lenoel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+synthesis+of+proteins+in+cell+suspensions+of+a+kinetin+dependent+tobacco.">Growth and synthesis of proteins in cell suspensions of a kinetin dependent tobacco.</a> Physiol. Plant. 20, 834-850 (1967).</li><li>Engelsberger, W. R., Erban, A., Kopka, J., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+labeling+of+plant+cell+cultures+with+K15NO3+as+a+tool+for+quantitative+analysis+of+proteins+and+metabolites.">Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites.</a> Plant Methods. 2, 1-11 (2006).</li><li>Arsova, B., Kierszniowska, S., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+heavy+nitrogen+in+quantitative+proteomics+experiments+in+plants.">The use of heavy nitrogen in quantitative proteomics experiments in plants.</a> Trends Plant Sci. 17, 102-112 (2012).</li><li>Rappsilber, J., Ishihama, Y., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stop+And+Go+Extraction+tips+for+matrix-assisted+laser+desorption/ionization,+nanoelectrospray,+and+LC/MS+sample+pretreatment+in+proteomics.">Stop And Go Extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.</a> Anal. Chem. 75, 663-670 (2003).</li><li>Olsen, J. V., Macek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+accuracy+mass+spectrometry+in+large-scale+analysis+of+protein+phosphorylation.">High accuracy mass spectrometry in large-scale analysis of protein phosphorylation.</a> Methods Mol. Biol. 492, 131-142 (2009).</li><li>Schroeder, M. J., Shabanowitz, J., Schwartz, J. C., Hunt, D. F., Coon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neutral+loss+activation+method+for+improved+phosphopeptide+sequence+analysis+by+quadrupole+ion+trap+mass+spectrometry.">A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.</a> Anal. Chem. 76, 3590-3598 (2004).</li><li>Frese, C. 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=Improved+peptide+identification+by+targeted+fragmentation+using+CID,+HCD+and+ETD+on+an+LTQ-Orbitrap+Velos.">Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos.</a> Journal of Protrome Research. 10, 2377-2388 (2011).</li><li>van Breukelen, B., vanden Toorn, H. W., Drugan, M. M., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=StatQuant:+a+post-quantification+analysis+toolbox+for+improving+quantitative+mass+spectrometry.">StatQuant: a post-quantification analysis toolbox for improving quantitative mass spectrometry.</a> Bioinformatics. 25, 1472-1473 (2009).</li><li>Zauber, H., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+wants+cRacker:+Automated+standardized+data+analysis+of+LC/MS+derived+proteomic+data.">Proteomics wants cRacker: Automated standardized data analysis of LC/MS derived proteomic data.</a> J. Proteome Res. 11, 5548-5555 (2012).</li><li>Raffaele, S., Mongrand, S., Gamas, P., Niebel, A., Ott, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+annotation+of+remorins,+a+plant-specific+protein+family:+Evolutionary+and+functional+perspectives.">Genome-wide annotation of remorins, a plant-specific protein family: Evolutionary and functional perspectives.</a> Plant Physiol. 145, 593-600 (2007).</li><li>Shah, M. B., Sehgal, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondetergent+isolation+of+rafts.">Nondetergent isolation of rafts.</a> Methods Mol. Biol. 398, 21-28 (2007).</li><li>Persaud-Sawin, D. -. A., Lightcap, S., Harry, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rafts+from+mouse+brain+tissue+by+a+detergent-free+method.">Isolation of rafts from mouse brain tissue by a detergent-free method.</a> Journal of Lipid Research. 50, 759-767 (2009).</li><li>Macdonald, L. J., Pike, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+the+preparation+of+detergent-free+lipid+rafts.">A simplified method for the preparation of detergent-free lipid rafts.</a> Journal of Lipid Research. 46, 1061-1067 (2005).</li><li>M&#252;hlhaus, T., Weiss, J., Hemme, D., Sommer, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+shotgun+proteomics+using+a+uniform+15N-labeled+standard+to+monitor+proteome+dynamics+in+time+course+experiments+reveals+new+insights+into+the+heat+stress+response+of+Chlamydomonas+reinhardtii.">Quantitative shotgun proteomics using a uniform 15N-labeled standard to monitor proteome dynamics in time course experiments reveals new insights into the heat stress response of Chlamydomonas reinhardtii.</a> Mol. Cell. Proteomics. 10, M110.004739 (2011).</li><li>Schulze, W., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+in+mass-spectrometry-based+proteomics.">Quantitation in mass-spectrometry-based proteomics.</a> Annu. Rev. Plant Biol. 61, 491-516 (2010).</li><li>Foster, L. J., de Hoog, C., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+quantiative+proteomics+of+lipid+rafts+reveales+high+specificity+for+signaling+factors.">Unbiased quantiative proteomics of lipid rafts reveales high specificity for signaling factors.</a> Proc. Natl. Acad. Sci. USA. 100, 5813-5818 (2003).</li><li>Lilley, K. S., Dupree, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+organelle+proteomics.">Plant organelle proteomics.</a> Curr. Opin. Plant Biol. 10, 594-599 (2007).</li><li>Sadowski, P. G., Groen, A. J., Dupree, P., Lilley, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-cellular+localization+of+membrane+proteins.">Sub-cellular localization of membrane proteins.</a> Proteomics. 8, 3991-4011 (2008).</li></ol>

作者宣称，他们有没有竞争的财务权益。 笔者维托尔德Szymanski的，是分子植物生理学的马克斯普朗克研究所的雇员。笔者瓦尔特劳德舒尔茨是雇员在霍恩海姆德国大学。

在本文提出的协议包含了许多步骤，所有的人都非常重要，以获得植物质膜到洗涤剂耐膜和清洁剂的可溶性组分的纯净和有代表性的分馏。因此，作为指示要按照每个步骤是很重要的。 质膜组分与非离子型去污剂（步骤3.2）的治疗有对的膜微区分馏质量最强的影响。以获得可再现的结果不同制剂之间，并以能够从相同的实验比较不同样品，精确地评估蛋白质在细胞膜的浓度?...

1972年，乔纳森歌手和加思尼科尔森提出的流体镶嵌模型细胞膜的结构模型，替换被普遍接受的20世纪60年代初的蛋白质 - 脂质 - 蛋白质三明治模型。歌手和尼科尔森推测，生物膜可以被认为是一个两维的液体，所有的脂质和蛋白质分子的自由和方便5扩散。自那时起，该膜组合物的质膜和知识的结构模型变得更加复杂。特别是，在质膜内，结构，例如蛋白质复合物和脂质/固醇基础结构紊乱的微区可以观察到。在仿真模型膜6,7，固醇和鞘脂可以横向与其他脂类物质分离，形成区域与改变的物理特性。细胞膜内偏析这主要是由甾醇及高度SA之间的自相关联的属性phopsho和鞘脂8的turated烃链。特别是，刚性甾醇环青睐与硬，直饱和脂质种类以及这些相互作​​用迫使邻近的烃链为更长时间的构象，增加膜的厚度和硬度的相互作用。 之一的甾醇富集的膜微区中通常观察到的特征是其不溶性，在治疗与非离子洗涤剂如曲通X-100或的Brij 35。这些组分被认为是相同的膜微区，并根据其生化制备方法2，被称为耐洗涤剂膜（DRM）。非离子型洗涤剂的DRM提取过程中使用接收到的一些批评作为生化DRM制剂可以不直接对应于活细胞9中的任何特定的膜隔室。特别是，所述洗涤剂蛋白质比率似乎在这类制剂是至关重要的，一s个不同的清洁剂，以及不同量的洗涤剂可以产生不同的耐洗涤剂的膜部分10的组合物。然而，有证据表明，特定蛋白的物种特异性与这些细胞的甾醇富集膜域相关联，并且这些蛋白质以及富含洗涤剂耐膜部分11的生化制剂。被发现在植物的DRM级分，并针对其存在的DRM中是固醇依赖性蛋白质的核心，是特别GPI锚定的蛋白质，如fasciclin状阿拉伯半乳聚糖蛋白（FLAS）和SKU蛋白家族的成员。也有一些信号蛋白，如受体样激酶或磷脂酶被发现11。这些结果与对哺乳动物的膜微区12,13许多蛋白质组研究是一致的。此外，在植物有越来越多的证据表明膜微的应激反应14方面的作用-16。 这里所描述的协议提供了一个可靠的方法对质膜微区分离，特别是使用蛋白质洗涤剂浓度，使我们能够描绘固醇丰富的膜隔4,11,14应激诱导的改变。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>Chemicals were ordered from Sigma-Aldrich unless noted otherwise</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonia (stock 25 % solution)</td>
			<td>WAKO</td>
			<td>010-03166</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TiO2 10 &mu;m</td>
			<td>GL-Science</td>
			<td>5020-75010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Empore Disk C18</td>
			<td>Varian</td>
			<td>12145004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethylene glycol(PEG 3350)</td>
			<td>Sigma</td>
			<td>88276</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Roth</td>
			<td>9219.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (PIC)</td>
			<td>Sigma</td>
			<td>P9599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K15NO3</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-765-PK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT </td>
		</tr>
		<tr>
			<td>Optima L-80 XP Ultracentrifuge</td>
			<td>Beckman</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EASY-nLC II nano-Liquid Chromatograph</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ-Orbitrap mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5417R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed Vac RVC 2-25</td>
			<td>Christ</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaker Unimax 2010</td>
			<td>Heidolph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

metabolic labeling and membrane fractionation for comparative proteomic analysis of arabidopsis thaliana suspension cell cultures

质膜微区是基于所述脂质和甾醇环境中的物理性质的功能，并有在信令进程特定角色。从植物细胞中提取甾醇富集的膜微区用于蛋白质组分析是一项艰巨的任务，主要是由于多个制备步骤和来源自其它细胞区室的污染。质膜构成大约只有5-20％的在植物细胞中的所有膜的，因而高度纯化的质膜组分的分离是具有挑战性的。经常使用的方法涉及水性两相分配中的聚乙二醇和葡聚糖，这将产生质膜囊泡为95％1的纯度。质膜内固醇丰富的膜微区是不溶性后用冷的非离子型洗涤剂在碱性pH下处理。此耐洗涤剂膜组分可以从散装质膜中作为分离通过超速离心ucrose梯度2。其后，蛋白质可以提取从由甲醇/氯仿沉淀的蔗糖梯度密度低频段。提取的蛋白质将被胰蛋白酶消化，脱盐，最后用LC-MS/MS分析。我们的提取协议甾醇富集微区被优化用于从拟南芥细胞培养清洁洗涤剂性膜级分的制备方法。 我们利用拟南芥悬浮细胞培养的全代谢标记为K 15 NO 3作为唯一的氮源为后续生物处理的利息3定量比较蛋白质组学研究。通过将标记和未标记的细胞培养物进行联合蛋白质提取的相等比例对最终定量结果的制备步骤的影响被保持在最低限度。还材料的提取过程中的损失将影响控制和以同样的方式处理的样品，一个ð因此光线和升沉肽的比率将保持不变。在所提出的方法无论是标记的或未标记的细胞培养物经历了一个生物处理，而另一作为控制4。

使用代谢标记的拟南芥细胞培养物中所提出的协议，可以从植物组织中（步骤2， 图2和图4）分离血浆膜，血浆膜中富集耐洗涤剂的膜部分（步骤3， 图3和图5）。其后，该协议允许提取从这些耐洗涤剂的膜部分（步骤4）和消化的蛋白质进行比较蛋白质组学分析（步骤5）的蛋白质。最后，磷酸（第8步）的可选的富集是研究细胞信号传导过程特别有趣。最后一步则是?...

Watch this Scientific Journal Video about Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures at JoVE.com

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

在这里，我们描述了植物细胞质膜的基础上，未标记的混合物分馏成耐洗涤剂和清洁剂的可溶性和膜在体内完全15 N标记的拟南芥细胞培养一个强大的方法。该过程被施加于比较蛋白质组学研究，了解信令进程。

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 ...

metabolic-labeling-membrane-fractionation-for-comparative-proteomic

Research

JoVE Journal

Biology

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

14.0K 次观看.  Max Plank Institute of Molecular Plant Physiology. 在这里，我们描述了植物细胞质膜的基础上，未标记的混合物分馏成耐洗涤剂和清洁剂的可溶性和膜在体内完全15 N标记的拟南芥细胞培养一个强大的方法。该过程被施加于比较蛋白质组学研究，了解信令进程。 

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

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

科研

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2:β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

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

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

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

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

不同光照条件下的拟南芥生长行为分析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦:研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...