援助的实验室方法的发展过程中所提供的斯科特·特里斯，凯特琳阿尔特，杰里米Ragsdell，托尼·拉马克詹姆斯，艾米菲舍尔，汉娜·布莱尔，和夏延杰兹。我们要感谢麦地那 - 玻利瓦尔实验室（路易斯·H.老圃奥拉沙宝;阿肯色州生物科学研究所），用于与薄层扫描密度援助。阿肯色州资产倡议P3中心（EPS-0701890）在阿肯色州生物科学研究所：MALDI-TOF MS用于该项目通过了NSF州EPSCoR，RII提供了依据。经费是由美国渔业和野生动物服务/阿肯色州野生动物格兰特，全国的洞穴协会和中心的北美蝙蝠保育研究在美国印第安纳州立大学提供。

注意：事先取得所有必要的州和联邦许可证装卸，运输，贮存和蝙蝠。认证也必须从你的机构的动物护理和使用委员会，以及从你的机构生物安全委员会获得的。如果要处理现场蝙蝠（或神经系统组织）是，动物管理人员应接种狂犬病。在目前的研究中使用蝙蝠是从奥索卡圣弗朗西斯国家森林公园，阿肯色州，按标准方法（阿肯色州州立大学的生物安全委员会批准＃135349-1）28收集2010年夏季期间。 1。组织处理和提取脂质<ol><li>之前和来自不同个体的组织收集的清洁与甲醇的所有仪器。修剪头发（约1.0克）从皮肤用剪刀和地点在125毫升锥形瓶中。从机翼表面的皮脂样擦洗皮肤，4 - 湿用氯仿6棉球：甲醇溶剂（C：M; 3点02体积/体积），并把这些在单独的烧瓶中。 </li><li>提取组织用10毫升的C：M（2:1体积/体积）含有0.5％的丁基化羟基甲苯（BHT）以防止氧化29。只用高效液相色谱质量溶剂。 </li><li> 2小时后，加入约0.5g无水硫酸钠到每个烧瓶中，简要地混合，并通过滤纸过滤收集溶剂。 </li><li>重复步骤1.2和1.3的两倍。一旦与1:1 C：M和顺序1:2 C：M。池滤液在一起。 </li><li>蒸发下的含有N 2的流汇集的滤液，测定干重，并溶解在3点02分C中的脂类残留物：M（含0.5％BHT），以10毫克/毫升的浓度。在玻璃小瓶在-20℃储存样品它通常是最好的样品收集后分析的一个月内的样本，并尽量减少冻融循环。 </li></ol> 2。脂质通过分离制备薄层色谱法<ol><li>提前准备溶剂洗1.5毫升microcentrifuge管填充用3:2 C：M，用丙酮漂洗，风干。这样做是为了除去增塑剂，可与以后质谱分析干涉。储存样品管在无尘的容器和处理这些管子只能用手套，以防止污染皮肤油脂。 </li><li> M.：先预开发它与3:2Ç激活薄层板上加入足够的溶剂中，在TLC室以1厘米的深度，然后将板在所述腔室（接近玻璃盖），并允许溶剂完全运行到盘的顶部。这大约需要45分钟。 </li><li>取出板并干燥在通风橱中，直到溶剂蒸发（约15分钟），然后在烘箱中放置至少10分钟，在120℃下放置一个铅笔标记在板的顶部，以保持定位时应用的样品。放置一个直铅笔线，从板的底部边缘1.5厘米，以记其中样品和标准将被放置在基线。 </li><li>通过切割准备TLC室一片滤纸足够大，以排列在两个短壁和一个长壁。放置滤纸衬入腔室。它会充分湿润时的溶剂加入到腔室中。 </li><li>制备100毫升流动相溶剂，这是己烷：乙醚：乙酸（H：E：A; 80:20:2 V / V / V）。倾溶剂进入腔室以得到约1厘米的深度。盖上玻璃盖，用硅脂密封沿着所述腔室的顶部边缘。允许室在使用前过夜平衡。 </li><li>用毛细管吸液管或作为连续的条纹从一个边长约1.5厘米至1.5厘米至另一边缘手动应用样品制备的板。一种自动化的样品施加器是优选的，因为它会加载样品中的更均匀的条纹。使用薄层板的外侧车道被发现甾醇约20微升的混合物，游离脂肪酸，标签和甾醇酯标准（使用10毫克/毫升;预制的混合物可以得到）。 </li><li>将加载薄层板上成在平衡室中，关闭的盖子，并开发板直至溶剂运行到上边缘。这需要大约45分钟，这里所描述的流动相。从腔室中取出板，并允许剩余的溶剂，以从该板在通风橱中蒸发约1分钟。 </li><li>用0.05％若丹明6G的95％乙醇喷雾。想象下一个长波紫外线灯脂带。马克用铅笔样品和标准在解决荧光波段的R F位置。照片记录，可采取在这一点上。 </li><li>识别对应的TAG标准的频带，通过用刮刀刮二氧化硅断到一个大型一张玻璃称量纸的从板中取出。转移硅石到1.5ml溶剂洗涤离心管中。 </li><li>加1.0毫升3:2Ç：M溶剂到样品管中，超声处理1分钟，沉淀二氧化硅通过离心，然后将溶剂转移到一个新的预先称重的试管中。重复次ë前一步骤，汇集的滤液，并减压蒸除溶剂下的氮气流。 </li><li>存储包含在黑暗中在4℃下在N 2下的TAG而附加的组织样品通过TLC分离干燥的残留物。若丹明6G的存在的样品，但它不溶于己烷中，并在紧接在MS分析之前样品的溶解除去。 </li></ol> 3。通过MALDI-TOF质谱分析TAG <ol><li>通过在1ml溶剂（49.5％乙醇，49.5％乙腈和1％的0.1％TFA水溶液）10mg溶解CHCA制备新鲜的α-氰基-4 - 羟基肉桂酸（CHCA）基质溶液。 </li><li>通过将1μl的促肾上腺皮质激素（1毫克/毫升）与39.5微升的0.1％三氟乙酸（TFA），得到10为pmol /μl仪器的分辨率和灵敏度测试;准备adrenocarticotropic激素（18-39片段，2465.1989沓ACTH）原液。这可以被存储在-20℃下以供将来使用。 </li><li>准备一个新鲜的工作促肾上腺皮质激素SOLUT离子（1皮摩尔/微升），通过取1微升的10皮摩尔/微升原液，用9微升0.1％TFA混合，然后混合（1:1）与10微升的CHCA基质溶液，得到500 fmol的/微升的浓度。 </li><li>准备TAG标准为10毫克/毫升3:2 C：M表示MALDI仪器校准（ 如 tricaprin [470.361达]，三辛酸甘油酯[554.455达]，trilaurin [638.549达]，TRIPALMITIN [722.642达]，tripalmitolein [800.689达] ，甘油三肉豆蔻酸酯[806.736沓]，三油酸甘油酯[884.783沓]，三-11-eicosenoin [968.877沓]和trierucin [1052.971沓]）。 </li><li>制备三油酸甘油酯在10毫克/毫升的3:2的C：M表示的外部锁定质量校准TAG标准。 </li><li>溶解在己烷中的所存储的标签样品至10毫克/毫升的溶液。 </li><li>制备一个0.5米（77.06 mg/1.0毫升）的2,5 - 二羟基苯甲酸（DHB）用90％甲醇用作样品和标准矩阵原液。还制备的NaOH的1.0M（2.0 g/50.0毫升）溶液中。使用铝政府信息公开覆盖管DHB解决方案<html>升避光。 </li><li>混合（预洗管）10.0微升DHB基质，10.0微升样品或标准，和5.0微升1.0 M氢氧化钠。混合并短暂离心​​管中，使混合物的底部。 </li><li>现货1.0微升的标准，样品或促肾上腺皮质激素到MALDI不锈钢靶板并将其放置在干燥器中干燥为止。将目标板放入仪器进行数据采集。 </li><li>进行MALDI-TOF MS分析，在正反射器模式。调整和校准与使用促肾上腺皮质激素和TAG标准溶液由仪器制造商所描述的仪器。 </li><li>获得光谱发现在目标板上每个样品（含仪器规格一致;对这项工作参数为5赫兹激光射速，每〜100点拍摄获得的平均光谱）。平滑和减去电离光谱背景后，手动流程离子峰的在线搜索引擎LIPID MAPS（/工具/ MS / glycerolipids_batch“目标=”_blank“&gt; www.lipidmaps.org /工具/ MS / glycerolipids_batch）。 </li><li>复制并粘贴清单谱成的母离子和强度框的列表。限制搜索到所需要的酰基构成。由质量/电荷（M / Z）从离子存在于光谱对每个样品比识别标记。如果脂肪酸甲酯（FAME）的百分数都是可从单独的GC / MS分析，将这些数据以获得当前的TAG的概率。 </li></ol>仪器：在本研究中所用的质谱仪是在Waters MALDI微MX（配有337 nm的20赫兹氮气激光器）。任何制造商的MALDI仪器具有正反射器模式的能力可以被使用。使用一般设置为脉冲电压，2,000 V，反射器，5,200 V;源，15,000 V，使用MassLynx软件（版本4.0）的数据采集。这些运行条件下提供质量分辨率大于12,000。

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作者宣称没有竞争的财务权益。

本文提出了分离分离哺乳动物珠被用制备TLC广泛的脂质类和MALDI-TOF MS测定TAG配置文件的简单而可靠的方法，而不脂质分子耗时衍生。生产标记与MALDI-TOF MS的谱图质量的关键步骤包括：1）成功提取以最小的污染或氧化的化合物; 2）足够的分离和隔离用色谱法，以及通过MALDI-TOF 3）高分辨率和质量精度MS。 本文从东方红蝙蝠的plagiopatagium提取分离中性脂质成分得到TAG MS配置文件说明了方法。虽然本研究采用了蝙蝠（哺乳纲：翼手），这些方法可以扩展到任何研究哺乳动物的外皮血脂。蝙蝠外皮的特征在于主要是胆固醇，用较少量的标记，游离脂肪酸，鳞状溶解乙炔和甾醇/蜡酯。在蝙蝠皮脂脂质比率从人类中角鲨烯的不同是存在于少量（相对于在人类中高达16％），而胆固醇发生在较大的比例（1 -在人类中的7％，但26 -蝙蝠62％）22 ，31。人的头发含有约3％的标签，而比例高达28％被发现在东方红蝙蝠的头发。脂质样品从蝙蝠的提取是相似的其他物种。而在本研究中浸泡在溶剂棉球用于去除皮脂，1也可以颠倒含有溶剂的小瓶或试样管上的珠被多次的表面上。专用胶带产品还提供其他方法来提取表面脂类32。适当的脂质提取的一个关键部分是从皮肤出油减少污染。这是很容易保持挤压瓶用甲醇和喷涂所有玻璃器皿和餐具，擦餐具与所有样品和戴手套检查之间的组织来完成。氧化Øf多不饱和酰基，防止通过加入BHT和它应该不管样本被存储在该温度下的使用。 生物分子分析通常需要一个色谱步骤，感兴趣的污染物分离分子。 TLC是用在此方法中，避免了对气体或液体色谱仪的要求和需要较少的技术经验来实现健壮的和可重复的结果。取决于感兴趣的脂质类，许多不同的流动相可被并入。此外，使用HPTLC板和扫描光密度的可用于实现定量结果。虽然薄层色谱方法的变化更是不胜枚举，在这里列出，在脂质薄层色谱中常用的一些流动相：氯仿：甲醇：水为磷脂和糖脂或异辛烷的分离：乙醚的非极性脂类28分。在分离等类脂质，其H TAG的方面：E：A这样lvent系统都能正常工作，并提供可比较的结果。 采用薄层色谱法的另一个优点是所关注频带可以用MALDI-TOF MS没有被分析物的衍生化之前被快速成型。在这项研究中，二氧化硅被从TLC板上除去，且分析物从它在一种溶剂和随后的离心分离，洗脱溶剂中的吸附剂和蒸发洗脱通过超声处理。或者，基质可以直接施加到分离的TLC板，然后直接通过MALDI-TOF MS分析33的分析物条带。分析成功通过MALDI-TOF MS有依靠足够的样品制备和操作者的熟练程度与调谐和校准仪器。仪器应每天用覆盖分子量范围适用于感兴趣的分子的标准进行校准。该设备的合适的灵敏度和分辨率（ 例如 ≥10,000）也应confirme每天用ACTHÐ。 哺乳动物的体壁表面上发现的皮脂腺脂质成分可能是由细菌/真菌病原体的定植起到一定的作用。因此，物种和个体间的化学成分的知识可以提供关于人类和野生动物的疾病过程的线索。患病和健康人之间的种内差异可能代表临床症状，援助在疾病检测和诊断。此外，如果能抑制微生物生长的特定化合物存在时，它们可以被识别为在疾病的治疗和预防用途。

哺乳动物的外皮组织包括表皮，角质层结构（ 例如头发和指甲）和外分泌腺。皮脂腺型外分泌腺与毛囊，它们统称为毛囊皮脂腺单元1相关联。皮脂腺释放油状渗出到被称为皮脂在皮肤表面。皮脂主要由甘油脂（ 如三酰甘油酯[标签]），游离脂肪酸酰基（远期运费协议），甾醇/蜡酯和角鲨烯。皮脂的化学成分是种属特异性2。除了 ​​是先天免疫系统的一部分，并提供抗菌功能3，皮脂腺的油脂会影响重要的生理过程，包括蒸发失水通过皮肤4，细胞的完整性和基因调控5，和药物的吸收6。皮脂腺脂质组合物还可以作为疾病标志物。改变比例和皮脂腺的B值道路脂质类的疾病，例如痤疮7，头皮屑8，脂溢性皮炎8，皮脂缺乏症9，其中包括10个临床体征。表皮和毛发组织包括含有甾醇及其衍生物，标记，游离脂肪酸，神经酰胺，磷脂，和其他小脂质成分变量配置文件。因为外皮脂类可以在疾病过程中发挥作用，决定在健康和患病个体之间的TAG的化学成分差异可能是疾病的临床诊断。 脂质一般定义为不溶于水的有机化合物与任何非极性或极性-极性取代基11。脂质结构可以是长链烃，含氧烷烃（包括蜡酯，游离脂肪酸，醇类，酮类和醛类），或复杂的环状结构，例如胆固醇12。有脂质基于结构八大类别（远期运费协议，glycerolipiDS [GL]，甘油[GP]，鞘脂[SP]，甾醇脂类[ST]，异戊烯醇脂[PR]，糖脂[SL]，和聚酮[PK]），其中展出范围广泛的化学性质取决于类13。由于脂类的化学性质变化范围很宽，直接仿形未经脂质分子前衍生化是需要的。在脂质研究一种新兴的方法是薄层色谱（TLC）结合飞行时间的基质辅助激光解吸/电离质谱（MALDI-TOF MS）14。 MALDI-TOF MS广泛用于蛋白质组研究，以确定蛋白质和它们的特定的氨基酸序列由于从胰蛋白酶消化的蛋白15产生的高度精确的肽离子的质量“指纹”相关联。 MALDI-TOF MS也可用于分析其他生物分子类，包括脂类如变量16-18。 MALDI需要使用一个矩阵，典型值售货机，ically包含芳族和共轭双键结构的有机化合物。基质分子起到激光的能量轻轻转移到分析物，促进质子转移，并产生单电荷的气相离子19-21。离子经受高压电场在高真空下和加速进入一个TOF质谱分析仪，其中离子随后通过在速度正比于它们的质荷比的差异分离。即使是非常大的生物分子可以与小碎片被电离，产生单电荷的分子离子物种简化频谱分析。直接分析脂质分子，恕不另行衍生的能力，促进了准备采用MALDI-TOF MS的脂质组研究18。 本文提出了一种常规方法，从头发分离和分析外皮脂质，皮脂分泌物和东方红蝙蝠plagiopatagium（Lasiurus BOREALIS）。这是用来确定在蝙蝠外皮间血脂变化，以阐明白鼻子综合症（WNS）22疾病的进程。 WNS是蝙蝠的甲真菌病，是由新近被描述的嗜冷物种Geomyces destructans 23-25。 WNS已造成超过500万的北美洞穴 ​​蝙蝠的死亡并威胁脆弱的蝙蝠物种的灭绝，随着数十亿美元的损失，以农业产业26,27潜在的经济影响。为了研究在G的步骤destructans感染，脂质，从东方红蝙蝠的头发和机翼组织中提取并分离为广义的类脂质薄层色谱分离的TAG部分以供后续分析通过MALDI-TOF MS分析。标记包含短酰基链，很容易通过MALDI-TOF质谱检测很少基质干扰。

<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>Butylated Hydroxytoluene</td>
			<td>MP Biomedicals, LLC</td>
			<td>101162</td>
			<td><a href="http://www.mpbio.com" target="_blank">www.mpbio.com</a></td>
		</tr>
		<tr>
			<td>TLC Flexible Plates</td>
			<td>Whatman</td>
			<td>4410-222</td>
			<td><a href="http://www.whatman.com" target="_blank">www.whatman.com</a></td>
		</tr>
		<tr>
			<td>ACTH</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>A8346-5X1VL</td>
			<td><a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a></td>
		</tr>
		<tr>
			<td>Triolein</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>44895-U</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC Lipid Standard</td>
			<td></td>
			<td>TLC 18-1</td>
			<td><a href="http://www.nu-chekprep.com" target="_blank">www.nu-chekprep.com</a></td>
		</tr>
		<tr>
			<td>MALDI TAG Standard</td>
			<td>Nu-Check Prep., Inc.</td>
			<td>NIH Code 53B</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI TAG Standard</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>17810-1AMP-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Vials with Teflon Cap</td>
			<td>U.S. National Scientific Co.</td>
			<td>B7800-2</td>
			<td><a href="http://www.nationalscientific.com/" target="_blank">www.nationalscientific.com/</a></td>
		</tr>
		<tr>
			<td>DHB</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>50862-1G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>CHCA</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>C8982-10X10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sebutape</td>
			<td>CuDerm</td>
			<td>S100</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling the triacylglyceride contents in bat integumentary lipids by preparative thin layer chromatography and maldi-tof mass spectrometry

哺乳动物的体壁包括分泌的油性材料涂敷到皮肤表面皮脂腺。皮脂分泌是先天免疫系统，它是保护性对抗病原微生物的一部分。不正常的皮脂分泌和化学成分也是特定的皮肤疾病的临床症状。皮脂包含脂类，包括三酰基甘油酯，它是物种特异性的复杂混合物。通过多样化的脂质类的展出广泛的化学性质阻碍了具体确定的皮脂成分。分析技术的脂类通常需要化学衍生化是劳动密集和提高样品制备成本。本文介绍了如何从哺乳动物的体壁，独立广阔的类脂质薄层色谱法提取油脂，并用飞行时间的基质辅助激光解吸/离子化质谱分析的三酸甘油酯含量。这一功能强大的方法使直接测定的物种和个体间的三酰基甘油酯的档案，并且它可以很容易地应用到哺乳动物的任何分类群组。

提取方法从Folch 30所述组织分离总脂质是一个简单的过程，它是在这里适应。组织萃取和溶剂蒸发后，将脂质通常显示为浅黄色薄膜。黄色的颜色最有可能是由蛋白质污染物，这可以通过执行一个液 - 液萃取除去。这个额外的样品处理是没有必要在此过程中，因为制备TLC分离这些杂质的TAG级分。在所有过滤阶段加入新鲜的无水硫酸钠有助于减少水质污染，影响到准确的脂肪重量测定。 哺乳动物的皮肤衍生物的脂质经制备薄层色谱分析用H：E：A为流动相，通常能够解决相应的（从原点开始）甾醇，游离脂肪酸，标签和甾醇酯/蜡酯/角鲨烯（ 图1）四个不同的频段。有时使用analyti时卡尔高性能（HP）TLC用H：E：A流动相，甾醇酯，蜡酯，角鲨烯和将分离并显示为三个独立的频带。下，在本研究中使用的条件下，甾醇/蜡状酯不会分开。如果这些频带是感兴趣的，流动相可以切换到异辛烷：乙醚（95:5 V / V），和HPTLC板上可以通过扫描光密度测定来分析。其他因素可能会导致分离度不佳。这些通常是通过将滤纸在室内，应用油脂的盖子密封，平衡室过夜，并保持清洁薄层色谱室，以获得稳定的质量薄层色谱法分离和数据消除。 对于标签从东方红蝙蝠中分离获得的代表MALDI-TOF质谱谱图见图2和3。这些光谱包含TAG离子峰M / Z 850之间的质量范围- 910，这是典型的标签从非水生哺乳动物中分离秒。在加入1.0M的NaOH促进单电荷的Na +离子是比H +离子更稳定。除了 ​​稳定性，不存在H +和K +离子的增加容易进行频谱分析。在M / Z 850 - 910离子峰对应16:0 18:0 18:1和18时02分足总杯部分是在标记中占主导地位的酰基成分（ 表1）。个变量的可能的FA部分可以通过本总的MALDI-TOF MS谱离子 m / z，并推导出物种和个体间的差异来初步确定的。然而，如果酰基含量的特定比例是必需的，那么MS / MS或气相色谱（GC / MS）必须被使用。上的酰基的比例进一步的信息可以通过观察在光谱的二酰基甘油的区域（ 图4）的峰来推导。二酰基甘油是从MALDI源标记碎片产生，并且可以在M / Z 590上找到- 650区域。 TAG碎片可以增加省略了加入1.0M的NaOH 16。在m / z 879.7和毛发组织与在m / z 881.8（ 图2和图3分别为）一个主峰东方红蝙蝠翼组织的特点是主峰。峰在m / z 907.8，879.7，和855.7（POP，优先提供者）是约即使在强度（〜50％）的头发组织中的峰值在853.7感〜40％。 <table border="1"><tbody><tr><td fo:width="2in"> 组成 </td><td fo:width="1in"> 元素组成 </td><td fo:width="1in"> 观察到的质量 </td></tr><tr><td> 的Na +标记 </td><td></td><td> 的Na +标记 </td></tr><tr><td> SSO </td><td> Ç57ħ108 O 6 </td><td> 911.8 </td></tr><tr><td> OOS，LSS </td><td> Ç57ħ106 O 6 </td><td> 909.8 </td></tr><tr><td> OOO，LNS时，LSO </td><td> Ç57ħ104外层6 </td><td> 907.8 </td></tr><tr><td> LOO，LLS </td><td> Ç57ħ102 O 6 </td><td> 905.8 </td></tr><tr><td> LLO，OOLn </td><td> Ç57小时100 O 6 </td><td> 903.7 </td></tr><tr><td> LLL </td><td> Ç57 H 98 O 6 </td><td> 901.7 </td></tr><tr><td> LLLn </td><td> Ç57高96 O 6 </td><td> 899.7 </td></tr><tr><td> LLnLn </td><td> Ç57ħ94 O 6 </td><td> 897.7 </td></tr><tr><td> LnLnLn </td><td> Ç57ħ92 O 6 </td><td> 895.7 </td></tr><tr><td> OSP </td><td> Ç55 H 104 O 6 </td><td> 883.8 </td></tr><tr><td> LSP的，面向对象的，索普</td><td> Ç55 H 102 O 6 </td><td> 881.8 </td></tr><tr><td> LOP，LnSP，LSPo </td><td> C 55小时100 O 6 &lt;/ TD&gt; <td> 879.7 </td></tr><tr><td>律师事务所，LNOP，洛波</td><td> C 55 H 98 O 6 </td><td> 877.7 </td></tr><tr><td> LnLP，LLPo，LnOPo </td><td> C 55高96 O 6 </td><td> 875.7 </td></tr><tr><td> LnLnP，LnLPo </td><td> Ç55 H 94 O 6 </td><td> 873.7 </td></tr><tr><td> LnLnPo </td><td> Ç55 H 92 O 6 </td><td> 871.7 </td></tr><tr><td> PPS </td><td> Ç53ħ102 O 6 </td><td> 857.8 </td></tr><tr><td> POP，PPOS </td><td> Ç53小时100 O 6 </td><td> 855.7 </td></tr><tr><td> OOM，PPL，PoPoS，POPO </td><td> Ç53 H 98 O 6 </td><td> 853.7 </td></tr><tr><td> PPLN，PPOL，PoPoO，MyOO </td><td> Ç53高96 O 6 </td><td> 851.7 </td></tr><tr><td>法学硕士，LnOM </td><td> Ç53ħ94 </sUB&gt; O 6 </td><td> 849.7 </td></tr><tr><td> PPP，SSLA </td><td> Ç51 H 98 O 6 </td><td> 829.7 </td></tr><tr><td> PPPO，人员法律援助办公室</td><td> Ç51高96 O 6 </td><td> 827.7 </td></tr><tr><td> PPoPo，PMyO </td><td> Ç51ħ94 O 6 </td><td> 825.7 </td></tr><tr><td> LnLnLa </td><td> Ç51ħ86 O 6 </td><td> 817.6 </td></tr><tr><td>彩信，巴掌，PPM </td><td> Ç49ħ94 O 6 </td><td> 801.7 </td></tr><tr><td> SLaPo，PPOM，PPMy </td><td> Ç49ħ92 O 6 </td><td> 799.7 </td></tr><tr><td> PoPoM，OOCa </td><td> Ç49 H 90 O 6 </td><td> 797.7 </td></tr><tr><td>基质金属蛋白酶</td><td> Ç47 H 90 O 6 </td><td> 773.7 </td></tr><tr><td> MMPo，OCAP </td><td> Ç47ħ88Ø6 </td><td> 771.7 </td></tr><tr><td> OCaPo </td><td> Ç47ħ86 O 6 </td><td> 769.6 </td></tr><tr><td> MMM，PPCA，PMLA </td><td> Ç45 H 86 O 6 </td><td> 745.6 </td></tr><tr><td> PoPCa，PoMLa </td><td> Ç45 H 84 O 6 </td><td> 743.6 </td></tr><tr><td> PoPoCa </td><td> Ç45 H 82 O 6 </td><td> 741.6 </td></tr><tr><td> LaLaP，MMLa，MCAP </td><td> Ç43 H 82 O 6 </td><td> 717.6 </td></tr><tr><td> LaLaPo </td><td> C 43 H 80 O 6 </td><td> 715.6 </td></tr><tr><td>面向对象</td><td> 39 H 72 O 5 </td><td> 643.5 </td></tr><tr><td> OL </td><td> C 39 H 70 O 5 </td><td> 641.5 </td></tr><tr><td> LL </td><td> 39 H 68 O 5 </td><td> 639.5 </td></tr><tr><td> SP </td><td> 37 H 72 O 5 </td><td> 619.5 </td></tr><tr><td> OP </td><td> 37 H 70 O 5 </td><td> 617.5 </td></tr><tr><td>唱片</td><td> 37 H 68 O 5 </td><td> 615.5 </td></tr></tbody></table> 表1中。脂肪酸组成，元素组成，并且三酰基甘油酯和二酰基甘油的sodiated加合物的同位素质量。LN =亚麻酸（18:3）中，L =亚油酸（18:2），O =油酸（18:1），S =硬脂酸（18:0），P =棕榈酸（16:0）中，PO =棕榈油酸（16:1）中，M =肉豆蔻酸（十四时），我=肉豆蔻脑酸（14:1）的La =月桂酸酸（12时00分），CA =癸酸（10点）。 <img alt="图1" src="/files/ftp_upload/50757/50757fig1.jpg" /> 图1。乙醚：醋酸广泛的脂质类分离己烷薄层色谱（80:20:2 V / V / V）作为流动相。固醇和FFA之间的频带不是由标准确定的，也可以是脂肪醇或蜡酯。 <img alt="图2" src="/files/ftp_upload/50757/50757fig2.jpg" /> 图2。 sodiated的TAG的MALDI-TOF质谱的扩展TAG地区-从东方红蝙蝠（L.北极光 ）翼组织（M / Z 700 950）确定在m / z 853.7（OOM，PPL，PoPoS，POPO） 和 m峰/ Z 879.7（LOP，LnSP，LSPo） <a href="https://www.jove.com/files/ftp_upload/50757/50757fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/50757/50757fig3.jpg" /> 图3。从东方红蝙蝠（L.北极光 ）毛发组织- sodiated标记（950 M / Z 700）的MALDI-TOF质谱的扩展TAG区域。峰确定为m / z 905.8（LOO，LLS） 和 m / z 907.8（000，LNS时，LSO）。 <a href="https://www.jove.com/files/ftp_upload/50757/50757fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/50757/50757fig4.jpg" /> 图4。 sodiated DAG片段的MALDI-TOF质谱（M / Z 530 - 730）的DAG区域。从东方红蝙蝠（L.北极光 ）翼组织认定为m / z 643.5（OO） 和 m / z 615.5（LP）的峰。 <a href="https://www.jove.com/files/ftp_upload/50757/50757fig4large.jpg" target="_blank">点击这里查看大图</a> 。

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Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

哺乳动物的外皮含有溶剂萃取的油脂，可以提供单个物种的化学成分特征。本文提出了分离外皮组织中分离出广阔的类脂质采用薄层色谱法，测定甘油三酯轮廓由飞行时间的基质辅助激光解吸/离子化质谱常规方法。

用制备薄层色谱和MALDI-TOF质谱分析的三酸甘油酯含量的蝙蝠外皮血脂

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

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21.1K 次观看.  Arkansas State University. 哺乳动物的外皮含有溶剂萃取的油脂，可以提供单个物种的化学成分特征。本文提出了分离外皮组织中分离出广阔的类脂质采用薄层色谱法，测定甘油三酯轮廓由飞行时间的基质辅助激光解吸/离子化质谱常规方法。 

视频: Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF 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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

基质辅助飞行的完整蛋白质（MALDI-TOF）质谱分析激光解吸/电离飞行时间大于100 kDa的

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

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 ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

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 ...

The Effect of Anodization Parameters on the Aluminum Oxide Dielectric Layer of Thin-Film Transistors

Aluminum-oxide (Al2O3) is a low cost, easily processable and high dielectric constant insulating material that is particularly appropriate for use as the dielectric layer of thin-film transistors (TFTs). Growth of aluminum-oxide layers from anodization of metallic aluminum films is greatly advantageous when compared to sophisticated processes such as atomic layer deposition (ALD) or deposition methods that demand relatively high temperatures (above 300 &#176;C) such as aqueous combustion or spray-pyrolysis. However, the electrical properties of the transistors are highly dependent on the presence of defects and localized states at the semiconductor/dielectric interface, which are strongly affected by the manufacturing parameters of the anodized dielectric layer. To determine how several fabrication parameters influence the device performance without performing all possible combination of factors, we used a reduced factorial analysis based on a Plackett-Burman design of experiments (DOE). The choice of this DOE permits the use of only 12 experimental runs of combinations of factors (instead of all 256 possibilities) to obtain the optimized device performance. The ranking of the factors by the effect on device responses such as the TFT mobility is possible by applying analysis of variance (ANOVA) to the obtained results.

Anodization parameters for growth of the aluminum-oxide dielectric layer of zinc-oxide thin-film transistors (TFTs) are varied to determine the effects on the electrical parameter responses. Analysis of variance (ANOVA) is applied to a Plackett-Burman design of experiments (DOE) to determine the manufacturing conditions that result in optimized device performance.

阳极化参数对薄膜晶体管氧化铝介电层的影响

Aluminum-oxide (Al2O3) is a low cost, easily processable and high dielectric constant insulating material that is particularly appropriate for use as the ...

Fabrication of Thin Film Silver/Silver Chloride Electrodes with Finely Controlled Single Layer Silver Chloride

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin film silver electrodes. Thin film silver electrodes sized 80 &#181;m x 80 &#181;m and 160 &#181;m x 160 &#181;m were sputtered on quartz wafers with a chromium/gold (Cr/Au) layer for adhesion. After passivation, polishing and cathodic cleaning processes, the electrodes underwent galvanostatic oxidation with consideration of Faraday&#39;s Law of Electrolysis to form smooth layers of AgCl with a designated degree of coverage on top of the silver electrode. This protocol is validated by inspection of scanning electron microscope (SEM) images of the surface of the fabricated Ag/AgCl thin film electrodes, which highlights the functionality and performance of the protocol. Sub-optimally fabricated electrodes are fabricated as well for comparison. This protocol can be widely used to fabricate Ag/AgCl electrodes with specific impedance requirements (e.g., probing electrodes for impedance sensing applications like impedance flow cytometry and interdigitated electrode arrays).

This paper aims to present a method to form smooth and well-controlled films of silver chloride (AgCl) with designated coverage on top of thin film silver electrodes.

薄膜银/银氯化银电极的制造，具有精细控制的单层氯化银

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin ...