DZ的支持，MD安德森癌症中心和美国国立卫生研究院拨款AI079232。 MD安德森癌症中心是支持部分由美国国立卫生研究院授予CA16672。

1。实验室安全问题<ol><li>所有的有机溶剂，必须存储在特定领域具有明确标记。请参阅生产所需的灭火器类型的标签。 </li><li>几种试剂的使用有潜在致癌性，可能会导致精神抑郁。必须处理这些试剂的化学与通风罩（特定的试剂和设备的细节，请参阅表）。 </li><li>我们建议，当用有机溶剂和细胞，个人防护装备，如手套，实验室外套，保护眼睛在任何时候都可以利用。 </li></ol> 2。鼠白血病细胞中提取的鞘糖脂<ol><li>商店RBL鼠白血病细胞的8（10万至100万美元），16x100毫米的玻璃管在-80°C的条件。台盼蓝染色后，用血细胞计数仪计数细胞。细胞存活率应该高于95％。 </li><li>提取的脂质广泛使用sonicat离子四次1-2小时，每一个与混合的极性溶剂。使用6毫升氯仿 - 甲醇= 1:1（体积/体积）作为第一个和最后一个溶剂。六毫升异丙醇：己烷：H 2 O的 55:25:20（体积/体积/体积，在使用前通过抽吸除去上层相），然后，可以使用的第二和第三溶剂。此制备溶剂：己烷：H 2 O中的55:25:20的比例通过混合异丙醇，用力摇晃，和允许的上部相出现，这将被删除。保持较低的阶段使用。 </li><li>超声处理离心沉淀不溶性物质。池的上清液。干燥它们在Speed​​Vac离心4小时。可以使用氮气流或旋转蒸发器中，如果Speed​​Vac离心是不可用。使用一个良好的冷阱，以保持蒸发的有机溶剂。到真空泵油中的有机溶剂的污染会降低真空泵的效率。 </li><li>使用高效薄层层析（HPTLC）执行初步分析。为了量化糖脂，制备出0.2％的地衣酚在50％的硫酸溶液在50毫升（100毫克）和半乳糖（40毫克在100毫升原液）标准。在30μl的反应体积的准备一系列的稀释，使用标准曲线（从0克/升20克/升）。每个系列稀释样品加20μl甲醇。每个糖脂的样品溶解在200μl的甲醇中，超声，并使用20微升糖脂的数量测量。带螺纹帽（萨尔斯塔特，72.692.005）在1.5ml Eppendorf管中，加入0.4毫升0.2％的地衣酚在50％的硫酸溶液，向每个样品中，与半乳糖-H 2 O-甲醇的解决方案，用于标准曲线的一系列。加热块中煮沸5分钟，在100℃阅读日出Tecan公司酶标仪在440 nm的光密度显色反应。要执行的HPTLC，使用氯仿：甲醇：H 2 O为60:35:8（体积/体积/体积）作为溶剂，用氯仿-甲醇= 1:1（体积/体积）处理的中性脂质。使用异丙醇：己烷：H 2 O的 55:25:20（体积/体积/体积）作为的溶剂的酸脂类（神经节苷脂）。在200μl溶剂氯仿：甲醇= 1:1（体积/体积）中溶解分离出的中性GSLS Merck硅胶薄层层析板德拉蒙德Microcaps上加载。板运行在溶剂氯仿：甲醇：H 2 O为60:35:8（体积/体积/体积）并干燥。鞘糖脂是可视化的地衣酚 - 硫酸在300℃下</li><li>为了分离出中性脂质从酸性脂类，馏分满分通过阴离子交换色谱法在一个小的DEAE葡聚糖凝胶A-25柱的中性和酸性脂类[DEAE交联葡聚糖A25树脂浸泡的Sephadex A-25树脂，可以通过以下来制备粉末在氯仿：甲醇：H 2 O的 30:60:8（体积/体积/体积）中过夜。吸出上清液，直到葡聚糖A25树脂是所有剩下的。加入新鲜的氯仿：甲醇：H 2 O的 30:60:8（体积/体积/体积）洗树脂。重复3次，以去除带负电荷的残基的交联葡聚糖A25树脂]。 </li><li>溶解，声处理（不超过10分钟），并在需要时从步骤2.1，氯仿：甲醇：30:60:8（体积/体积/体积）H 2 O重悬的脂质。 </li><li>准备一个小的DEAE交联葡聚糖A25（表格）氯列通过使用玻璃棉和一个9“巴斯德吸管包装玻璃棉仔细使树脂粉末不通过色谱柱。加入DEAE交联葡聚糖A25（以Cl形式）树脂”颈“9”巴斯德吸管没有让列干。请确保您没有看到任何树脂洗脱。溶解的样品溶解在氯仿：甲醇：H 2 O的 30:60:8（体积/体积/体积）。中性脂质组分是流过级分。 </li><li>洗脱酸性用8毫升醋酸钠在甲醇中的脂质成分（绑定到树脂）。干中性和酸性组分，淡化他们的透析，干Speed​​Vac离心和分析他们的HPTLC。 </li></ol>酸性馏分含有神经节苷脂，它可以为3步反应直接透析。中性馏分包含不仅中性鞘糖脂，但也磷脂（磷脂酰胆碱，鞘磷脂），通过乙酰化反应必须除去。 根据我们的经验，该方法的透析盒，比一个透析管或反相C18柱色谱法是更方便快捷的。 <ol start="9"><li>下一个步骤是除去中性鞘糖脂，磷脂从乙酰化和peracetylation反应。擦干DEAE葡聚糖凝胶A-25直通馏分在Speed​​Vac离心（冷却下）4小时。后的样品是干的，把它们放回Speed​​Vac离心和干燥为另一个4小时。确保这些样品是完全干燥的，任何残留的水会防止peracetylation反应。 </li><li> Peracetylate样品与1毫升吡啶和0.5ml乙酸酐在黑暗中，在室温或37℃培养过夜。吡啶和乙酸酐的量是适当的，最多以200μg的鞘糖脂。该反应可以在37℃下进行，作为GSLS有较好的溶解性。 </li><li>干燥过酰化材料通过Speed​​Vac离心3小时，加入1毫升的甲苯中，3×（共蒸发），以确保完全蒸发。如果样本是还没有完全干，Speed​​Vac离心干燥为止。 </li><li>巴斯德吸管准备一个弗罗里硅土（Sigma-Aldrich公司）柱（30〜60目，10×80毫米），带玻璃毛，用硅酸镁载体珠。硅酸镁珠应完全在使用前干燥，在110℃的烘箱中孵育。填充珠到巴斯德吸管到脖子上，在1,2 - 二氯乙烷 - 己烷4:1（体积/体积）和平衡。 </li></ol>从弗罗里硅土柱洗脱，速度非常快。快速波动的溶剂，可能会导致柱干燥。因此，所有的溶剂的混合物前要准备好运行的列，因为它是不可能停止期间洗脱柱。 <ol start="13"><li>应用全乙酰化ated样品在1毫升的1,2 - 二氯乙烷 - 己烷4:1（体积/体积）中，并用6ml的相同的溶剂中，然后由10毫升1,2 - 二氯乙烷洗柱。洗脱中性的过酰化GSLS用6毫升1,2 - 二氯乙烷 - 丙酮= 1:1（体积/体积）。此步骤将删除过酰化磷脂，糖脂，，因为过酰化糖脂结合的弗罗里硅土柱，而过酰化磷脂不要。 </li><li>擦干馏分Speed​​Vac离心2小时，脱乙酰用2ml 0.5M的甲醇钠[Sigma-Aldrich公司，403067]在2毫升甲醇中，在室温下的3小时。 </li><li>中和该混合物用3ml甲醇乙酸[乙酸/甲醇15/85体积/体积]，干燥通过Speed​​Vac离心，经透析脱盐[将干燥后的产品溶解在3毫升的水，并注入到幻灯片-A仪透析盒，透析对水24小时。改变水的至少两次]。将透析的GSLS（水溶液）由Speed​​Vac离心干燥，直到样品完全干燥。 </li&gt; </ol> 3。化学修饰（Permethylation）的糖脂13 <ol><li>到一个玻璃管用螺丝帽和聚四氟乙烯内衬介绍GSLS（1-20微克），并加入二甲亚砜​​（150微升），而无需使用特殊的干燥条件下或惰性气体气氛。 </li><li>准备粉状氢氧化钠（40-60毫克）从氢氧化钠颗粒通过研磨它们在化学砂浆用杵。请一定要在一个非常干燥的环境中，存储的粉末仔细研磨的化学罩，以避免吸入粉末的。 </li><li>往下氢氧化钠粉末添加到样品溶液，用刮勺​​。加入碘甲烷（80微升）与100微升Halmilton注射器，[或100微升VWR校准移液器挂钩到注射器通过橡胶管，可以使用]，并在室温下摇动混合物1小时。 </li><li>淬灭与水（2毫升）中的甲基化反应。提取全甲基化产品的3倍加入二氯甲烷（2毫升）[糖脂是在二氯甲烷相，这是较低的相位，所以上层相可以被转移到新的玻璃管，并再次用二氯甲烷萃取]。 </li><li>洗净合并的二氯甲烷提取3次，用2毫升水[上层相，它是水，然后可以被布置...]。在最后一次洗涤后，样品转移到一个新的试管中，并干燥使用Speed​​Vac离心。 </li><li>然后，样品可以被溶解在100至200微升甲醇ESI-MS分析。 </li></ol> 4。 MALDI-TOF分析的全甲基Gycosphingolipids <ol><li>执行上的MALDI TOF-TOF质谱仪（应用生物系统公司，加利福尼亚州福斯特城，4700）MALDI-TOF-的MS和MALDI-TOF/TOF-MS/MS实验。准备的10毫克/毫升羟基苯甲酸（DHB）在乙腈和0.1％三氟乙酸（TFA）在水中的50％体积的50％体积的混合矩阵。 </li><li> DHB基质的MALDI TARGE可以发现吨（1微升），接着由1微升（含100毫微克GSLS）点的样品溶解在甲醇中。慢慢地，申请允许它干之前分析。 </li></ol> 5。离子阱质谱分析全甲基鞘糖脂<ol><li>质谱线性离子阱质谱仪（LTQ，ThermoScientific，圣何塞，加利福尼亚州）使用纳升喷雾源，0.30μl/ min的流速毛细管温度在230°C在正离子模式下，用30-50％的碰撞能量。将留下一个微小残留丰度的前体离子的碰撞能量。检查所有的离子如钠加成物。 </li><li>确定GB3（Galα4Galβ4Glcβ1Cer）的和iGb3（Galα3Galβ4Glcβ1Cer）标准等压混合iGb3（Galα3Galβ4Glcβ1Cer）通过比较不同模式的MS产物离子从该sodiated的分子离子聚糖片段的m / z 667和终端双糖1 -烯离子m / z 445（ 即X→667→445→，X指的分子离子ESI-LIT-MS与全甲基GB3（Galα3Galβ4Glcβ1Cer）的的MS 1），的纯，全甲基iGb3（Galα3Galβ4Glcβ1Cer）标准通过检测。 </li></ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congruent+strategies+for+carbohydrate+sequencing.+2.+FragLib:+an+MSn+spectral+library.">Congruent strategies for carbohydrate sequencing. 2. FragLib: an MSn spectral library.</a> Anal. Chem. 77 (19), 6263-6270 (2005).</li></ol>

DZ是一个顾问BIOTEX，休斯敦，得克萨斯州，在这篇文章中提到的技术相关的专利，发行或在应用程序的发明者。

糖组，相似的基因组和蛋白质组提出的一个术语，指的是所有的糖结构的有机体。要完全领会多方面的功能的糖基化将需要一个综合的方法，包括功能和结构的糖组学研究。两者是复杂的由非模板驱动的性质聚糖的生物合成，得到的聚糖结构的复杂性和多样性，糖苷配基的结构的调制糖链在分子水平上的配体 - 受体相互作用的频繁参与，和低丰度配位体的功能的重要性。 人们普遍接受，质谱（MS）是一种不可缺少的结构糖组学的研究，特别是用于识别和表征低丰度配位体的方法。观察聚糖或糖复合物的分子种类，我们经常使用的一种高效，低能量的离子化方法，称为电喷雾电离（ESI）。欲了解更多DETailed聚糖结构表征，它是必不可少的选择个别分子物种和打破聚糖成更小的碎片。这通常是通过碰撞诱导解离，其中涉及通过与惰性气体分子碰撞激活聚糖。增加能量导致键断裂，产生的碎片系统的分析提供信息的糖链分子结构。通常情况下，特别是糖链的结构特点，诊断为“签名”的片段，可以产生。这些片段与分子质量一起，有时可以是足够用于识别聚糖，但在过去已被要求更多的信息完全表征它们，尤其是如果该结构是新颖的。这些方法包括糖组成分析，气相色谱-质谱分析用于连锁分析的部分甲基化的醛醇乙酸酯，和特定的糖苷酶消化17。 18日至19日 ，多轮的低能量碎片，这可以有效地进行离子阱质谱仪（IT-MS），大大提高了信息的产量聚糖质谱分析。四个或五个轮碎片（这可以没有被完成与其他MS文书），可以区分的异构体聚糖包含的相同的糖组件布置在不同的糖的联系，即使当这些是本一起的组件的混合物中具有相同的相对分子质量（等压线）。对于这种分析，聚糖衍生，免费更换所有的羟基甲基使用permethylation的。虽然聚糖的结构分配是可能的没有permethylation，的permethylation增加灵敏度17。 Levery，周，结合了IT-MS方法的潜在优势，包括结构性的同分异构体的检测ctures使用签名诊断离子，只观察到在MS和MS 5光谱，高度敏感的鉴定和定量的形式的多个同量异序的混合物7-9本鞘糖脂。从理论上讲，我们可以能够识别每一个现有的糖脂结构，以待的可用性标准的糖脂，可以通过化学方法合成的。 的分析结果的关键步骤是GSLS从生物样品中的回收率。通常为80微克GSL可以恢复肿瘤细胞如RBL从100万。以产生足够的分子离子的多个轮中的碎片离子阱质谱仪，至少10微克肿瘤GSLS是必需的。在纯化过程中，会导致低收率GSLS低丰度的离子，不能进行进一步破碎。分析的成功依赖于GSLS回收量。通常情况下，最终浓entration的全甲基GSLS应达到1毫克/毫升，溶解在甲醇中，在被分析之前在纳米电源。作详细的MS n分析的极限是依赖于特定的离子在MS 1档案中的丰度。一个典型的电子离子丰度需要进行彻底的MS 分析 。可以引起的损失GSLS pe​​racetylation步骤期间（除去磷脂），时每-乙酰化GSLS必须被绑定到硅酸镁载体树脂层析柱，洗涤，并随后洗脱GSLS在纯化过程中的低收率。为了确保每个-乙酰化GSLS硅胶的结合，样品应被重新加载两次色谱柱后流经。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>1,2 Dichloroethane</td>
 <td>Sigma-Aldrich</td>
 <td>34872</td>
 <td>Carcinogenic </td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>VMR</td>
 <td>JT9524-0</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic anhydride</td>
 <td>Fluka</td>
 <td>45830</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fischer</td>
 <td>A18P-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform </td>
 <td>Fischer</td>
 <td>C606-1 </td>
 <td>Carcinogenic</td>
 </tr>
 <tr>
 <td>DEAE Sephadex A25 (Cl Form)</td>
 <td>GE Healthcare Biosciences</td>
 <td>AB 17&mdash;170-01 </td>
 <td>100 gram</td>
 </tr>
 <tr>
 <td>Decane (anhydrous): </td>
 <td>Sigma-Aldrich</td>
 <td>457116 </td>
 <td>100 ml</td>
 </tr>
 <tr>
 <td>Dichloroacetic acid</td>
 <td>Sigma</td>
 <td>D54702 </td>
 <td>Carcinogenic</td>
 </tr>
 <tr>
 <td>Dialysis cassette </td>
 <td>Fischer(Pierce) </td>
 <td>66110 </td>
 <td>Slide-A-Lyzer, 3500 MWCO, 3-12 ml </td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Thermo Scientific </td>
 <td>20684 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl acetate </td>
 <td>Fischer</td>
 <td>UN1173 </td>
 <td></td>
 </tr>
 <tr>
 <td>Florisil</td>
 <td>Fluka</td>
 <td>46384 </td>
 <td>for packing chromatography column</td>
 </tr>
 <tr>
 <td>Hexanes </td>
 <td>EMD</td>
 <td>HX0290-6 </td>
 <td></td>
 </tr>
 <tr>
 <td>Iodomethane </td>
 <td>Riedel de Haen, Germany</td>
 <td>03810 </td>
 <td>stabilized with silver foil Carcinogenic</td>
 </tr>
 <tr>
 <td>Methanol </td>
 <td>VWR/EMD</td>
 <td>MXO475-1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Neutral glycosphingolipid Qualmix </td>
 <td>Matreya</td>
 <td>1505</td>
 <td></td>
 </tr>
 <tr>
 <td>Monosialganglioside mixture</td>
 <td>Matreya</td>
 <td>1508</td>
 <td></td>
 </tr>
 <tr>
 <td>Disialoganglioside mixture </td>
 <td>Matreya</td>
 <td>1509</td>
 <td></td>
 </tr>
 <tr>
 <td>Lactosyl ceramide and sialosylderivatives </td>
 <td>Matreya</td>
 <td>1510</td>
 <td></td>
 </tr>
 <tr>
 <td>Gangliotetraosyl ceramide and sialosyl derivatives </td>
 <td>Matreya</td>
 <td>1511</td>
 <td></td>
 </tr>
 <tr>
 <td>Pasteur pipettes</td>
 <td>Fischer</td>
 <td>13-678-6A </td>
 <td>9"''</td>
 </tr>
 <tr>
 <td>Pyridine: </td>
 <td>Sigma</td>
 <td>270407 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Hydroxide: </td>
 <td>BDH </td>
 <td>0292 </td>
 <td>500 gram</td>
 </tr>
 <tr>
 <td>Sodium methoxide </td>
 <td>Sigma </td>
 <td>404367 </td>
 <td>0.5 M solution in methanol</td>
 </tr>
 <tr>
 <td>Toluene </td>
 <td>J.T. Baker</td>
 <td>9351-03 </td>
 <td>4 L</td>
 </tr>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Pasteur pipettes</td>
 <td>Fischer</td>
 <td>13-678-6A </td>
 <td>9"''</td>
 </tr>
 <tr>
 <td>Fiber glass (glass wool)</td>
 <td>Corning Incorporated</td>
 <td>3950 </td>
 <td>Pyrex 9989 glass</td>
 </tr>
 <tr>
 <td>12x75 mm glass tubes</td>
 <td>Fischer</td>
 <td>14-962-10B</td>
 <td></td>
 </tr>
 <tr>
 <td>Calibrated pipettes (100 microlitter)</td>
 <td>VMR</td>
 <td>53432-921</td>
 <td></td>
 </tr>
 <tr>
 <td>16x100 mm disposible borosilicate tubes x1,000 </td>
 <td>Fischer</td>
 <td>14-961-29 </td>
 <td>With screw caps</td>
 </tr>
 <tr>
 <td>Caps for glass tubes</td>
 <td>Kimble</td>
 <td>40566C </td>
 <td>size/13-415 </td>
 </tr>
 <tr>
 <td> Merck TLC plates</td>
 <td>Sigma</td>
 <td>Z 29, 301-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass tank for TLC</td>
 <td>Sigma</td>
 <td>Z 12,619-5 </td>
 <td></td>
 </tr>
 <tr>
 <td>Drummond Microcaps </td>
 <td>Drummond scientific company</td>
 <td>1-000-0100 </td>
 <td>10 &mu;l, for loading of TLC samples</td>
 </tr>
 <tr>
 <td>Sunrise Microplate Absorbance Reader</td>
 <td>Tecan</td>
 <td>A-5082</td>
 <td></td>
 </tr>
 <tr>
 <td>Whatman Chromatography Paper</td>
 <td>Fischer</td>
 <td>05-716-3E</td>
 <td>18 cm x 34 cm For TLC Tank</td>
 </tr>
 <tr>
 <td> Orcinol ferric chloride spray reagent</td>
 <td>Sigma</td>
 <td>O7875</td>
 <td>For detecting glycosphingolipids on TLC plates</td>
 </tr>
 <tr>
 <td>Prevel Spray Unit</td>
 <td>Sigma</td>
 <td>Z 36,555-6 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sonicator</td>
 <td>Fischer</td>
 <td>FS20 </td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Sorvall</td>
 <td>Legend RT </td>
 <td></td>
 </tr>
 <tr>
 <td>Speedvac</td>
 <td>Savant</td>
 <td>AS160 </td>
 <td>With chemical trap for organic solvants</td>
 </tr>
 <tr>
 <td>MALDI TOF-TOF Mass spectrometer</td>
 <td>Applied Biosystems</td>
 <td>Proteomics 4700 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ion Trap Mass spectrometer</td>
 <td>Thermo</td>
 <td>LTQ </td>
 <td></td>
 </tr>
 </tbody>
</table>

mass spectrometric analysis of glycosphingolipid antigens

鞘糖脂（GSL）属于生物大分子的糖缀合物类，其中承担重要的生物过程，如胚胎发育，信号转导，免疫受体识别1-2的结构信息。它们包含复杂的糖部分中的异构体形式，和脂质部分的变化，包括脂肪酰基链长，不饱和度，和羟化。碳水化合物和神经酰胺部分可能是生物学意义的基础。例如中，三正hexosylceramides包括globotriaosylceramide（Galα4Galβ4Glcβ1Cer）和isoglobotriaosylceramide（Galα3Galβ4Glcβ1Cer），它们具有相同的分子量，但不同的糖联系的碳水化合物部分，负责为完全不同的生物功能3-4。在另一个例子中，它已被证明，变形例的神经酰胺部分的α-半乳糖神经酰胺，不变性一种有效的激动剂配体的蚂蚁NKT细胞，改变细胞因子的分泌分布和功能的动物模型的癌症和自身免疫疾病5。异构体的免疫器官和细胞中的困难进行了结构分析作为确定多种生物学功能的障碍。 在这里，我们提出了一个可视化的版本的方法比较简单，快速，灵敏的分析免疫细胞糖脂配置文件中7-9。这种方法是基于对提取和化学改性（permethylation，请参见下面的图5A，所有的OH基的己糖被替换后的MeO反应permethylation）的鞘糖脂10-15，然后通过随后的分析，使用基质辅助激光解吸/电离飞行时间质谱 - 飞行质谱（MALDI-TOF/MS）和离子阱质谱的。这种方法需要50万的免疫细胞为一个完整的分析。在一个星期内就可以完成的实验。可以划定的相对丰度的各种糖脂合成标准进行比较。此方法具有的灵敏度的测量之间GB3异构体的1％iGb3，当2总iGb3/Gb3混合物的fmol的是本9。 可以使用离子阱质谱法分析的异构体。例如，分析globotriaosylceramide和isoglobtriaosylceramide的存在下，在同一样品中，一个可以使用的碎片鞘糖脂分 ​​子在结构上区分的两个（见下面的图5）。此外，化学改性的糖部分（通过permethylation反应）提高更高的灵敏度和特异性的电离和分散的效率，并且增加的唾液酸残基的稳定性。可以执行在一个典型的认证化学罩的提取和化学修饰的鞘糖脂，并且可以由核心进行质谱离子阱MS仪器与设施。

自大鼠白血病细胞线RBL（NKT细胞的鞘糖脂抗原的抗原呈递细胞类型）的鞘糖脂的MALDI MS分析的一个例子如图3所示。的离子可以被分配到特定的鞘糖脂结构（ 图3A）。由NB-DGJ 16，一种药物，抑制鞘糖脂的合成，观察到显着减少的所有鞘糖脂（ 图3B）在RBL细胞。不能区分的结构异构体。 Applied Biosystems公司4700的MS / MS的能力允许MS1离子到MS 2片段的碎片，使有限的结构信息来生成。然而，MS2分析往往是不足够的歧视寡糖异构体。 离子陷阱鞘糖脂使MS分析研究的异构体，如iGb3（Galα3Galβ4Glcβ1Cer）和GB3（Galα4Galβ4Glcβ1Cer）可能存在作为的trihexosylceramide离子检测到在图3A。为了检测这样的异构体，标准iGb3和GB3分析了由多个回合的碎片，直到发现差异（ 图4A和图4B）。在这里给出的例子中（ 图5C）iGb3 GB3可以通过比较标准iGb3和GB3判别。 <img alt="图1" src="/files/ftp_upload/4224/4224fig1.jpg" /> 图1。鞘糖脂提取程序的流程图，从细胞中提取的有机溶剂和化学改性的鞘糖脂（permethylation）鞘糖脂。要删除非糖脂脂质过酰化和脱乙酰化。其次，鞘糖脂化学改性由permethylation反应，从而提高了的灵敏度和特异性的MS detectioN。最后，糖脂配置文件使用MALDI-TOF质谱和离子阱质谱测定。 <img alt="图2" src="/files/ftp_upload/4224/4224fig2.jpg" /> 图2。鞘糖脂由地衣酚硫酸法的量化 ：量化使用0.2％的地衣酚在50％的硫酸溶液和葡萄糖标准曲线的鞘糖脂。 <img alt="图3" fo:content-width="4.75in" fo:src="/files/ftp_upload/4224/4224fig3highres.jpg" src="/files/ftp_upload/4224/4224fig3.jpg" /> 图3。大鼠白血病细胞。glycosphingolipidomics A.写照MALDI质谱，每个离子表示一个特定的鞘糖脂结构，结构同分异构体不能区分。B. NB-DGJ，一种药物，抑制鞘糖脂合成，S;ignificantly降低糖脂在RBL细胞产生。 <img alt="图4" fo:content-width="4in" fo:src="/files/ftp_upload/4224/4224fig4highres.jpg" src="/files/ftp_upload/4224/4224fig4.jpg" /> 图4。串联质谱异构体的鞘糖脂。 A.正离子模式离子阱质谱的全甲基的GSLS千兆3 CER和igb 3 CER的特征分解途径。 GB 3和igb 3清楚地分开他们的分裂模式。联动的差异反映在MS 4产品一致的同量异序的m / z 445的前体离子。 乙 。代表性的结果示出iGB3可以测量的iGb3/Gb3异构体的混合物中。星号表示诊断离子只为iGb3。 <a href="http://www.jove.com/files/ftp_upload/4224/4224fig4large.jpg" target="_blank">点击这里</a>查看大图。 <img alt="图5" fo:content-width="6in" fo:src="/files/ftp_upload/4224/4224fig5highres.jpg" src="/files/ftp_upload/4224/4224fig5.jpg" /> 图5。离子阱质谱允许糖脂同 ​​分异构体的分析。A.标准iGb3和GB3显示不同的碎片轮廓经过4轮的碎片在离子阱LTQ质谱仪。B.特性MS 4个人来自iGb3或GB3。C.离子Trihexosyleramides的iGb3和GB3异构体的混合物，可以通过离子阱质谱法（图大鹏周） <a href="http://www.jove.com/files/ftp_upload/4224/4224fig5large.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Mass Spectrometric Analysis of Glycosphingolipid Antigens at JoVE.com

Mass Spectrometric Analysis of Glycosphingolipid Antigens

一个特异和敏感的方法来深入了解糖脂抗原的免疫器官和细胞的表达谱。该方法利用离子阱质谱可以逐步碎裂的糖脂分子结构分析，比较，化学​​合成的标准。

鞘糖脂抗原的质谱分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

mass-spectrometric-analysis-of-glycosphingolipid-antigens

Research

JoVE Journal

Immunology and Infection

16.4K 次观看.  Rice University. 一个特异和敏感的方法来深入了解糖脂抗原的免疫器官和细胞的表达谱。该方法利用离子阱质谱可以逐步碎裂的糖脂分子结构分析，比较，化学​​合成的标准。

视频: Mass Spectrometric Analysis of Glycosphingolipid Antigens

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

纯化膜隔间跨演示文稿中的外源抗原内质网关联降解

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

非遗传表面修饰的素-生物素系统在牛分枝杆菌卡介苗中的外源抗原表达

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

作为酶和抗原的非重组显示系统的孢子吸附

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

链球菌中高分子质量蛋白的纯化

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...