这项工作是由美国能源生物科学研究所授予OO0G01。

OLIMP方法是体现在双子叶植物，xyloglucan的细胞壁中存在的主要半纤维素，使用的是内切葡聚糖酶的glycosylhydrolase。该方法是证明整个拟南芥植物组织源。酶和细胞外基质材料上使用相同的步骤取决于所需的分析，可替代。 1。细胞墙隔离<ol><li>收获五个为期5天的老整个拟南芥幼苗或您所需材料的同等数额，并转移到1.5ml的反应管。确保该材料置于试管底部。 </li><li>反应管在液氮中的样品和管理单元冻结顶部添加两个3毫米金属球。 </li><li>研磨冷冻样品使用一个球磨（下午2时30分至2分钟，25HZ）。 </li><li>加入1ml 70％的含水乙醇样品。删除使用磁铁的金属球。涡彻底。 </li><li>离心14000 RPM为10分钟，以颗粒不溶于醇残留物含有细胞壁物质的样品。 </li><li>小心取出上清液吸。确保不打扰颗粒。 </li><li>氯仿/甲醇（1:1 V / V）溶液，加入1毫升。涡彻底悬浮颗粒。 </li><li>在10分钟14000转离心样品，并小心取出上清液吸。确保不打扰颗粒。 </li><li>使用浓缩干燥样品。 </li></ol> 2。溶低聚糖<ol><li>对于增溶在其低聚糖的形式，尤其是多糖悬浮在一个适当的缓冲液25μl的干燥颗粒。内切葡聚糖酶50MM酸铵，PH4.5，是使用。新增的内切葡聚糖酶0.2U。涡的悬挂和自旋液体下来。 </li><li>孵育16h后的样品在37 ° C的孵化器，在300RPM晃动。 </li><li>取出的精华（水）使用一个集中的溶剂。 </li></ol> 3。 MALDI - TOF分析发布低聚糖<ol><li> BioRex MSZ 501阳离子交换树脂用于去除盐类和酶消化的样品。条件放置在一个空列等分和大量的水冲洗珠小珠。 </li><li> 5-10 BioRex珠消化和干燥样品。然后添加加入10μl水，可用于数额较小的水材料5μL。溶液中浸泡简要纺纱管珠。在室温下孵育至少10分钟。 </li><li>现货2μL矩阵（2,5 - 二羟基苯甲酸（DHB），在水中10mg/ml的）到MALDI靶盘。下导致同质矩阵化学晶体的真空溶剂蒸发矩阵。 </li><li>脱盐，目标板上的矩阵晶体样品溶液点2μL。 如果你想现场的大量样品，只保留了3分钟的最大的斑点。这一时限保证，不会干，还首次发现样品。等待一个额外的2分钟，以确保重新溶解在去年发现样品基质和样品寡糖分子充分混合。再用干的真空状态下的目标板。 样本/矩阵结构应在不到1分钟，使同质结晶结晶。 </li><li>放入一个的MALDI - TOF质谱仪的目标板。机应积极reflectron模式，加快20000V的电压和延迟为350 nm;选定的质量范围应达500-3000（可能会改变依赖于预期的低聚物）。开始发射激光，并收集每个样品100-200光谱，这是编译生成一个具有代表性的平均光谱（图2A）。代表特定寡糖的离子可以由他们过充质量（M / Z）的比例确定。所有感兴趣的离子的离子强度（峰高）应增加高达100％，在每个样品中的低聚糖的相对丰度（图2B）。 </li></ol> 原位 OLIMP分析4。 OLIMP也可用于直接省略任何墙壁准备步骤的组织。作为一个例子黄化拟南芥胚轴用于植物组织源。同样，一个内切葡聚糖酶是用来确定的半纤维素xyloglucan的结构。酶和组织材料可以代替所需的分析，根据使用相同的步骤。 <ol><li>收获一个黄化苗和它放到一个空的MALDI靶盘，让苗干。 </li><li>添加在所需的位置上干苗木0.5μL内切葡聚糖酶（0.4U/μl，50MM酸铵溶解，PH4.5）。确保酶下降触及部分目标板。 </li><li>放置在密闭容器中，饱和湿度的MALDI靶盘，以避免酶的下降变干。在会议厅内孵育1板6H在37 ° C。 </li><li>干MALDI靶盘，在真空条件下的植物组织和酶下降。 </li><li>加入0.5μL矩阵（道亨银行;为10mg / L）的每个干酶点之上。静置2分钟。 </li><li>在真空条件下干燥MALDI靶盘。 </li><li>分析样品的MALDI - TOF。地方组织和酶/矩阵（S）现货成的MALDI - TOF质谱仪的目标板。机应积极reflectron模式，加快20000V的电压和延迟为350 nm;选定的质量范围应达500-3000（可能会改变依赖于预期的低聚物）。 </li><li>启动激光发射到基质/样品的晶体组织旁边。确保你不打组织本身，在这种情况下，分子的飞行时间将关闭。收集20-50％左右谱斑，这是编译生成一个代表性的平均光谱（图3）。代表特定寡糖的离子可以由他们过充质量（M / Z）的比例确定。离子强度（离子身高），可报告和所有感兴趣的离子应增加高达100％，在每个样品中的低聚糖的相对丰度。 </li></ol> 5。代表性的成果在拟南芥中的半纤维素xyloglucan OLIMP分析的一个例子是如图2所示。由于离子质量差异和特点已知酶（内切葡聚糖酶）的特异性的离子可以被分配到具体的寡糖结构（图2A）。显然无法区分，结构异构体。各种低聚糖的相对丰度测定的基本假设（图2B）是他们的质谱响应因子这些低聚糖是非常类似的。如下所示，OLIMP量化高度重复性。然而，请注意，该方法的鲁棒性是高度依赖的信号信噪比的各种低聚糖离子。例如低聚糖的盐或较低的数额污染可以减少这个比例。 该组织本身（原位分析）的OLIMP分析，可以非常小和定义地区的研究涉及很少的样品制备。这里介绍的例子（图3）没有质的（相同的离子），但数量差异（离子强度的变化）之间的拍摄和拟南芥幼苗根组织的观察。 OLIMP方法的排列，从而导致组织的“成像”。 <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="图1" /> 图1。OLIMP使用作为一种植物源的整个拟南芥幼苗的过程流程图。首先，该组织是浸渍和细胞壁物质准备（图片修改野等10）。然后释放一个特定的壁多糖寡糖使用特定的水解酶。最后，可溶性低聚糖的相对丰度使用MALDI - TOF质谱测定。 <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="图2" /> 图2。OLIMP确定在拟南芥中的黄化苗xyloglucan低聚糖的相对丰度。一）代表xyloglucan寡糖的质谱，每个离子代表一个特定的寡糖结构，结构异构体无法加以区分。 B）为低聚糖的相对丰度测定相应条形图。 <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="图3" /> 图3。 原位 OLIMP使用为例，分析拟南芥黄化苗。每种酶/消化液滴（彩色圆圈）可以独立进行分析，和一个可以得到相应的质谱和分析。 <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="图4" /> 图4。OLIMP，消化，用木聚糖酶（Megazyme）芒草叶材料获得的半纤维素木聚糖谱。

<ol>
	<li>Lerouxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+structural+phenotyping+of+plant+cell+wall+mutants+by+enzymatic+oligosaccharide+fingerprinting.">Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting.</a> Plant Physiology. 130 (4), 1754-1754 (2002).</li><li>Obel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microanalysis+of+plant+cell+wall+polysaccharides.">Microanalysis of plant cell wall polysaccharides.</a> Molecular Plant. 2 (5), 922-922 (2009).</li><li>Mouille, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+analysis+of+primary+cell+wall+composition+in+Arabidopsis.">Quantitative trait loci analysis of primary cell wall composition in Arabidopsis.</a> Plant Physiology. 141 (3), 1035-1035 (2006).</li><li>Bauer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+suite+of+polysaccharide-degrading+enzymes+for+analyzing+plant+cell+walls.">Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls.</a> PNAS. 103 (30), 11417-11417 (2006).</li><li>Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+xyloglucan-specific+endo-beta-1,4-glucanase+from+Aspergillus+aculeatus:+expression+cloning+in+yeast,+purification+and+characterization+of+the+recombinant+enzyme.">A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme.</a> Glycobiology. 9 (1), 93-93 (1999).</li><li>Aboughe-Angone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+carbohydrates+from+fruit+pulp+of+Argania+spinosa:+structural+analysis+of+pectin+and+xyloglucan+polysaccharides.">Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides.</a> Carbohydr Res. 343 (1), 67-67 (2008).</li><li>Brown, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+xylan+synthesis+mutants+reveals+new+insight+into+the+mechanisms+of+xylan+synthesis.">Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis.</a> Plant Journal. 52 (6), 1154-1154 (2007).</li><li>Lee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARVUS+gene+is+expressed+in+cells+undergoing+secondary+wall+thickening+and+is+essential+for+glucuronoxylan+biosynthesis.">The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis.</a> Plant Cell Physiol. 48 (12), 1659-1659 (2007).</li><li>Obel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectin+may+hinder+the+unfolding+of+xyloglucan+chains+during+cell+deformation:+implications+of+the+mechanical+performance+of+Arabidopsis+hypocotyls+with+pectin+alterations.">Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations.</a> Molecular Plant . , (2009).</li><li>Cavalier, D. M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+xyloglucan+xylosyltransferases+catalyze+the+addition+of+multiple+xylosyl+residues+to+cellohexaose.">Two xyloglucan xylosyltransferases catalyze the addition of multiple xylosyl residues to cellohexaose.</a> Journal of Biological Chemistry. 281 (45), 34197-34197 (2006).</li><li>Leonard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+Arabidopsis+gene+encoding+a+GH95+alpha1,2-fucosidase+active+on+xyloglucan+oligo-+and+polysaccharides.">Identification of an Arabidopsis gene encoding a GH95 alpha1,2-fucosidase active on xyloglucan oligo- and polysaccharides.</a> Phytochemistry. 69 (10), 1983-1983 (2008).</li><li>Cavalier, D. M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+xyloglucan+xylosyltransferases+catalyze+the+addition+of+multiple+xylosyl+residues+to+cellohexaose.">Two xyloglucan xylosyltransferases catalyze the addition of multiple xylosyl residues to cellohexaose.</a> Journal of Biological Chemistry. 281 (45), 34197-34197 (2006).</li><li>Lee, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+irregular+xylem9+mutant+is+deficient+in+xylan+xylosyltransferase+activity.">The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity.</a> Plant and Cell Physiology. 48 (11), 1624-1624 (2007).</li><li>Iglesias, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+glycosidases+active+against+xyloglucan+oligosaccharides+of+Arabidopsis+thaliana.">Apoplastic glycosidases active against xyloglucan oligosaccharides of Arabidopsis thaliana.</a> Plant and Cell Physiology. 47 (1), 55-55 (2006).</li><li>Fujino, T., Sone, Y., Mitsuishi, Y., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cross-links+between+cellulose+microfibrils,+and+their+occurrence+during+elongation+growth+in+pea+epicotyl.">Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl.</a> Plant Cell Physiol. 41 (4), 486-486 (2000).</li><li>Vanzin, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mur2+mutant+of+Arabidopsis+thaliana+lacks+fucosylated+xyloglucan+because+of+a+lesion+in+fucosyltransferase+AtFUT1.">The mur2 mutant of Arabidopsis thaliana lacks fucosylated xyloglucan because of a lesion in fucosyltransferase AtFUT1.</a> PNAS. 99 (5), 3340-3340 (2002).</li><li>Cavalier, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+two+Arabidopsis+thaliana+xylosyltransferase+genes+results+in+plants+deficient+in+xyloglucan,+a+major+primary+cell+wall+component.">Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component.</a> Plant Cell. 20 (6), 1519-1519 (2008).</li><li>Hilz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+liquid+chromatography,+capillary+electrophoresis,+and+mass+spectrometry+methods+to+determine+xyloglucan+structures+in+black+currants.">A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants.</a> Journal of Chromatography A. 1133 (1-2), 275-275 (2006).</li><li>Pauly, M. <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 (1), 67-67 (2001).</li></ol>

OLIMP这里介绍的方法，使一个非常敏感和快速的分析，在细胞外基质聚合物。 OLIMP结合酶的释放与随后的MALDI - TOF分析的低聚物。一个的MALDI - TOF谱的生成需要不到一分钟，因此，OLIMP是适合的应用范围广泛，包括高通量的研究，如突变屏幕。 OLIMP不仅限于植物多糖，但有可能被应用于广泛的聚合物，只有通过具体的水解酶限制。然而，OLIMP限制，不能得到的聚合物的绝对丰度。 正如前面提到OLIMP可用于研究多种，在细胞外基质的物种多样性多糖的结构。作为一个例子，如图4代表主要草种的半纤维素，聚糖OLIMP频谱。在这里，来自温带草芒草的细胞壁物质消化使用木聚糖酶。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

oligo mass profiling (olimp) of extracellular polysaccharides

介导的细胞对环境的直接接触是在许多生物细胞外基质。细胞外基质的一个共同点是，它们包含在复杂形式的糖蛋白糖基，蛋白多糖，和/或多糖。例子包括人类和动物细胞组成的纤维状蛋白和蛋白多糖或多糖的植物和真菌的细胞壁主要的细胞外基质，蛋白多糖/糖脂细菌的细胞壁。所有这些glycostructures都在细胞与细胞和细胞环境的通信和信号中扮演重要的角色。 一个非凡的细胞外基质的复杂的例子是在目前较高的植物细胞墙。他们的墙几乎完全是由糖，干重的75％，这个星球上最丰富的生物聚合物组成。因此，研究如何最好的利用这些材料作为一个碳中性的可再生资源，以取代化石燃料衍生石化进行。燃料转换的主要挑战仍然是酶解或化学降解，由于在目前这种独特的生物复合材料的独特glycostructures墙顽抗。 在这里，我们目前的植物细胞壁glycostructures的快速，灵敏的分析方法。这种方法OLIGO质谱分析（OLIMP）是基于从墙体材料促进特定glycosylhydrolases和后续分析的可溶性寡糖混合物使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/MS的低聚糖的酶释放1）（图1）。 OLIMP要求墙壁只有5000细胞为一个完整的分析，可以对组织本身2的执行，并适合高通量分析 3 。虽然由OLIMP溶解低聚糖的绝对数额不能确定的各种寡糖离子的相对丰度，可以划定从大众给予在墙上的原生多糖的替代模式的见解光谱。 OLIMP可用于分析的墙聚合物的种类繁多，仅由 4个特定酶的可用性的限制。例如，在植物细胞壁的酶聚合物的分析可用来分析的半纤维素xyloglucan 使用xyloglucanase 5，11，12，13，木聚糖使用远藤-β-（1-4），木聚糖酶6,7 ，或使用相结合的一个多聚半乳糖醛酸酶和methylesterase 8果胶多糖。此外，使用OLIMP glycosylhydrolase，甚至糖基转移酶活动相同的原则，可以监测和确定9。

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

描述一个快速的方法是争取到在细胞外基质多糖结构的见解。该方法采用glycosylhydrolases特异性，灵敏度和质量允许微量的材料加以分析法的优势。这种技术直接用于组织本身的适应性。

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 ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K 次观看.  University of California, Berkeley. 描述一个快速的方法是争取到在细胞外基质多糖结构的见解。该方法采用glycosylhydrolases特异性，灵敏度和质量允许微量的材料加以分析法的优势。这种技术直接用于组织本身的适应性。

视频: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

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, ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...