This work was supported by the grant P01HL107151 from the NHLBI/NIH. The authors also gratefully acknowledge the support and access to instrumentation provided through grant P41GM103490 from the NIGMS/NIH.

注：实验室安全问题 符合标准的实验室的最佳实践，请遵守下列规定。存放在合适的位置所有的有机溶剂。保留所有废旧物资的化学废物容器的化学成分明确的标签。如在这些协议中使用几种试剂是潜在的致癌物质或产生挥发性可燃气体，处理所有的试剂在通风橱中与通风。用有机溶剂工作时穿戴个人防护装备，如手套，实验室外套，保护眼睛。 <img alt="图1" src="/files/ftp_upload/51840/51840fig1highres.jpg" width="450px" /> 图1.程序在胶O型糖组学。感兴趣的（A） 的蛋白通过SDS-PAGE分离，通过合适的染色程序（考马斯亮蓝或银）来检测，并切下。切除的凝胶块被切成小方块，脱色，并用乙酸乙酯洗涤，以减少与随后的MS分析干涉污染物。凝胶切片的一部分可通过LC-MS / MS保留用于在凝胶胰蛋白酶消化和随后的蛋白质鉴定。 （B）的 O-连接的聚糖是由凝胶还原性β-消除释放出解决的糖蛋白。基本步骤包括脱盐和硼酸去除共沸物用甲醇。 （C）的 O-连接的通过还原性β-消除释放聚糖全甲基化，并随后分成水相和有机相。 Sulfoglycans定量回收上层（水相），而中性和唾液酸化聚糖的分区到下部（DCM）的层。 <img alt="图2" src="/files/ftp_upload/51840/51840fig2highres.jpg" width="700px" /> 图2详细的流程图，用于在凝胶糖组学。每个experimen在图1所示河谷步骤被单独示出。 （A）凝胶脱色和乙酸乙酯提取，（B）直接在凝胶还原β-取消对O连接聚糖的释放，以及（C）全甲基和阶段划分。 <a href="/files/ftp_upload/51840/51840fig2highres.jpg" target="_blank">请点击这里查看图的放大版本。</a> 1.凝胶切除，并去除凝胶衍生污染物<ol><li>在开始程序之前，在准备材料清单所列化学品和试剂。 </li><li>使用标准的Tris-甘氨酸缓冲系统解决在SDS-PAGE凝胶上的蛋白质。染色解决了既考马斯亮蓝或银染蛋白质。在一般情况下，从银染色的凝胶O-连接的聚糖的恢复是从考马斯取得的恢复约80-90％染色的凝胶。 </li><li>电泳后，将凝胶在玻璃板上和消费吨用干净的手术刀或刀片，他感兴趣的区域。 </li><li>以增加O型聚糖产量，感兴趣的凝胶带转移到另一玻璃板上，并切断了切除的凝胶片成约2mm的立方体。避免使凝胶碎片小于1mm的立方体因为聚糖回收率降低。传送小凝胶片成螺旋盖的玻璃试管（13×100mm）上用不锈钢microspatula。 </li><li>加入1毫升25mM的碳酸氢铵（AMBIC，NH 4 HCO 3），向样品管中，盖上玻璃管有聚四氟乙烯内衬的螺纹顶帽，并通过轻弹管让静置10分钟，然后轻轻地混合。不使用剧烈搅拌以避免撕裂的凝胶片。 </li><li>小心地使用一个巴斯德玻璃吸管或配有超长的塑料尖吸管玻璃管删除AMBIC。避免粉碎和转移的微小凝胶块，同时消除了AMBIC解决方案。 </li><li>加入1毫升乙腈（ACN）的向玻璃管，帽，然后轻轻地将通过让支架10分钟前弹管混匀。确保了凝胶片被完全覆盖溶剂。如果有必要，推凝胶块脱落管壁进入溶剂枪头。 </li><li>移液器关闭ACN从玻璃管并重复步骤1.5-1.7，直到亮蓝色被消除。如有必要，重复至少五个连续的AMBIC / ACN洗涤。对于银染凝胶，使用脱色试剂盒。进行到下一个步骤，当凝胶片变成均匀的白色，而在ACN。 </li><li>移液器关闭任何液体从玻璃管，重新盖上试管，加入1 ml AMBIC并静置5分钟。 </li><li>除去AMBIC并加入2毫升乙酸乙酯（EtOAc）在玻璃管。重述管有聚四氟乙烯衬里的盖并置于4℃过夜终过端搅动或备选地执行三个的EtOAc洗涤液在室温下每次30分钟。越长的EtOAc洗涤，更有效地去除污染物。 </li><li>清除最终的EtOAc洗涤，并执行各洗涤三次用2ml H 2 O完全除去乙酸乙酯，将确保一个强大的还原β消去反应。 </li><li>移液器关H 2 O和1毫升乙腈洗涤脱水后的凝胶。 注：干燥后，将脱水的凝胶是准备用于凝胶β消去。脱水的凝胶碎片可以储存在-20℃直到使用。 </li><li>阻止洗涤过 ​​程中，在任何步骤，并存储凝胶片在4℃下过夜，无论哪个缓冲液或溶液加到凝胶的（AMBIC，乙腈，H 2 O）。在随后两天随时重新启动的样品制备。 </li><li>用于通过标准的蛋白质组方法的蛋白质的ID使用的脱色和干燥凝胶片的一部分上。 </li></ol> 2.凝胶O-聚糖通过还原β-消除发行<ol><li>制备的，用50％NaOH或固体NaOH，并储存于4℃直至使用1M氢氧化钠（NaOH）中的储备液。 <ol&gt; <li>取5.2毫升的50％的氢氧化钠（或4克固体NaOH中），并调整体积至100ml，使1M的NaOH溶液。稀释的1 M氢氧化钠原液至100mM的NaOH，它可以被存储在4℃下长达6个月无效率的在还原性β消除反应的损失。 </li></ol></li><li>作的2M硼氢化钠（加入NaBH 4）在100mM的NaOH。溶解38毫克硼氢化钠的0.5ml的100mM的NaOH中和。通常，可以使用0.5〜1.0毫升各样品的硼氢化溶液。变化对在第1步制备的干燥的凝胶片的量的反应溶液的体积期望使用0.5的硼氢化钠溶液中1-2切取的凝胶片（步骤1.3）。 </li><li>加入500微升100mM NaOH中的脱水凝胶片，让以平衡的凝胶到其增强聚糖恢复的基本条件放置在冰上3-5分钟。 </li><li>加入500微升的2M的NaBH 4在100mM的NaOH，产生最终浓度为1M的硼氢化钠<sub&gt; 4在100mM NaOH中。轻轻混合试管，孵育在45℃下18小时。 <ol><li>在孵化的第一个小时，轻轻混合试管，每15分钟。避免强烈的情绪激动，因为它可能会片段的凝胶块。执行中的一个平衡良好的培养箱/烘箱中或在加热块中的反应。确保该凝胶片覆盖有反应溶液中。 </li></ol></li><li>停止反应，从孵化器中或加热块取出样品管中，并放置在冰上。 注：使β消去反应进行18小时后，脱盐过程必须在同一天内进行。 </li></ol> 3.脱盐的阳离子交换层析<ol><li>同时保持样品管在冰上，以防止热的形成过剩，慢慢加入10％乙酸（AcOH中）滴加至中和碱。轻轻涡旋添加乙酸之间的样品管中。小心地慢慢加酸和降明智的additi于酸时会产生泡沫的“火山”。离心管，以消除泡沫，防止溢出，如果需要的话。 <ol><li>为了除去在反应混合物中的大量的钠，通过一个小的阳离子交换柱（的Dowex或AG-50W-X8树脂，H +型 ）通过将反应混合物。 </li><li>用1M NaOH和冲洗阳离子交换树脂1M的HCl中，以便用它来删除与MS的分析，干扰，即使树脂是分析纯的污染物之前。 </li><li>浸泡在树脂中的1M NaOH和通过倾析除去溶液。加入去离子水，除去水分，再加入1M的盐酸。 </li></ol></li><li>重复这些步骤，直到树脂上面的解决方案是无色的。在4℃下于5％AcOH的洗涤清洗的树脂用离子交换水和存储。 </li><li>使一个小的玻璃柱，划痕和使用陶瓷刀具，使得移液管尖的锥大约为1厘米长打破玻璃巴斯德吸管尖。逗除了玻璃棉塞和推向前端形成用于树脂床的支持体。固定用夹具或衣夹和地点在玻璃试管中的吸移管列。 </li><li>用1毫升甲醇（MeOH中）和3毫升5％AcOH的洗空吸管。漩涡的H +的Dowex AG50或阳离子交换树脂浆液储存在5％AcOH中进行传送的浆料，足以产生在巴斯德移液管塔1毫升床体积。 </li><li>冲洗用五卷5％乙酸的列。检查流动通过对树脂粒子的外观和不使用该列是否被检测到的树脂。 </li><li>将列在一个新的螺旋盖的玻璃试管（16×125毫米）和负载样本。收集通过流量和洗脱聚糖与至少3倍体积的5％AcOH的进入同一管中。 </li><li>覆盖用Parafilm管和使微小的孔，用针。将样品管在-80℃下或在干冰上。一旦冻结，干燥样品冷冻干燥。存储该干燥材料在-20℃C，直到使用。 </li></ol> 4.硼酸盐去除<ol><li>准备10％乙酸的甲醇。取10毫升冰醋酸中，并调整体积至100毫升，用甲醇。存储此试剂在玻璃瓶用特氟隆衬盖长达6个月。 </li><li>在MeOH中添加300μl的10％AcOH中，以干燥的，冷冻干燥的样品管和涡流。除去所得到的硼酸三甲酯通过蒸发下在N 2流中。 注：在酸性条件下混合硼酸盐，用甲醇产生的硼酸三甲酯是一种挥发性化合物。 </li><li>在N 2流在37℃下干燥。干燥后，如在步骤4.2中所述的甲醇加入AcOH中的另一个等分试样。干燥的液体在氮气下5分钟（N 2），在37℃的流。 <ol><li>重复再悬浮和干燥至少3倍。存储该干燥材料在-20℃直到使用。 </li></ol></li></ol> 5. C18清理<ol><li>一个平衡C18墨盒列（大小为1毫升，100毫克树脂），使用三册ACN和五卷5％的乙酸。通过应用温和的正气压的加速平衡的解决方案通过。 </li><li>加入500微升5％的AcOH的脱盐和硼酸盐的无样品管中，并通过涡旋溶解。 </li><li>再悬浮的样品加载至平衡的C18柱，并通过收集流入的玻璃螺旋盖管（13×100毫米）。洗脱O型聚糖用3毫升5％AcOH的进入同一管中。 </li><li>封口膜管，使小孔用小针或使用松散的闭合的PTFE衬里的盖来覆盖管，冷冻的样品在-80℃下或在干冰上。通过冷冻干燥除去溶剂。储存在-20℃直到使用干燥的样品。 </li></ol> 6.基本准备全甲基注：为了实现强大的全甲基，准备氢氧化钠浆新鲜。用于全甲基反应，所有的玻璃器皿，应广泛地清洗。 <ol><li>为了制备碱试剂对全甲基，加入400μl的50％的NaOH于洁净的玻璃螺旋盖管（13×100毫米）。加入800微升的无水甲醇和旋涡。 </li><li>加入4毫升无水二甲亚砜（DMSO）和涡流产生白色沉淀。 </li><li>离心机在600×g离心1分钟以沉淀的沉淀物。移液器关闭上清并丢弃。添加额外的4毫升无水DMSO中的沉淀。旋涡。 </li><li>重复步骤6.2和6.3的最小的3次以上，或直至没有形成白色沉淀。 </li><li>溶解在3毫升无水DMSO中的沉淀碱和通过吸取用干净的巴斯德吸管向上和向下轻轻混匀。 </li><li>立即对下面的全甲基反应用的碱，因为它不能被存储。 </li></ol> 7.全甲基发布聚糖<ol><li>加入200μl无水DMSO为C18-纯化样品和涡流或超声重悬。 </li><li>加入300微升的resusp的端基泥浆的样品，并立即加入100微升碘甲烷（MEI）。密封管有聚四氟乙烯衬里的盖和剧烈通过涡旋混合5分钟。 </li><li>要停止全甲基反应，在冰和涡加2 ml 5％的乙酸。吸液上下5次通过蒸发来降低尾的剩余量。 注：乙酸中和该溶液，并在逐步分区的过程中除去的MeI的增加​​硫酸化聚糖的提取效率到水相中。 </li><li>加入2毫升二氯甲烷（DCM）并涡旋。离心机在600×g离心1分钟，在室温下以分离水相和有机相。 </li><li>转移的顶层（含有大部分全甲基化的硫酸化多糖的水相）到一个新的玻璃试管（13×100mm）上。 </li><li>加入2毫升的H 2 O连接至有机相并涡旋。离心机在600×g离心1分钟，以分离水相和有机相，并结合该第二水相用冷杉圣水相（步骤7.5）。 </li><li>重复步骤7.6和7.5 3次以上，但没有必要保存从后续分区所得的水相。 </li><li>除去最后的上层（水相）尽可能多地从所述底部层（有机相）和下层（有机相）转移到用清洁巴斯德吸管单独的新的玻璃试管中。 </li><li>干燥在N 2流中的有机相在42℃。 </li><li>覆盖管用Parafilm并储存在-20℃直至使用。 </li></ol>从水相8的C18清理的全甲基硫酸O型聚糖<ol><li>平衡与三卷ACN和五卷5％乙酸的C18墨盒列。 </li><li>加载收集并从步骤7.5和7.6上柱结合的水相。 </li><li>用10毫升H 2 O代表脱盐洗柱。 </li><li>洗脱全甲基硫酸化O-聚糖成用2ml新的玻璃管的50％ACN。使用一个额外的洗脱85％ACN，以提高较大的低聚糖全甲基硫酸化O-聚糖恢复。 </li><li>在氮气流下，在42℃下干燥洗脱液。 </li><li>于-20℃下向上的干燥材料存储到之前MS分析6个月。 </li></ol>的全甲基O型聚糖9质谱<ol><li>全甲基-O-聚糖通过直接注入到分析用纳电喷雾源在0.40-0.60μl/ min的在210℃的毛细管温度注射器流速适当质谱仪。对于碎片通过CID在MS / MS和的离子阱仪器级质谱，应用30-40％的碰撞能量。 </li><li>对于负离子模式，重构全甲基硫酸化O-聚糖在50μl甲醇/ 2-丙醇/ 1-丙醇/ 13毫摩尔乙酸铵水溶液（16：3：3：2，体积比）。对于正离子模式，重构在50μl的1 mM的钠水电全甲基中性/唾液酸化的O型聚糖西德在甲醇/水（1：1）。 </li><li>使用的总离子映射的Xcalibur软件2.0版（TIM）和中性丢失扫描（NL扫描）功能的MSN分析表征单个O型聚糖结构13,19。 </li></ol>

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The authors declare no competing financial interest.

Combining in-gel reductive &#946;-elimination with aqueous-organic extraction enhances the sensitivity and depth of structural data that can be acquired for characterizing sulfated and non-sulfated O-linked glycans harvested from small amounts of mucin-type glycoproteins resolved from other proteins by SDS-PAGE. The essential advances of the technical approaches presented in this study are: (a) facile removal of gel derived contaminants by simple washing steps; (b) quantitative recovery of permethylated sulfoglycans in t...

糖基化是一个重要的蛋白质翻译后修饰，有助于有机体生理学，组织病理学和细胞识别1-3。尽管在分析glycoscience重大进展，在一个特定的蛋白聚糖特征的完整的多样性仍然是一个极具挑战性的任务，特别是对蛋白质的主要生物来源分离出来。然而，糖蛋白的聚糖的微观不均经常会影响与其它蛋白质功能性相互作用。因此，聚糖多样性的表征对于理解细胞和组织中的糖基化4,5-生理意义是必不可少的。为了了解糖蛋白的糖基化对组织的生理与病理生理学，健壮，灵敏，全面的glycomic分析技术的贡献已经变得越来越重要。在蛋白质组分析，蛋白质的鉴定是通过LC-MS通常实现/胰蛋白酶肽6的MS分析。蛋白消化可使用以下在凝胶内消化以蛋白酶如胰蛋白酶7-9纯化的蛋白或蛋白通过SDS-PAGE解析来进行。预富集通过SDS-PAGE的蛋白质混合物的增强的蛋白质的ID的深度和准确性。类似的战略糖蛋白的糖基化glycomic分析的发展，关键在glycoscience的前列。 聚糖的两类主要是通过是N-连接或O-键附着到蛋白质骨架。 N-连接的聚糖附着到天冬酰胺（天冬酰胺）中所定义的的Asn-X-丝氨酸/苏氨酸/半胱氨酸一序列子的部分残基（X为任意氨基酸除脯氨酸外的），并且可以通过酶消化与肽N为释放-glycanase（PNGaseF或A），无论是在溶液中，在-凝胶法，或对印迹10-12。 O-连接的糖链主要附着到丝氨酸（丝氨酸）或苏氨酸（Thr）残基。但是，只有一个酶已经IDENtified，其能够从糖蛋白释放的O-联聚糖的，它有一个极其有限聚糖特异性，只释放最简单的O-连接的聚糖。化学释放策略仍首选从糖蛋白全面释放的O-连接的糖链的方法。还原性或非还原性β-消除，或肼是良好表征的化学释放技术和目前最常用的方法为从糖蛋白13,14释放O-连接的聚糖。虽然还原性β-消除已被用于从糖蛋白，通过SDS-PAGE分离释放O-连接的聚糖，先前的方法需要HPLC分离用于随后的分析15-17。 多维质谱（MS n）的分析，目前提供的数据结构为特征的糖蛋白中分离出大多数生物源预计的数额发布聚糖最丰富的来源。 MS的深度基于结构特征是由permethylating释放聚糖之前，他们的分析大大便利。全甲基增强离子化，容易在广泛的聚糖结构18,19的均衡摩尔信号的响应。此外，全甲基明确标记终端和取代的单糖部分具有鲜明的群众，从而提高结构解析20-23。例如，酸性聚糖一般难以检测作为非全甲基化物种被MS。虽然酸性聚糖可以在负离子模式通过质谱进行检测，就不可能检测酸性和中性聚糖在同一离子模式。聚糖全甲基的一个主要优点是，所有的聚糖的单糖取代基的游离羟基（OH）的将甲基（OCH 3或OMe），因此唾液酸化聚糖的电荷被中和被封端的，这使得它们作为可检测如全甲基化中性（亚洲LO）聚糖。然而，硫酸盐部分的上sulfoglycans的羟基是抗全甲基，导致保留阴离子电荷，从而抑制离子化和降低灵敏度。这种抑制目前防止的非常复杂的糖蛋白，如粘蛋白，它携带高丰硫酸化多糖24-26综合glycomic分析。 上纯化的硫酸化聚糖的最近的报道用过充电，反相层析，纯化和之前的MALDI分析单独的全甲基化的聚糖。此方法依赖于使用用于洗脱不同的流动相，这是我们发现比有机相分配不那么严格的硫酸化和非硫酸化聚糖的完全分离。因此，适合于sulfoglycans的检测和富集新技术是这里提出。这些技术允许的硫酸化聚糖的以下水的定量回收在水相中：DCM（二氯甲烷）提取，这是经常在聚糖全甲基反应27结束时执行。重要的是，从全 ​​甲基非硫酸化多糖的混合物，这种强大的分离全甲基sulfoglycans的伴随丰富的带电物质，同时也简化了MS 2碎裂模式。一个全面的协议，用于改善胶O连接聚糖分析也提出了。改进协议增强聚糖恢复，增加了获得的通过级质谱全甲基化聚糖的分析的结构信息，并提高了施加到从生物来源分离的必不可少的糖蛋白sulfoglycomic分析的灵敏度。 此协议旨在用于通过SDS-PAGE分离感兴趣的特定糖蛋白的全提取物的糖蛋白O-连接的糖链的分析或与由三个实验程序; A）凝胶清理，B）的凝胶还原β-淘汰，C）聚糖permethy分页。我们的目标是获得全面的O连接glycomic数据糖蛋白生物的兴趣（ 图1）的主要来源收获。糖蛋白通过SDS-PAGE分离是通过染色和感兴趣的条带切下，将所得的凝胶带被切成小块的可视化。将凝胶片被脱色，并进行乙酸乙酯洗涤以除去凝胶污染物（ 图2A）。聚糖释放是通过在凝胶还原性β-消除（ 图2B）来实现与所释放的糖链进行全甲基化。水-有机萃取下面的全甲基定量分割阴离子硫酸化聚糖远离非硫酸化的中性聚糖（ 图2C）。在凝胶还原β-消除耦合到水 - 有机萃取使的O-连接的聚糖，以及少量的糖蛋白，通过SDS-PAGE分离的释放sulfoglycans表征。该战略概述研究概述zed有在图1中，其细节示于图2中 。此外，可用于通过标准的LC-MS / MS的蛋白质组技术蛋白的ID的脱色和洗涤凝胶片的一部分上。

<table border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Material/ Equipment</td>
			<td style="width:155px;">Company</td>
			<td style="width:124px;">Catalog Number</td>
			<td style="width:303px;">Comments/Description</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:231px;">Water, deionized water</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>270733-4L</td>
			<td style="width:303px;">CHROMASOLV, for HPLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Acetic acid, Glacial</td>
			<td style="width:155px;">Fisher</td>
			<td>A38-500</td>
			<td style="width:303px;">Certified ACS&#8805;99.7 w/w %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Methanol</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>34860-4L-R</td>
			<td style="width:303px;">CHROMASOLV, for HPLC, &#8805;99.9%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ammonium bicarbonate</td>
			<td style="width:155px;">Fluka</td>
			<td>09830-500G</td>
			<td style="width:303px;">BioUltra, &#8805;99.5% (T), Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Acetonitrile</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>34998-4L</td>
			<td style="width:303px;">CHROMASOLV Plus, for HPLC, &#8805;99.9%, Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ethyl acetate</td>
			<td style="width:155px;">Fluka</td>
			<td>34972-1L-R</td>
			<td style="width:303px;">LC-MS CHROMASOLV, Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium borohydride</td>
			<td style="width:155px;">Aldrich</td>
			<td>213462-25G</td>
			<td style="width:303px;">ReagentPlus, 99%, Step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Iodomethane</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>289566-100G</td>
			<td style="width:303px;">ReagentPlus, 99.5%,&#160; Step 6.&#160; Store at 4 &#176;C until use.&#160; Sit at room temperature before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Sodium hydroxide solution, 50% w/w</td>
			<td style="width:155px;">Fisher</td>
			<td>SS254-1</td>
			<td style="width:303px;">Certified, Step 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dimethyl sulfoxide, anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>276855-1L</td>
			<td style="width:303px;">Anhydrous, &#8805;99.9%, Step 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Methanol, anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>322415-100ML</td>
			<td style="width:303px;">Anhydrous, 99.8%, Step 6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Dichloromethane</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>34856-4L</td>
			<td style="width:303px;">CHROMASOL&#174;, for HPLC, &#8805;99.8%, contains amylene as stabilizer, Step 7</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:231px;">Dowex 50WX8 hydrogen formhydrogen form 100-200&#160;mesh</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>217506-500G</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AG 50W-X8 Resin&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td>142-1441</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:231px;">BAKERBOND spe 1 ml x 100 mg Solid Phase Extraction Column, PP, Octadecyl (C18) Reverse Phase</td>
			<td style="width:155px;">JT Baker</td>
			<td>JT-7020-01</td>
			<td style="width:303px;">Step 5 and 8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">7.5% Mini-PROTEAN TGX Precast Gel&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td></td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Bio-Safe Coomassie Stain&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td>161-0786</td>
			<td style="width:303px;">G-250, Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Silver Stain Kit for Mass Spectrometry</td>
			<td style="width:155px;">Pierce</td>
			<td>24600</td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Oligosaccharides Kit (Maltotriose, Dp3: R474140)</td>
			<td style="width:155px;">Supelco&#160;</td>
			<td>47265</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Oligosaccharides Kit (Maltotetraose, Dp4: R474135)</td>
			<td style="width:155px;">Supelco&#160;</td>
			<td>47265</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Disposable Pasteur Pipets, Glass, Short Tip</td>
			<td style="width:155px;">VWR</td>
			<td>14673-010</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">PYREX 13 x 100 mm Disposable Round Bottom Threaded Culture Tubes</td>
			<td style="width:155px;">Corning</td>
			<td>99447-13</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">PYREX 16 x 125 mm Disposable Round Bottom Threaded Culture Tubes</td>
			<td style="width:155px;">Corning</td>
			<td>99447-16</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td>
			<td style="width:155px;">Kimble</td>
			<td>45066C-13</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td>
			<td style="width:155px;">Kimble</td>
			<td>45066C-15</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81265</td>
			<td style="width:303px;">volume 500&#160;&#956;L, needle size 22 ga&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81165</td>
			<td style="width:303px;">volume 250&#160;&#956;L, needle size 22 ga</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81065</td>
			<td style="width:303px;">volume 100&#160;&#956;L, needle size 22s ga&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>80965</td>
			<td style="width:303px;">volume 50&#160;&#956;L, needle size 22s ga</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton Calibrated Syringes</td>
			<td style="width:155px;">HAMILTON</td>
			<td>80300</td>
			<td style="width:303px;">volume 10&#160;&#956;L, needle size 26s ga&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">Petri Dish Glass 100mm x 15mm</td>
			<td style="width:155px;">GSC INTERNATIONAL INC</td>
			<td>1500-4</td>
			<td style="width:303px;">Wash before use, Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Bard-Parker Surgical Blades</td>
			<td style="width:155px;">Fisher</td>
			<td>371310</td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Reacti-Therm Heating/Stirring Module</td>
			<td style="width:155px;">Pierce</td>
			<td>18870</td>
			<td style="width:303px;">Step 1, 4 and 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Heating Blocks</td>
			<td style="width:155px;">Fisher</td>
			<td>125D</td>
			<td style="width:303px;">Step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Pyrex fiber glass wool borosilicate pore size 8&#160;&#956;m</td>
			<td style="width:155px;">Aldrich</td>
			<td>CLS3950</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fused Silica CutterTubing cutter</td>
			<td style="width:155px;">alltech</td>
			<td>3194</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Multi-tube vortexers&#160;</td>
			<td style="width:155px;">VWR</td>
			<td>444-7063</td>
			<td style="width:303px;">Step 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Lyophilizer 25EL Freezemobile</td>
			<td style="width:155px;">Virtis</td>
			<td>25EL</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Centrifuge</td>
			<td style="width:155px;">VWR</td>
			<td>Clinical 50</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;"></td>
			<td style="width:155px;"></td>
			<td></td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">LTQ Orbitrap Discovery</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td></td>
			<td style="width:303px;">0</td>
		</tr>
	</tbody>
</table>

improved in-gel reductive &#946;-elimination for comprehensive o-linked and sulfo-glycomics by mass spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

醋酸乙酯处理对此前在凝胶还原β-消除 从牛用凝胶还原性β-消除粘蛋白释放的全甲基化O-连接的糖链样品的代表性质谱示于图3中 。在凝胶片的乙酸乙酯洗涤有效地除去SDS和聚丙烯酸污染物，它们与随后的MS分析27干涉。 <img alt="图3" src="/files/ftp_upload/51840/51840fig3highres.jpg" /> 聚丙烯酰胺凝胶?...

Watch this Scientific Journal Video about Improved In-gel Reductive Î²-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry at JoVE.com

Improved In-gel Reductive &amp;#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

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Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Unit 1

Nucleophilic Substitution and Elimination Reactions of Alkyl Halides

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视频: Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

科研

化学

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

汞（II）与Dicysteinyl四肽的络合的电喷雾质​​谱研究

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

寡 peptoids 的合成及质谱分析

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

综合结构质谱分析蛋白质结构和蛋白质配体配合物

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

聚类诱导解吸/电电质谱法对复杂分子及其在表面的反应分析

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

纳米材料获得的蛋白质和代谢物科罗纳的毛细电电量谱方法

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

通过质谱学研究蛋白质高阶结构，与二乙基碳酸酯共价标签

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

使用质谱法表征单个神经元中的RNA修饰

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...