作者要感谢马萨纳罗梅乌 - 纳达尔在大鼠研究的贡献。

1。动物规程<ol><li>所有的动物工作应按照内政部动物（科学程序）法（1986）进行。 </li><li>交配10周龄以一夫一妻制繁殖旧Wistar大鼠，并通过阴道塞的外观确认怀孕。记录这是怀孕的第1天，并开始试验性的饮食。对于处女女，分别容纳各组大鼠，并开始试验性的饮食。 </li><li>喂养试验饲料20天安乐死后大鼠用CO 2窒息颈椎脱位。 </li><li>用解剖镊子和剪刀以暴露腹腔，并通过切割韧带，其中肝脏连接至该膜片，腹部的前壁，胃和十二指肠切除肝。存放于-80°C。前PBS洗肝，冷冻在液氮中</li></ol> 2。提取的总脂的制备9 <ol><li>添加moleculAR筛（填写溶剂容器的1/10）所有溶剂打造&#39;干&#39;的溶剂。 在通风橱内进行所有的溶剂的工作 。 </li><li>削减约100毫克冻结肝脏和权衡。将组织成筒在一个冰桶，并添加0.8毫升冰冷的0.9％NaCl溶液。均质化的组织。 </li><li>添加溶解在无水氯仿中的1毫升/毫克内部标准：甲醇（2:1，V / V）含丁基化羟基甲苯（BHT; 50毫克/升）作为抗氧化剂。对大鼠肝100毫克加入100微克的CE标准（十七烷胆固醇十七时00分） 注意 ：氯仿和BHT是危险的。 </li><li>加5.0ml无水氯仿：甲醇（2:1，V / V）含BHT（50毫克/升）。 </li><li>加入1.0毫升1 M氯化钠，涡旋充分混合，直到混合均匀的外观。样品可以封盖并储存在-20℃下在此阶段长达一周。 </li><li>离心机在1000×g离心10分钟，在室温下低速制动器。 </li><li>低收用玻璃巴氏吸管相，在40℃下转移到新螺丝帽玻璃管和干燥氮气下样品可以封盖并储存在-20℃下在此阶段长达一周。 </li></ol> 3。固相萃取分离脂质类的（SPE）10 <ol><li>在SPE水箱连接到真空泵，并把氨丙基硅胶固相萃取柱的坦克。 </li><li>将新的螺旋盖玻璃管标签TAG和CE在列下的水箱架，收集第一部分。 </li><li>溶解总脂提取物1.0ml无水氯仿和旋涡。 </li><li>申请样品用玻璃巴氏吸管的列，并允许通过滴落到螺纹管在重力作用下。如果没有进一步的水滴落下，通过真空除去残留的液体。 </li><li>洗脱TAG和CE分数在真空下，用干氯仿2×1.0毫升洗洗柱。 </li><li>当所有的液体被除去，干燥的TAG和CE fractio在40℃下的氮（N）样品可以封盖并储存在-20℃下在此阶段长达一周。 </li><li>将新的螺旋盖玻璃管标签的PC变成栏下的坦克托盘。 </li><li>洗脱，在真空下加入2×1.0毫升无水氯仿中的PC级分：甲醇（60:40，体积/体积），直到所有的液体从柱中除去。 </li><li>在40℃下在氮气氛下取出和干燥的PC级分样品可以封盖并储存在-20℃下在此阶段长达一周。 </li><li>放置一个新的螺旋盖玻璃管标记的PE入罐托盘和洗脱级分的PE与另外1.0毫升下真空干燥甲醇。 </li><li>在40℃下在氮气氛下取出并干燥聚乙烯级分样品可以封盖并储存在-20℃下在此阶段长达一周。 </li><li>把新的螺旋盖玻璃管标记NEFA入罐托盘和洗脱真空下通过加入2×1.0毫升洗涤干燥氯仿的NEFA分数：甲醇：冰醋酸（100:2:2，V / V / V） 注意 ：冰醋酸是危险的。 </li><li>在40℃下在氮气氛下除去收集的NEFA馏分和干样品可以封盖并储存在-20℃下在此阶段长达一周。 </li><li>将新的氨丙基硅胶固相萃取柱在SPE坦克，并放置一个螺丝帽玻璃管在坦克托盘墨盒下的垃圾收集。 </li><li>用干燥己烷在真空条件下洗涤3次，然后在重力作用下最终1.0毫升洗洗列。不要让墨盒变干（转筒柱通道关闭位置时，正己烷水平靠近盒矩阵） 注意 ：正己烷是危险的。 </li><li>更换废液管与新的螺丝帽玻璃管标记CE。 </li><li>溶解干豪和CE分数（步骤3.6制备）的1.0ml无水正己烷和旋涡。将此运用到使用玻璃巴斯德吸管列，并允许根据克至滴水通过ravity。 </li><li>如果没有进一步的水滴落下，取出在真空条件下剩余的液体。 </li><li>在真空条件下，用2×1.0毫升洗涤干燥己烷洗柱在氮气下，以洗脱CE和干燥收集到的馏分，在40℃下样品可以封盖并储存在-20℃下在此阶段长达一周。 </li><li>把新的螺旋盖玻璃管标记TAG在罐托盘和洗脱TAG与另外2×1.0毫升洗涤干燥己烷：甲醇：乙酸乙酯（100:5:5）在真空条件下。 </li><li>在40℃下在氮气下干燥收集到的馏分样品可以封盖并储存在-20℃下在此阶段长达一周。 注意 ：乙酸乙酯是危险的。 </li></ol> 4。从CE 10 FAME的制备<ol><li>加入0.5毫升无水甲苯分离的CE部分（收集步骤3.19）和涡注意 ：甲苯是危险的。 </li><li>制备的甲基化试剂（干燥甲醇与2％（体积/体积）的H 2 SO 4），其为1.0毫升每采样必需的。免除无水甲醇的体积为玻璃或带盖合适的塑料容器中，加入所需的H 2 SO量4滴，然后颠倒混匀注意 ：硫酸是危险的。 </li><li>加入1.0毫升甲基化试剂的溶解于无水甲苯的样品中，盖上管牢固，并轻轻混匀。 </li><li>加热该样品2小时，在50℃下</li><li> 2小时后离火管。冷却后加入1.0毫升的中和液（0.25M KHCO 3 0.5MK 2 CO 3） 注意 ：碳酸氢钾和碳酸钾是危险的。 </li><li>加入1.0ml无水正己烷和旋涡。 </li><li>离心机在250×g离心2分钟，在室温下低速制动器。 </li><li>收集上层相，其中包含了名气，并转移到一个新的非螺旋盖一次性玻璃管。 </li><li>干下收集到的FAME氮气在40℃下样品可以封盖并储存在-20℃下在此阶段长达一周。 </li></ol> 5。从CE FAME 14去除游离胆固醇污染 （免费胆固醇会污染样品，参见图1为例色谱痕迹和不游离胆固醇去除）。 <ol><li>将废管进入固相萃取槽，然后将硅胶固相萃取柱到坦克。 </li><li>在真空下3×1毫升洗涤干燥己烷和在重力作用下1×1毫升洗柱。 </li><li>消除浪费清洗和添加新的废物管插入罐。 </li><li>溶解CE成名于1ml无水正己烷，旋涡。 </li><li>适用于使用玻璃巴斯德吸管列，并允许在重力作用下通过滴。 </li><li>在真空下3×1毫升洗涤己烷洗柱。 </li><li>消除浪费清洗，并放置新的非螺丝帽管插入油箱标示CE名声大噪。 </li><li>洗脱CE FAME用2×1毫升无水己烷：乙醚（95:5体积/体积）洗涤。 </li><li>在氮气氛下干燥，在40℃下样品可以封盖并储存在-20℃下在此阶段长达一周。 注意 ：乙醚是危险的。 </li></ol> 6。转移FAME进入气相色谱自动进样瓶<ol><li>加入75微升的干燥己烷来样，旋涡，并转移至GC自动样品瓶。 </li><li>添加进一步的75微升的干燥己烷样品，旋涡和转移到相同的GC自动样品瓶。样品可以封盖并储存在-20℃下在此阶段长达一个月。 </li></ol> 7。分析使用气相色谱仪14 <ol><li>在气相色谱分析FAME。例如设置：30米×0.25微米×0.25毫米BPX-70随温度的协议熔融石英毛细管柱： 初始温度115℃，保持2分钟，斜10°C / min升至200℃，保持18.5分钟，斜60°C / min升至245°C，H老4分钟。 专栏：氦气，流速1.0，压力14.6和速度29。 进样器：温度= 300℃。 检测器：氢流量40.0，空气流量184.0，弥补气氦气，流量45.0，温度为300°C。 </li><li>设置分流比为适当（ 如 25:1获得CE FAME分析）。 </li><li>确定区域使用适当的软件每个峰下，并通过与标准比较确定的FAME。参见图2例如色谱图。 </li><li>使用该区域下的峰值的数据来计算的单个脂肪酸占总脂肪酸的百分比贡献。 </li><li>由量除以内标面积计算脂肪酸绝对含量添加。由这一结果除以各脂肪酸的面积，以组织的使用量范围内得到每种脂肪酸的绝对浓度。 </li></ol>

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作者宣称没有竞争的财务权益。

气相色谱法是一种精确的技术来用于脂肪酸分析，以及其高重复性认为该技术适于临床分析。适当的气相色谱柱，必须使用以使得能够识别的感兴趣的脂肪酸，具有变化的固定相，柱的长度和内径的极性可用的列。在分析这种方法的使用石英毛细管柱提供了良好的热稳定性和高的，由于其高的表面惰性和良好的分辨率8保留时间的重现性。 本协议?...

气相色谱（GC）是一种用于识别和量化的脂肪酸掺入脂质池和细胞膜1,2期间补充或生理条件，例如肥胖（及其相关的疾病，如糖 ​​尿病），或者怀孕3行之有效的技术- 5。它也适合于分析中的食物的种类和脂肪的数量。当表征实验饲料，以及确保食品行业符合规定，这是很有用的。例如，GC可以用来确认商品如膳食补充剂中的身份和脂肪酸的量，以确保标签是正确和法规粘附到6,7。脂肪酸的分析可以提供有价值的见解在健康和疾病，饮食变化的影响，并改变生理状态8的影响脂质代谢。使用GC在怀孕期间，研究的样本提供了重要的变动脂肪酸和复合脂质稳态3的信息。 提前的色谱分离的，脂质通常从样品用脂质中的溶解度在氯仿和甲醇的混合溶剂萃取。加入氯化钠，以促进该混合物的成含有相9,10水溶液和有机脂质的分离。感兴趣的复合脂质类可以从总脂提取物固相萃取（SPE）分离。这种分离方法洗脱根据其极性或结合亲和力的脂质类。 Triacyglycerols（TAG）和胆甾醇酯（CE）是首先被洗脱的馏分合并，进一步类，磷脂酰胆碱（PC），磷脂酰乙醇胺（PE），和非酯化脂肪酸（NEFA）通过增加洗脱溶剂的极性洗脱。 TAG的从CE分离利用的TAG只能到一个新的SPE cartri绑定DGE，允许CE被洗脱。 TAG然后可以通过增加洗脱溶剂9,10的极性进行洗脱。这种方法允许多个样品有较高的产率比同时分离与薄层层析来实现，这意味着相对小尺寸的样品（ 如 &lt;100微升的血浆或血清，&lt;100 mg组织）可以分析11,12。 GC是一个行之有效的技术在20世纪50年代首次描述，有人提出，在当时的液 - 液系统中的流动相可以被替换为蒸气。它最初是用于石油分析，但迅速扩大到其他领域，如氨基酸分析和脂类生物化学，这仍然是主要的利益。前进中的GC设备和技术，例如从以前使用填充柱的毛细管柱的发展导致了我们目前的技术，其中的脂肪酸是能够被在导致GC被常规用于鉴定和定量脂肪酸在广泛调查13的较低的温度下更有效地分离。 GC需要的脂肪酸，使他们有可能成为充分挥发在合理的温度被洗脱不热分解进行衍生化。这通常涉及到含有氢以形成酯，硫酯或酰胺的用于分析的官能团的取代。甲酯通常被研究的衍生物，它是由甲基化制备。在该方法中复合脂质的酯键被水解以释放游离脂肪酸，它们transmethylated以形成脂肪酸甲基酯（FAME）。 FAME的所得到的轮廓，由GC测定，被称为的脂肪酸组成和可能不同的实验组9,10之间容易地进行比较。该技术允许individ两者的比例UAL脂肪酸和它们的浓度进行测量。 除了使用气相色谱，用于分析在营养学和食品工业中的脂肪酸，该技术可以在宽范围的分析领域中使用。例如，使用气相色谱环境分析包括通过杀虫剂和土壤测量水质污染分析测定氯苯的内容。在毒理学，GC也被用于识别非法物质在尿和个体的血液样品，这样的运动性能增强剂12和分离烃类的复杂混合物的能力，使得在石油工业中这种技术流行的用于石油化学分析12。 妊娠与显著改变母体组织中的脂肪酸组合物，特别是ω-3的含量（N-3）和ω-6（N-6）多不饱和脂肪酸ACI相关DS（PUFA）3。在目前的研究中，我们通过举例说明其使用从饲喂低，高脂肪的饮食不同油源和处女孕鼠取肝组织中脂肪酸组成的分析中脂肪酸的测定采用气相色谱。这里提供的试验日粮是一种低脂肪的大豆油为主的饮食，高脂肪的大豆油为主的饮食习惯（130.9克总脂肪/公斤总脂肪）或高脂肪的亚麻子油为主的饮食习惯（130.9克总脂肪/公斤饮食），提供了20天。这些饮食的全部营养和脂肪酸组合物先前已被14所述。豆油日粮中含有丰富的亚油酸（18:2的n-6）和含有一些α-亚麻酸（18点03的n-3），而亚麻油饮食中富含α-亚麻酸。这些高脂肪的饮食代表的亚油酸与α-亚麻酸（8:1和1:1分别口粮）不同的口粮。该方法对于个别脂质类和用GC分析的隔离是我们LL建立和验证，以及先前已10但没有在此找到详细的技术说明公布。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Methanol:</td>
			<td>Fisher Scientific</td>
			<td>M/4056/17</td>
			<td>&#39;CAUTION&#39; Fumes - HPLC Grade</td>
		</tr>
		<tr>
			<td>Chloroform:</td>
			<td>Fisher Scientific</td>
			<td>C/4966/17</td>
			<td>&#39;CAUTION&#39; Fumes - HPLC Grade</td>
		</tr>
		<tr>
			<td>BHT:</td>
			<td>Sigma- Aldrich</td>
			<td>W218405</td>
			<td>&#39;CAUTION&#39; Dust fumes - Anhydrous</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma- Aldrich</td>
			<td>S9888</td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher Scientific</td>
			<td>H/0406/17</td>
			<td>&#39;CAUTION&#39; Fumes - HPLC Grade</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma- Aldrich</td>
			<td>695084</td>
			<td>&#39;CAUTION&#39; Burns - 99.85%</td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma- Aldrich</td>
			<td>339741</td>
			<td>&#39;CAUTION&#39; Burns - 99.999%</td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Sigma- Aldrich</td>
			<td>209619</td>
			<td>99% ACS Reagent grade</td>
		</tr>
		<tr>
			<td>Potassium bicarbonate</td>
			<td>Sigma- Aldrich</td>
			<td>237205</td>
			<td>99.7% ACS Reasgent grade</td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Fisher Scientific</td>
			<td>10204340</td>
			<td>&#39;CAUTION&#39; Fumes - 99+% GLC SpeciFied</td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Scientific</td>
			<td>T/2300/15</td>
			<td>&#39;CAUTION&#39; Fumes</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma- Aldrich</td>
			<td>309966</td>
			<td>&#39;CAUTION&#39; Fumes</td>
		</tr>
		<tr>
			<td>Nitrogen (oxygen free) cylinder</td>
			<td>BOC</td>
			<td>44-w</td>
			<td>&#39;CAUTION&#39; Compressed gas - explosion risk</td>
		</tr>
		<tr>
			<td>Aminopropyl silica SPE cartridges</td>
			<td>Agilent</td>
			<td>12102014</td>
			<td>Cartridge - Bead mass 100mg</td>
		</tr>
		<tr>
			<td>Silica gel SPE cartidges</td>
			<td>Agilent</td>
			<td>14102010</td>
			<td>Cartridge - Bead mass 100mg</td>
		</tr>
		<tr>
			<td>Molecular seives</td>
			<td>Sigma- Aldrich</td>
			<td>334324</td>
			<td>Pellets, AW-300, 1.6mm</td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>FB50251</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass test tube</td>
			<td>Fisher Scientific</td>
			<td>FB59527</td>
			<td>Non-screw cap</td>
		</tr>
		<tr>
			<td>Screw thread glass test tubes</td>
			<td>Fisher Scientific</td>
			<td>FB59555</td>
			<td>13mm</td>
		</tr>
		<tr>
			<td>Caps for screw thread test tubes</td>
			<td>Fisher Scientific</td>
			<td>FB51353</td>
			<td>To fit 13mm tube</td>
		</tr>
		<tr>
			<td>Solida phase extraction (SPE) tank</td>
			<td>Agilent</td>
			<td></td>
			<td>VacElut 20 Manifold</td>
		</tr>
		<tr>
			<td>Luer stopcocks for SPE tank</td>
			<td>Agilent</td>
			<td>12131005</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Sigma- Aldrich</td>
			<td>2656-194GB-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>GC vials</td>
			<td>Kinesis</td>
			<td>STV 12-03TS</td>
			<td>Short thread 9mm, TPX 0.2ml fused glass insert</td>
		</tr>
		<tr>
			<td>GC vial lids</td>
			<td>Kinesis</td>
			<td>SCC09-0.2B</td>
			<td>Short thread 9mm blue</td>
		</tr>
		<tr>
			<td>GC inlet liners</td>
			<td>SGE Analytical science</td>
			<td>092002</td>
			<td>Split/splitless Focus liner ID 4mm, OD 6.3mm length 78.5mm</td>
		</tr>
		<tr>
			<td>GC septa</td>
			<td>SGE Analytical science</td>
			<td>041856</td>
			<td>11mm, MN material</td>
		</tr>
		<tr>
			<td>GC column</td>
			<td>SGE Analytical science</td>
			<td>054612</td>
			<td>Length 30m, ID 0.22mm, Film thickness 0.25&#181;m</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td></td>
			<td></td>
			<td>HP 6890 series</td>
		</tr>
	</tbody>
</table>

the use of gas chromatography to analyze compositional changes of fatty acids in rat liver tissue during pregnancy

气相色谱（GC）是用于从组织，细胞，血浆/血清识别和量化脂质的脂肪酸含量，得到具有高精度和高再现性的结果高度敏感的方法。代谢和营养研究的GC允许更改下列措施中的脂肪酸浓度或在改变生理状态，如妊娠的评估。固相萃取（SPE），使用氨丙基硅胶管允许各大类脂质包括甘油三酯，磷脂不同，和胆固醇酯（CE）的分离。气相色谱结合固相萃取是用来分析的变化，行政长官在部分已被送入各种高和低脂肪饮食的处女和孕鼠的肝脏中脂肪酸的组成。有肝经行政长官的ω-3和ω-6脂肪酸含量显著饮食/妊娠互动效应，这表明怀孕的女性有不同的反应，饮食manipulat离子比被认为是处女的女性之一。

这种方法的成功依赖于精确的按照协议和有关使用清洁溶剂和试剂，以减少“噪声”，并且可以出现在色谱污染。污染的样品是更具挑战性的分析，从而降低了计算曲线下的面积的精度。如果该协议被成功地遵循的色谱图清晰对称的，明确定义的峰值和最小的背景噪声， 如图3应获得。如果已发生污染的色谱将显示额外的峰，并表现出如图4所示的非对称的（倾斜）的峰。...

Watch this Scientific Journal Video about The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy at JoVE.com

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

妊娠导致显著改变母体组织中的脂肪酸组合物。血脂可通过气相色谱法来获得允许在怀孕期间大鼠中识别和个别类脂质的脂肪酸定量饲喂各种高，低脂肪饮食。

使用气相色谱怀孕期间分析脂肪酸组成的变化在大鼠肝组织

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

the-use-gas-chromatography-to-analyze-compositional-changes-fatty

Research

JoVE Journal

Chemistry

29.9K 次观看.  University of Southampton. 妊娠导致显著改变母体组织中的脂肪酸组合物。血脂可通过气相色谱法来获得允许在怀孕期间大鼠中识别和个别类脂质的脂肪酸定量饲喂各种高，低脂肪饮食。 

视频: The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

寡肽测定气相酸性

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

科研

化学

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

通过超性能液相色谱对细菌细胞壁进行隔离和制备，进行组成分析

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

定量检测痕量爆炸物蒸气通过程序升温脱附 - 气相色谱 - 电子捕获检测器

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

的4-取代喹唑啉衍生物简便制备

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

利用毛细管电泳从植物组织量化有机酸：一个测试用例检查<em&gt;小粒种咖啡</em&gt;种子

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

拉曼光谱和红外光谱电化学法作为分析共轭有机化合物的工具

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps.

This method for two-step pyrolysis online coupled to gas chromatography with mass spectrometric detection and data evaluation protocol can be used for multi-component analysis of tattoo inks and discrimination of counterfeit products.

一种用于检测纹身油墨成分和假冒产品的两步热解-气相色谱法

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. ...

Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is crucial. There are significant challenges to overcome when measuring trace compounds in a complex matrix such as fuels. Background interferences and matrix effects can create limitations to routine analytical instrumentation, such as GC-MS. In order to facilitate specific and quantitative measurements of trace nitrogen compounds in fuels, a nitrogen-specific detector is ideal. In this method, a nitrogen chemiluminescence detector (NCD) is used to detect nitrogen compounds in fuels. NCD utilizes a nitrogen-specific reaction that does not involve the hydrocarbon background. Two-dimensional (GCxGC) gas chromatography is a powerful characterization technique as it provides superior separation capabilities to one-dimensional gas chromatography methods. When GCxGC is paired with a NCD, the problematic nitrogen compounds found in fuels can be extensively characterized without background interference. The method presented in this manuscript details the process for measuring different nitrogen-containing compound classes in fuels with little sample preparation. Overall, this GCxGC-NCD method has been shown to be a valuable tool to enhance the understanding of the chemical composition of nitrogen-containing compounds in fuels and their impact on fuel stability. The % RSD for this method is &#60;5% for intraday and &#60;10% for interday analyses; the LOD is 1.7 ppm and the LOQ is 5.5 ppm.

Here, we present a method utilizing two-dimensional gas chromatography and nitrogen chemiluminescence detection (GCxGC-NCD) to extensively characterize the different classes of nitrogen-containing compounds in diesel and jet fuels.

多维气相色谱在燃料中的氮化合物表征

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is ...