这项工作得到了资助AI064184和AI76322从卫生研究院（NIH）全国学院和格兰特61789-MA-MUR由美国陆军研究办公室的MST研究也支持韦尔奇基金会资助F1155和美国国立卫生研究院授予R01GM103655到JSB支持

所有的解决方案应该用超纯水和HPLC级甲醇和氯仿来制备。含有有机溶剂，如甲醇，氯仿或吡啶并浓缩的酸或碱制备的溶液应准备并在化学通风橱中使用。所有的解决方案可以被存储在RT。溶剂应在有刻度的玻璃汽缸被测量并存储在玻璃溶剂瓶带有PTFE内衬的盖子。对于长期储存的含有氯仿溶剂应存放在有色琥珀色玻璃瓶中，以避免在生产光气，反应性高的酸性氯化物。聚四氟乙烯离心管中，旋转蒸发器烧瓶中，应用甲醇和氯仿后使用。处理溶剂和/或放射性废物时，请遵循必要的联邦，州和/或机构废物处理规定。 1。大型脂质A萃取（50毫升至1.5升） <ol><li>准备一个单相布莱 - 代尔的混合物：氯形式 - 甲醇-1X磷酸盐缓冲盐水（PBS），pH为7.4; 1:2:0.8 V / V）。结合200ml氯仿中，在1升溶剂瓶400毫升PBS中的甲醇和160毫升盖瓶，颠倒混匀（&gt; 30倍）。混合发泄和密封储存期间定期拧开瓶盖。 </li><li>制备预平衡的两相的Bligh-代尔混合物 - 氯仿：甲醇：水（2:2:1.8 V / V）。结合400毫升氯仿，400毫升甲醇和1升溶剂瓶180ml水。盖瓶，颠倒混匀，确保混合发泄期间定期松开盖子。让它平衡的O / N和储存密封。 </li><li>制备温和酸水解缓冲液（50mM醋酸钠，pH 4.5，1％十二烷基硫酸钠（SDS））。为500毫升弱酸水解缓冲液中，称量2.05克乙酸钠和转移到500ml烧杯中。加水至〜350毫升搅拌体积，然后加入50毫升10％的SDS。混合并调节pH值至4.5，转移至量筒，并加水至500毫升。 </li><li>准备氯仿：甲醇（4:1，V / V）：量取100毫升氯仿中，并转移至溶剂瓶。测量25ml甲醇中，并用氯仿混合。存储在琥珀色玻璃瓶。 </li><li>从单个菌落接种将5ml培养基（Luria培养基或其他）。成长O / N在37℃，或在需要℃下生长。该萃取过程的图示于图2。 </li><li>第二天，测量OD 600，并使用5毫升O / N培养物接种200毫升培养至0.05的起始OD 600。细胞生长直到OD达到600 0.8-1.0。 </li><li>通过离心以10,000×g离心10分钟收获细胞。再旋转，可能需要该沉淀不良的菌株。倒掉培养基上清液。 </li><li>用50ml的1X磷酸盐缓冲盐水（PBS）洗细胞沉淀物。重复离心沉淀细胞。倒掉上清，店内细胞沉淀在-20°C或继续执行步骤1.9。 </li><li>将细胞再悬浮于40毫升的1X PBS及二份各250ml聚四氟乙烯离心管（得20 ml的细胞悬液/管）之间的鸿沟。加入25毫升氯仿和50毫升甲醇中，每个管，为单相的Bligh-代尔（氯仿：甲醇：水; 1:2:0.8 V / V）（ 图2）。颠倒混匀并在室温下孵育&gt; 20分钟，以确保完整的细胞裂解。 </li><li>在2000×g离心离心混合物20分钟。 LPS将沉淀连同蛋白质和核酸（ 图2），但是，磷脂，异戊二烯基的脂质，以及小的，疏水性肽将留在上清液中。弃上清。 </li><li>用约100 ml单相布莱 - 代尔混合洗的内毒素沉淀。离心机在2000×g离心20分钟。弃上清液。当从大肠杆菌中分离脂质A 大肠杆菌或沙门氏菌 ，只有一洗涤是必需的，但是，可能需要额外的洗涤步骤从一些生物体中分离脂质A时，以减少污染的磷脂。 </li><li>添加27毫升英里的ld的酸水解缓冲液（50mM醋酸钠，pH 4.5，1％SDS中，参见步骤1.3），以LPS沉淀，并且通过移液器上下吹吸混合直到只有小颗粒保持。超声均匀地悬浮颗粒的LPS与探头尖端超声发生器解决方案在一个恒定的占空比为20秒，50％的输出。重复样品2倍（20秒/连拍，〜5之间爆发秒）的超声。 </li><li>煮沸样品在水浴中30分钟。注意：请确保瓶盖紧，但不是完全密封。一些生物，如霍乱弧菌（霍乱弧菌）需要更长的培养时间（1小时），增加脂质A的总收率从水浴中取出瓶子，并允许样品，然后再继续冷却至室温。 </li><li>水解后提取脂质，转换SDS溶液成两相的Bligh-代尔（ 图2）的混合物，加入30ml的氯仿和30毫升甲醇中，对于一个氯仿：甲醇：水（2:2:1.8，V / v）的混合物。通过颠倒混合并在2离心样品10分钟，000×g的。提取下相成使用玻璃移液管清洁聚四氟乙烯离心管中。 </li><li>通过加入30ml下层相从预平衡的两相的Bligh-代尔混合物（步骤1.2），以从步骤1.14的上层相进行​​第二次萃取。混合，然后离心以2,000 xg离心10分钟。提取下层相，并用在步骤1.14中提取的低相位池。 </li><li>甲醇：通过添加114毫升预平衡的两相的Bligh-代尔上层相（步骤1.2），以建立一个两相的Bligh-代尔混合物（氯仿洗涤合并的下部相（60毫升总）水; 2:2:1 1.8，V / V）。混合。离心机在2000×g离心10分钟。 </li><li>除去下层相用旋转蒸发器（ 图2）清洁玻璃旋转蒸发器烧瓶中，并干燥样品。 </li><li>加入5 ml的氯仿：甲醇（4:1，V / V）到旋转烧瓶中，浴超声处理（&gt; 30秒）从烧瓶的两侧中悬浮的脂质以帮助。使用玻璃移液管脂转移到一个干净玻璃试管（13×100毫米或更大）上铺衬四氟酚醛螺丝帽。在氮气氛下用氮气干燥机气流干燥样品。 </li><li>重悬干燥的脂质在1ml的氯仿：甲醇（4:1，V / V）。转移到小玻璃样品瓶（12×32毫米），锥形底座采用小体积更加定量悬浮（见表设备/试剂）。用干燥氮气机。 </li><li>干燥后的样品可以储存在-20℃直到随后的TLC（方案2）或质谱分析（协议3，4或5）。 （注：材料隔离并在随后的协议中使用的金额是根据什么足以满足大多数生物的提议相同的脂质样品可在2-5协议可以使用，服用％的材料去除值得注意见讨论更多的细节。） </li></ol> 2。通过薄层色谱脂质A种可视化<ol><li>制备薄层色谱法流动相溶剂系统（氯仿：吡啶：88％甲酸：水; 50:50:16:5体积/体积）。 COMBINE 200毫升氯仿，200毫升吡啶中的64毫升88％的甲酸，并毫升水20在1升溶剂瓶。盖瓶，颠倒多次混合，并发泄。 </li><li>使用TLC罐，可容纳20×20厘米板。行TLC罐约40公分层析纸。 </li><li>至TLC罐加200ml氯仿：吡啶：88％甲酸：水（50:50:16:5，v / v）混合物。让坦克预平衡时间&gt; 3小时，常常O / N是首选，更方便。 </li><li>从冰箱中取出干脂质样品（步骤1.20），并让温至室温。 </li><li>用剃刀从硅胶60 TLC板的上边缘除去二氧化硅。使用无光泽铅笔，画出平行于该板的底部，2厘米从底部的线。这条线是产地为察觉的样品。马克沿着这条参考线增量当场试样1 cm距离。 </li><li>溶于干燥的脂质从在200μl氯仿步骤2.4：甲醇（4:1，V / V），涡旋和超声处理（3次，15〜每一秒），收率〜100 -500纳克/微升脂质A，如果摘录自大肠杆菌大肠杆菌 。 </li><li>用微细的玻璃吸管，发现十分之一的体积（20μL）的到薄层板上标示在步骤2.5。允许样品风干的薄层色谱板〜15分钟。 （注：在小玻璃瓶中的样品可以使用氮气干燥机进行干燥处理并储存在-20℃，在​​3-5协议的后续使用，请注意％的材料去除）。 </li><li>放置装有样品斑点TLC板放入预平衡罐。一旦溶剂前沿到达TLC板（〜2.5-3小时）的顶部，取出板并空气干燥（&gt; 60分钟）。一个寒冷的空气枪可以用来确保完全干燥。 </li><li>而板干燥，开启热板，在250℃下进行炭化。 </li><li>在通风良好的通风橱，对于炭化脂质解决在二氧化硅TLC板上制备10％的硫酸 - 乙醇混合物（浓硫酸：100％乙醇，1:9 V / V）。测量10毫升硫酸慢慢加到90毫升的100％乙醇。关怀充分混合并转移到玻璃层析试剂雾化器。 </li><li>在通风橱中，用玻璃层析试剂雾化器喷雾干燥的薄层板上，以10％硫酸乙醇混合物。喷雾均匀地分布在板混合。 </li><li>将薄层板在250℃的热板上，直到烧焦的脂质样品显示为黑色/棕色斑点（&lt;1分钟）。不要曝光过度，板，因为这将导致整个板块变成褐色，使脂质A种可视化的困难。 </li></ol> 3。脂质A通过MALDI-TOF质谱结构表征<ol><li>从步骤1.20，（或步进2.7）从冰箱中取出脂质A的样品，让温暖至室温。干重悬在脂质〜样品20μL氯仿：甲醇（4:1，V / V）和涡流如果摘录自大肠杆菌获得〜1-5微克/微升脂质A的解决方案大肠杆菌 。 （注意：如果从步骤2.7所使用的材料10％浓度较低）。 </li><li>准备ATT基质成分。饱和的6 - 氮杂基-2 - 硫代胸腺嘧啶在50％乙腈：加入500μl的水和加入500μl乙腈到1.5ml微量离心管中，然后添加&gt; 10毫克6 - 氮杂-2 - 硫代胸腺嘧啶，使得50％的乙腈是超饱和的。三盐基饱和柠檬酸铵溶液：添加&gt; 1毫克至500μl水，沉淀物应该是显而易见的。使用前涡和离心机的解决方案，只有饱和的上清液使用。 </li><li>准备ATT基质混合物：饱和的6 - 氮杂-2 - 硫代胸腺嘧啶在50％乙腈，饱和三元柠檬酸铵（20:1，V / V）。通过加入20μl的ATT溶液1μl，以500μl的微量离心管中，涡旋混合，并应用到MALDI板上之前离心机饱和三元柠檬酸铵溶液混合基质组分一起。 </li><li>加入0.5微升校准混合物的MALDI板上靠近那里的样本将被存入一个点，为客户提供最准确的质量/电荷比决心准备MALDI板。 </li><li>存款0.5微升的ATT基质上的MALDI盘上的每个点，其中类脂A样品会沉积。 </li><li>存款0.5微升样品（总样本的2.5％）到ATT基质的现场，混上板，并获得通过光谱扫描样品的最佳离子信号（ 图3）。注：金额为最优化的MS信号可以与生物体而变化，并且需要根据经验确定。要添加更多的材料可以当场0.5微升多次让斑点的混合物干燥，除了更多的样本之间。样品也可以集中（干重悬在较小的体积）或稀释是必要的。更多的原料（起始细胞培养物的体积），也可用于（见讨论）。 </li></ol> 4。电喷雾质谱和脂质A的碰撞诱导解离<ol><li>准备一个氯仿：甲醇的混合溶剂（1:1，V / V）混合的HPLC级甲醇200μl的库存用200ml HPLC级氯仿中的玻璃溶胶发泄瓶。 </li><li>转移200μL的氯仿：甲醇混合溶剂的小瓶脂质A（ 如从步骤1.20，2.7，或协议3后晒干）和超声处理脂质5分钟或直到所有材料被溶解的解决方案。 </li><li>设置质谱仪负离子模式电喷雾电离。 </li><li>用250微升注射器和注射器泵，直接注入稀释的脂质A样品在2.0-3.5微升/分钟的流速。 </li><li>通过调整离子光学优化和提升类脂A离子信号。一个完整的质谱现在可以收集到的脂质A种（ 图4）。 </li><li>隔离并通过选择CID作为MS / MS方法激活目标类脂A。 </li><li>增加CID电压（或归一化的碰撞能量，NCE），直到前体脂质A物种是约10％的相对丰度相比的最高产物离子。 </li><li>获得和平均谱，直到足够的信号与噪声的实现为日E产品离子。所需的扫描次数是依赖于原始的前体的信号强度和范围可以从3-300扫描（ 图5A）。 </li></ol> 5。 MS / MS对脂质A通过紫外线光解<ol><li>准备样品和质谱仪按照步骤4.1-4.5。 </li><li>打开激光接口到质谱仪。质谱仪配备有一个193纳米准分子激光和修改，以允许UV活化在仪器的HCD细胞。光解被以类似的方式实现先前描述的35。我们使用修改后的真空歧管与CaF2 光学窗口发射光子转化为更高的能量C-阱解离（HCD）细胞。该激光是由一个晶体管 - 晶体管逻辑（TTL）信号从质谱仪的脉冲/延迟发生器中的MS / MS触发。 </li><li>设置仪器软件使得激光将触发受激准分子激光时的离子进入细胞HCD。这需要一个软件的适度变形。 </li><li>打开脉冲发生器，使得激光是脉冲，每2毫秒（500赫兹）。 </li><li>通过选择HCD如MS / MS法分离脂质A的前体离子和调节碰撞能量为1％罗富国教育学院。这使得前体离子的隔离的HCD细胞内的紫外线分解。虽然对HCD细胞时，该软件被修改，以该时间间隔期间执行UVPD。 </li><li>通过增加激光能量，并调整激光脉冲的数目激活分离的脂质A离子。一个典型的UVPD实验将使用10个6毫升脉冲来进行。 </li><li>如在步骤4.8，获取和平均光谱，直到有足够的信号与噪声，实现了UVPD产物离子。所需的扫描次数是依赖于原始的前体的信号强度和范围可以从3-300扫描（ 图5B）。 </li></ol> 6，32 P标记脂质A和后续的分离<ol><li>从一个单菌落接种将5ml培养基（Luria培养基或其它介质）。成长O / N，37°C或在规定的温度。 </li><li>第二天，测量外径600和使用O / N培养接种7毫升文化的〜0.05起始外径600，在一个标准的20×150毫米的一次性玻璃培养管增长。添加无机32 P的2.5微居里/毫升细胞生长直到OD达到600 0.8-1.0。请遵守联邦，州，和/或放射性材料的安全，妥善处理体制的指导方针。 </li><li>收获的细胞在16 x 125毫米的玻璃离心管聚四氟乙烯采用固定角度的临床离心机在1500×g离心10分钟衬盖。除去上清液到适当的放射性废物容器。在后续步骤中产生的所有放射性废物（ 如液体，玻璃，生物危害）应按照联邦，州进行处置，及/或机构GUIdelines。 </li><li>用5毫升的1X磷酸盐缓冲盐水（PBS）洗细胞沉淀物。离心10分钟，在1500×g下。弃上清液。 </li><li>将细胞再悬浮于5ml单相的Bligh-代尔的混合物（ 图2）由氯仿：甲醇：水（1:2:0.8，V / V）。旋涡混合，并在室温下孵育&gt; 20分钟，以确保完整的细胞裂解。 </li><li>离心机在临床离心机在1500×g离心20分钟。轻轻倒出上清液，其中包含磷脂和异戊二烯脂质。 </li><li>重悬沉淀LPS 1.8毫升50 mM的醋酸钠，pH值4.5; 1％SDS缓冲液涡旋。用水浴超声直至沉淀均匀地分散声处理样品（〜30秒）。 </li><li>在沸水浴中孵育样品30分钟。确保上限紧张，但不密封。 </li><li>从水浴中取出，并允许样品在室温下冷却5〜10分钟。 </li><li>加入2毫升氯仿和2毫升该溶液转变成两相的Bligh-代尔混合物甲醇，得到氯仿：甲醇：水（2:2:1.8 V / V）混合物中。旋涡混匀，离心1500×g离心分离阶段10分钟在临床离心机。 </li><li>提取下相成使用玻璃移液管（第一提取）干净的玻璃离心管中。 </li><li>通过加入2ml预平衡的两相的Bligh-代尔下层相（其制备如上述步骤1.2），以从步骤11中的剩余的上部相中进行样品上的第二次萃取。涡和离心10 min。除去下层相，并结合从第一萃取下层相。 </li><li>向合并的下层相（〜4毫升总体积）中，添加7.6预平衡的上层相（步骤1.2）中的溶液，得到一个两相的Bligh-代尔溶液（氯仿：甲醇：水; 2:2:1.8，V / v）中。涡旋并离心分离10分钟。 </li><li>除去下层相到一个干净的玻璃管中，干燥用氮气干燥。干燥的样品可以储存在-20℃直至进一步使用。 </li></ol> 7。可视通过薄层色谱的32 P标记的脂质A种zation <ol><li>制备32 P标记的脂质样品，TLC罐，和TLC板按照步骤2.1-2.8中所述。 </li><li>溶解32 P-标记的样品中加入500μl4:1氯仿-甲醇（体积/体积）。涡街流量计和超声浴使其完全溶解脂类物质。添加5微升的样品，以含有5ml闪烁液的闪烁瓶中。伯爵在闪烁计数器，计算样品的总次数/分钟。 </li><li>当可视化放射性标记的脂质物种，10×20厘米或20×20厘米的TLC板都可以使用。为10×20厘米板，样品应沿标记的步骤2.5的原点被察觉，使得板的最长的尺寸是垂直的（ 即发现在原点的样品标记为2厘米10厘米边缘段）。 </li><li>使用微细吸管，当场就板10,000-20,000 CPM /样品，并允许斑点干燥（&gt; 15分钟）。样品可能需要进行浓缩，干燥，根据氮，悬浮在适当的音量，以达到10,000-20,000计数/分/点（在时间&lt;10微升共2微升或2微升）。 </li><li>放置TLC板含有放射性标记的样品加入到预平衡罐。一旦溶剂前沿到达TLC板（〜3小时）的顶部，取出板并空气干燥（&gt; 60分钟）。注意：在吡啶的存在下进行TLC板必须在化学通风橱中彻底干燥，如吡啶中的痕量会损坏磷光屏。一个寒冷的空气枪可以用来确保完全干燥。 </li><li>敷板保鲜膜暴露于磷屏屏幕在放射自显影暗盒的O / N。第二天早上，扫描屏幕来获得图像（ 图6）。使用图像光密度分析软件，这种方法允许脂质A种准确，相对定量。 </li></ol>

没有利益冲突的声明。

在这个协议中，我们有详细的脂质A种细菌全细胞的分离，并描述了薄层色谱或质谱为基础的分析方法，以化学表征这个与世隔绝的材料。串联质谱法是一种强大的战略生物化合物的从头结构表征，并且是无价的，在自然界中观察到的脂质A分子的一整套的化学特性。 CID和UVPD是创建不同类型的产物离子，提供关键的指纹类脂A分子的两个互补的激活方法。同时使用CID和UVPD MS / MS碎片可以澄清?...

脂多糖（LPS）是几乎所有的革兰氏阴性菌的主要外表面的分子和由三个结构域分子：远侧O-抗原多糖，核心寡糖，以及膜结合的脂质沉积的外部小叶域外膜双层1,2。的脂质A结构域由3 -脱氧-D-甘露-辛-2 - ulosonic（KDO）残基和脂质A分子物质，其中脂质A可以被定义为LPS时温和酸水解1的氯仿可溶部分2。标准的脂质A分子可以化学定义为diglucosamine骨干即六酰化和双 -磷酸化;与模型生物体大肠杆菌（大肠杆菌）1,2中观察到的主要脂质A物种一致。九个组成型表达的基因，在整个革兰阴性菌的保守，有负责生产的脂质A结构域（ 图1）1,2。大多数细菌有另外一组基因，这变化在进化保守性程度，参与脂质A 3的进一步的化学修饰。去磷酸化，除去酰基链的，并且另外的化学基团如氨基糖（ 例如 aminoarabinose）和/或磷酸乙醇胺的是最常观察到的活性（ 图1）。许多负责脂质A修饰酶的直接由环境信号，如二价阳离子激活，或者它们的表达是由两个组分的反应调节系统3调节。 脂质A物种的宿主先天免疫系统的识别是由Toll样受体4/myeloid分化因子2（TLR4/MD2）共受体介导4。 MD2和脂质A酰基链，以及TLR4和脂质A的1和4&#39;磷酸基团促进唇部的强关联之间的疏水力id一个与TLR4/MD2 4,5。从而改变酰化状态或脂质的负电荷一个影响TLR4/MD2基于脂质修饰的识别和先天免疫反应的下游刺激活化剂NF-κB和炎症介质如TNFα和IL 1-β6,7。掩盖脂质A的负电荷的修改还可以防止杀菌阳离子抗菌肽的结合，对革兰氏阴性细胞表面3,8。许多类脂A的修改假设特定的环境条件，如内部的人类宿主中或生态位之下，以增加细菌的健身。出于这个原因，许多修饰酶抗菌化合物的合理开发有吸引力的目标。类脂A结构的化学多样性，对于生物体和/或环境，这些不同结构的生物学意义使脂质A的结构特征在T一个重要的努力他研究的革兰氏阴性细菌。 从整个细菌脂质A分子的分离涉及脂多糖从细菌细胞表面上的提取，水解步骤以释放脂质A，接着是最终的纯化步骤9-11。最经常提到的脂多糖提取过程是热酚水提取过程中，首先由韦斯特法尔和JANN 10推出。后提取全部的LPS进行温和酸水解，其中化学上从6&#39;-羟酰脂质A的末端葡糖胺糖（ 图1）的分离KDO。无数陷阱的热酚水的程序，包括使用高危险试剂的存在，需要降低共同提取的核酸和蛋白质，并且好几天都必须完成的协议10。 我们的实验室进一步发展的脂质A的提取和分离首先由Caroff和雷茨12,13研制。相对于热酚水程序，这里介绍的方法更快速，高效，适应范围广泛培养体积的5毫升到多升。此外，与热酚水提取，我们的方法不适用于粗糙或光滑型LPS的选择，提供类脂A物种的最佳恢复。在我们的协议，使用氯仿，甲醇和水，其中LPS可以通过离心沉淀的混合物进行全细菌细胞的化学裂解。轻度酸水解，溶剂萃取（布莱 - 代尔）的组合是用来解放脂质A的共价连接的多糖。布莱和代尔的方法中首先被施加到脂质物种的提取从各种动物和植物的组织14，这里修改为从脂质A。在这最后的分离步骤中分离的水解多糖，氯仿可溶的类脂选择性地划分成下有机阶段。以进一步纯化脂质A，反相或阴离子交换柱层析可用于12。 脂质A物种从整个细胞的分离后，可以使用若干分析方法来表征的分离的材料，例如核磁共振，薄层色谱法，和基于MS的分析物的化学结构。核磁共振允许非破坏性的结构解析，并提供了详细的结构与糖苷键，酰基链位置明确赋值，并附着位点脂质A修改，比如aminoarabinose或磷酸乙醇胺15-17的分配。脂质A的NMR分析是不是我们的协议中所讨论的，但已经充分说明别处15,16。对于快速分析TLC为基础的方法是经常使用的，但未能提供有关精细化学结构的直接信息。质谱为基础的协议是最经常使用的方法来表征类脂A结构18,19。相关矩阵的激光解吸电离（MALDI）-MS通常被用于最初勘测完好脂质A的种类。从分析物根据我们的提取方法制备产生单电荷离子。随着越来越多的精细结构分析是必需的，MS / MS为基础的方法证明比MALDI-MS的更多信息。耦合电喷雾电离（ESI）单独或多个带电脂质A的前体离子是进一步由分散的碰撞诱导解离（CID）或紫外线光解（UVPD），生成结构信息的产物离子18,20,21。也经常在ESI-MS产生中性丢失的产品从类脂A的前体离子提供的结构信息的附加层。 串联质谱（MS / MS）已被证明是对脂质A结构的阐明中不可缺少的和通用的方法。在MS / MS离子被活化，以产生可被用来阐明该前体离子的结构的诊断裂解谱。最广​​泛AVAilable MS / MS方法是CID。这种方法产生的碎片离子通过选定的前体离子的碰撞与惰性气体的目标，从而在通向离解的能量沉积。 CID已被证明在类脂A结构的分配，适用范围广的细菌种类22-33的一个重要工具。 虽然CID是最普遍实施MS / MS方法，它生成的子离子的有限阵列。 193纳米UVPD是一种替代和补充的MS / MS方法。此方法使用的激光照射离子和光子的吸收导致离子和随后的离解的通电。这种高能量的MS / MS技术生产的产品比离子的CID更多样化，从而提供更多的信息碎片化的模式。特别是，UVPD能提供有关基于分裂的糖苷，胺，酰和CC挂钩债券18,21,34中脂质A种微妙的变化信息。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Chloroform</td>
			<td>Thermo Fisher Scientific</td>
			<td>C607</td>
			<td>HPLC Grade</td>
		 </tr>
		 <tr>
			<td>Methanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A452</td>
			<td>HPLC Grade</td>
		 </tr>
		 <tr>
			<td>Teflon FEP Centrifuge Bottles</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-562-21</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Silica Gel 60 TLC Plates</td>
			<td>EMD Biosciences</td>
			<td>5626-6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Grade No. 3MM Chromatography Paper</td>
			<td>Whatman</td>
			<td>3030700</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Orbitrap Elite</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mass Spectrometer</td>
 <td></td>
 <td></td>
 <td></td>
		 </tr>
		 <tr>
			<td>ExciStar XS Excimer Lasrer</td>
			<td>Coherent Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PicoTip Nanospray ESI emitters</td>
			<td>New Obectives</td>
			<td></td>
			<td>&ge; 30 &mu;m to reduce clogging</td>
		 </tr>
		 <tr>
			<td>Model 505 Pulse/Delay Generator</td>
			<td>Berkeley Nucleonics Corporation</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hot Plate Thermoylne 2200</td>
			<td>Barnstead/Thermolyne</td>
			<td>HPA2235MQ</td>
			<td></td>
		 </tr>
		 <tr>
			<td>16x125 mm GPI 15-415 Threaded Disposable Borosilicate Culture Tubes</td>
			<td>Corning Pyrex</td>
			<td>99449-16X</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Reusable Threaded PTFE screw caps GPI 45-415</td>
			<td>Corning </td>
			<td>9999-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Personal Molecular Imager System (phosphorimager)</td>
			<td>BioRad</td>
			<td>170-9400</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Autoradiography Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>FBCS810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphorscreen SO230</td>
			<td>Kodak</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Peptide Mass Standards Kit</td>
			<td>Sequazyme</td>
			<td>P2-3143-00</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sonifier S250-A</td>
			<td>Branson</td>
			<td>101063196</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1.5 ml 12x32 mm Tapered Base Screw Thread Vial</td>
			<td>Thermo Fisher Scientific</td>
			<td>C4000-V1</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation and chemical characterization of lipid a from gram-negative bacteria

脂多糖（LPS）是革兰氏阴性细菌的主要细胞表面分子，沉积在外层膜双分子层的外层小叶。 LPS可以细分为三个结构域：远侧O-多糖，核心寡糖和脂质组成的脂质A结构域的分子种类和3 - 脱氧-D-甘露 - 辛-2 - ulosonic氨基酸残基（KDO）。该类脂A结构域是细菌细胞生存所必需的唯一组成部分。以下的合成，脂质A是化学修饰的响应于环境压力，如pH值或温度，以促进耐抗生素化合物，并通过宿主先天免疫反应的介体，以逃避识别。下面的协议的细节由革兰氏阴性细菌的脂质A的小型和大型隔离。分离的材料然后化学用薄层色谱（TLC）或质谱（MS）。除了基质辅助激光解吸/女的电离时间光（MALDI-TOF）质谱，我们还描述了使用电喷雾离子化（ESI）耦合到碰撞诱导解离（CID）和新聘紫外线光解（UVPD）方法分析类脂A分子种类串联质谱协议。我们的MS协议允许明确的决心，化学结构，极为重要的类脂A分子，含有独特或新颖的化学修饰的表征。我们还描述了放射性同位素标记，并随后分离，脂质A从细菌细胞通过TLC分析。相对于基于MS的协议，薄层色谱法提供了一种更经济的和快速的表征方法，但不能被用来明确指定脂质A的化学结构，无需使用已知的化学结构的标准。在过去的二十年间隔离脂质A和表征导致了许多令人兴奋的发现，提高了我们对革兰阴性菌，抗生素电阻机制的生理学的理解距离，人的先天免疫应答，并已在抗菌化合物的发展提供了许多新的目标。

E的规范脂质A 大肠杆菌和沙门伤寒是葡糖胺的六酰化二糖与磷酸基团的1 -位和4&#39; -位。在生长过程中的富媒体（ 如卢里亚肉汤）的脂质A部分包含焦磷酸组1位产生了三磷酸化的36种（ 图1）。 KDO（3 -脱氧-D- 甘露 -辛酮糖酸），附着在6&#39;-羟基并作为一个桥梁，脂质A链接到LPS的剩余碳水化合物域（ 即核心寡糖和O-抗原域）。虽?...

Watch this Scientific Journal Video about Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria at JoVE.com

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

隔离脂多糖（LPS）由革兰氏阴性菌的类脂A结构域和表征提供了洞察抗生素耐药性的细胞表面为基础的机制，细菌的生存和健康，以及如何化学性质不同的脂质分子种类差异调节宿主先天免疫反应。

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

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31.5K 次观看.  The University of Texas at Austin. 隔离脂多糖（LPS）由革兰氏阴性菌的类脂A结构域和表征提供了洞察抗生素耐药性的细胞表面为基础的机制，细菌的生存和健康，以及如何化学性质不同的脂质分子种类差异调节宿主先天免疫反应。 

视频: Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

自由基化学生物学的化学行为，生物标记开发

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

科研

化学

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

折叠的DNA折纸一个生物反应机器人与表征

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

核苷三磷酸 - 从合成到生化特征

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

一个反分析方法化工运输的油漆表征

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

功能化的合成与表征金属 - 有机骨架

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

超分子胶体的合成与表征

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

微流控干纺及再生丝素纤维的表征

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

钌线粒体钙吸收抑制剂的合成与评价

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.