C.Y.H. was supported by an Ontario Graduate Scholarship from the Government of Ontario. This article was made possible by funding held by R.J.B. from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and Ryerson University.

注:文中详细描述了一个在酵母来衡量PtdInsPs方法。它提供了用于标记的酵母细胞用 3 H- 肌醇 ，提取和脱酰化脂质和的HPLC洗脱协议以分级分离和量化去酰基化PtdInsPs的实验细节。请注意，标签，脱酰化，PtdInsPs在哺乳动物细胞分辨率和量化需要优化和更长的HPLC-洗脱曲线。这些细节可以找到其他地方，虽然我们讨论一些在讨论方面。总体而言，该方法提取脂质，脱酰化并提取水溶性格罗-InsPs从酵母中给出并示于图1。 1.协议标签和浅析PtdInsPs酵母<ol><li> 细胞培养及放射性标记 <ol><li>生长的酵母菌株SEY6210的20ml液体培养在30℃下以恒定振荡中完成合成媒体数中期或在约0.6-0.7 600nm的值（OD 600）的光密度。 注:请不要使用YPD媒体，因为这可能会影响3 H- 肌醇结合。这种文化的大小应为2-3 HPLC运行提供了足够的材料。 </li><li>沉淀总共10-14外径的酵母细胞（注意1 ml培养用的OD 600 = 1〜包含1×10 7细胞）通过离心，在800×g离心5分钟，在12毫升的圆底管中。重悬用2ml肌醇的培养基（IFM）（ 见表 1 IFM组合物）。 </li><li>重复离心和吸媒体。重悬带440微升IFM的沉淀并在室温下孵育15分钟。 </li><li>添加60微升3 H- 肌醇 （1微升= 1微居）向每个样品，孵育在30℃的生长温度进行1至3小时以恒定振荡。 注意:3 H- 肌醇是一种放射性伴侣里亚尔应经过适当的培训来处理。此外，所有的消耗品，使用3 H-含材料接触应适当设置，并指定为放射性废物。 注:生长温度是可以改变的，只要所有菌株进行比较生长在相同的温度下必需的。 </li><li>转移细胞悬液（500微升）到含有500微升9％高氯酸和200微升酸的离心管中洗涤玻璃珠。通过颠倒混合并在冰上孵育5分钟。 </li><li>涡样品以最高速度进行10分钟，并使用凝胶加载提示，以避免在玻璃珠的裂解物转移到新的离心管中。 </li><li>沉淀将样品在12000×g离心10分钟，在4℃，吸出上清液。 </li><li>悬浮颗粒通过浴超声处理在1ml冰冷的100mM EDTA中。再次颗粒和以前一样。 </li></ol></li><li> 去酰基化和提取 <o升&gt; <li>新鲜通过组合2.3毫升甲醇，1.3 ml的40％甲胺的，0.55毫升1-丁醇和0.80毫升水制备5.0毫升脱酰试剂。倒置混合。 </li><li>吸出EDTA和超声处理将沉淀于50μl水中。 </li><li>加入500μl脱酰试剂和超声处理混合。在室温下孵育20分钟。 </li><li>热脂质在53℃下在热块50分钟。完全干燥的样品通过真空离心超过3小时或O / N。 <ol><li>确保化学捕集器安装在真空离心机和真空泵，以防止进入空气的蒸气之间。 </li></ol></li><li>通过浴声处理重悬用300μl水的颗粒和在室温下孵育20分钟。干燥真空离心样品3小时或O / N。再一次重复此步骤。 </li><li>超声处理以450微升水沉淀。这是提取期间水相。 </li><li>刚准备将10毫升提取中的Ñ​​试剂通过加入8.0中毫升乙醚，1-丁醇，1.6 ml和0.40毫升甲酸乙酯。倒置混合。 </li><li>添加300微升提取试剂的水相。涡旋以最高速度混合物5分钟并通过离心以最高速度（18,000 xg离心）2分钟，分层。 </li><li>收集底部的水层到一个新的离心管，同时避免了上面的有机层，界面和沉淀。用新鲜的萃取剂重复萃取水相两次。 </li><li>完全干燥收集的水层通过真空离心。通过浴声处理分散在50μl水中沉淀。 </li><li>添加2微升各样品到4ml闪烁液在6 ml的聚乙烯闪烁瓶中。确定每个样品中计数（在CPM）的通过液体闪烁利用开窗口的数量。 </li><li>储存在-20℃的样本，直到准备用于HPLC。 </li></ol></li><li> 分离3 H-甘油基肌醇通过HPLC <ol><li>通过过滤1升超纯水一瓶顶级0.22微米真空过滤机，随后脱气准备的缓冲液A。 </li><li>通过使1M的磷酸铵二元（APS，MW 132.06）的水1升溶液制备的缓冲液B和将pH调节至3.8，用磷酸。过滤磷酸铵用瓶子顶端0.22微米真空过滤，随后脱气。 </li><li>通过舾装一个2毫升用弹簧加载的250微升小体积插入小瓶组装注射小瓶中。加载千万CPM，加水为55微升的总体积。准备一张空白小瓶55微升的水。 </li><li>帽与配备的PTFE /有机硅隔膜螺丝帽的小瓶（红色面朝所述小瓶的内部）。将每个小瓶中的HPLC的自动采样托盘，开始与空白小瓶。 </li><li>使用HPLC和相关的软件，用于控制缓冲液流，degasses缓冲器与控制样品injecti上。使用在线的流动闪烁器及其相关软件来调节闪烁体流量和监视3 H-衰变信号。 <ol><li>使用带有250毫米×4.6毫米尺寸并含有在HPLC 5微米二氧化硅树脂一个SAX液相层析柱。装备有一列卫，以防止注射污染物列。 </li><li>安装在流动闪烁体3 H-兼容500μl的流动池。 注:分馏可以用由化学工作站软件或与其它可商购的HPLC系统控制的Agilent 1200系列无穷HPLC系统来完成。高效液相色谱洗脱剂的流动闪烁可与β-RAM的流动闪烁体由劳拉软件或另一可商购的流动闪烁体或兼容的软件控制来完成。 </li></ol></li><li>初始化四元泵以1.0毫升/分钟用100％缓冲液A的流速</li><li>设置一个平衡配置文件的HPLC控制缓冲器A和B的梯度（称这为" 议定书甲平衡 "）:1％缓冲液B进行5分钟，1-100％B进行5分钟，100％B为5分钟，100-1％B保持5分钟，1％B，20分钟。在400巴，1.0ml /分钟的流速和30分钟运行时间设置压力极限。 </li><li>设置一个洗脱曲线（图2）上的高效液相色谱法，以控制缓冲器A和B的梯度（称之为" 议定书A"）:1％B保持5分钟，1〜20％乙​​40分钟，20-100％ B洗脱10分钟，100％B为5分钟，100-1％B，20分钟，1％B，10分钟。在400巴，1.0ml /分钟的流速，以及90分钟运行时间设置压力极限。 </li><li>设置在流闪烁体的检测协议（称这为" 议定书的检测 "）运行60分钟，用2.5毫升/分钟的闪烁流体流速和一个8.57秒的停留时间。 </li><li>方案的HPLC的自动进样序列，开始与水空白的" 协议，一种平衡 "，然后通过电子邮件ACH放射性标记的" 协议"样本 。初始化序列准备时。 </li><li>虽然运行仍然在平衡协议，创建一个批处理对流动闪烁测量所有的样品的" 协议的检测 。"每一个新样品的注入将初始化" 议定书"关于高效液相色谱而引发的流动闪烁，开始" 协议的检测 。" </li><li>在完成所有试验中，冲洗性HPLC，柱和1.0ml /分钟流速流动的闪烁体用100％缓冲液A为30分钟。 </li></ol></li><li> 数据分析 <ol><li>使用劳拉软件或可量化层析谱的任何其它软件。在本节中描述的步骤被示于图3。 </li><li>通过点击"文件"，"打开"，打开文件，并选择每个样品的原始数据文件。 </li><li>在"色谱图"选项卡中，莫宁嗡在伸展每个中的较小的峰（约1000计数[y轴]），同时保留时间分辨率。如果需要放大到每个人的峰值。 </li><li>突出每个使用"添加投资回报率"工具分析的峰值。洗脱时间确定峰:父母格罗宏在10分钟，GRO-Ins3P 18分钟，GRO-Ins4P 20分钟，格罗宏（3,5）P2在29分钟和格罗宏（4,5 ）P 2，在32分钟。 </li><li>在"地区表"选项卡，记录每一个峰的"区域（计数）"。请注意，"启动（MM:SS）的"时间和"结束（MM:SS）"各峰的时间。 </li><li>背景扣除，突出显示邻近于峰跨越的时间相同的区域。从相应的峰减去计数的数目。 </li><li>正常化针对亲格罗宏每个峰（表示为"％总磷脂酰肌醇的"）的区域。然后，归一化每个峰中的每个实验条件下对控制条件（表示为"n倍增加相比，控制"）。 注:各峰的正常化也可以对总计数完成，但由于父母格罗宏是比其他所有PtdInsPs丰富得多的结果往往是相似的。 </li><li>按"文件"，导出了色谱仪中的数据"另存为..."，并选择"CSV（逗号分隔）"文件格式。绘制数据的电子表格，并根据需要提出。 </li></ol></li></ol>

The authors declare that they have no competing financial interests.

这篇文章详细描述了从酵母HPLC-耦合流动闪烁量化细胞PtdnsPs水平所需要的实验方案。该方法使PtdInsPs 用 3 H- 肌醇 ，随后脂质处理和提取的水溶性的3 H-GRO-InsPs，HPLC分级和分析的代谢标记。使用这种方法，PtdInsPs在细胞在各种条件下的相对水平可以被量化，作为示出了用于磷脂酰肌醇（3,5）P 2在野生型，vac14 D和atg18ð酵母细胞（图4）。 有一些关键步骤和修改，可以做，以优化结果和/或排除。一个实现PtdInsPs的可靠的量化的最重要的参数是总放射性的信号电平。通常情况下，注射3-5百万计数进入高效液相往往不足以可靠定量变化喜gher丰PtdInsP物种如磷脂酰肌醇（4,5）P2。然而，注射高达1000万计数可能是必要的低丰度品种的量化，如磷脂酰肌醇（3,5）P 2。有可能影响放射性信号的电平实现的，包括用于制备样品，标记的时间和温度，3 H- 肌醇的浓度，PtdInsPs的周转率的细胞的总数和内源性合成的各种参数的肌醇，后者将与放射性标记的肌醇竞争。例如，放射性标记的酵母细胞用于至少一个倍增时间允许的3 H- 肌醇代谢掺入PtdInsPs，但延长的时间标记用于酵母突变体生长较慢或具有较慢的新陈代谢活动可能是必要的。此外，这里描述的方案使用了酵母培养，其中细胞生长至对数中期然后浓缩FO的diauxic移- [R标记超过倍增时间。然而，我们可以在初期和中期，数期和更长时段的时间减少diauxic通过转变细胞标记如以前做过32。在这一过程中，这些作者观察到较高水平的磷脂酰肌醇（3）P，磷脂酰肌醇（4）P和磷脂酰肌醇（4,5）P2不是细胞经历diauxic移位32。 同样地，哺乳动物细胞的标记时期，可能需要优化，因为一些哺乳动物细胞像的RAW 264.7巨噬细胞线加倍，每12小时，而另一些可以划分慢得多，或者作为在神经元的情况下，不是在所有20,35,39。此外，一个可以增加3 H- 肌醇的浓度，以获得理想的放射性信号电平。然而，请注意，部分3 H- 肌醇打包在90％的乙醇; 加入过量的 3 H- 肌醇将增加在媒体中的乙醇含量，并可能有一个意想不到EFFE克拉细胞上。最后，延长放射性标记也可以饿死肌醇的细胞。这在完全DMEM中含有40μM的肌醇 ，相比0.5μM3 H- 肌醇时，用10微居里/毫升标记哺乳动物细胞培养尤为明显。因此，如果需要的话，仅增加标记期间哺乳动物细胞的数量，建议。 蜂窝裂解和脂质加工步骤也用于获得一个最佳的产率，以及维持所述HPLC柱的健康至关重要。裂解用高氯酸允许脂质和其它大分子的沉淀，首先用于组织培养并稍后用于酵母28,33的方法。与使用传统的甲醇的总脂质的提取/盐酸/氯仿法，使用高氯酸的增强PtdInsPs的恢复，包括磷脂酰肌醇（3,5）P 2，与实验28之间较小的差异。亚甲胺的序贯应用打破了甘油骨架和两个疏水酰基链之间的酯键，而使水溶性格罗-INSP是相分离游离脂肪酸和完好脂质28,34。特别必须小心，以避免在提取步骤结转不需要的有机溶剂和不溶性材料。这可堵塞系统，引起压力尖峰和/或化学损坏色谱柱。此外，列必须在每天开始时由列暴露于高浓度的铵磷酸盐（缓冲液B）在使用前的为条件;因此，短暂的" 平衡 "的协议运行实验样品前就业。未能在所运行的第一个实验样本中的GRO-InsPs峰延迟洗脱曲线做调整步骤的结果。 有相关的分析和量化以考虑以及几个关键点。首先，第quantification本身应信号和每个峰的面积的积分，而不仅仅是峰高。此外，内部控制通常是必要的，因为对于每个峰的绝对积分信号可以显著样品和实验之间，但不反射在PtdInsP丰度的变化在细胞中而变化。亲本格罗宏峰值通常用作正常化反对每个GRO-督察峰值内部控制。在格罗宏峰值通常持有近90％的放射性信号，并且不容易的条件之间切换。可替代地，人们可以选择正常化针对总计数，特别是如果怀疑亲磷脂酰肌醇遭受在实验条件显著变化。最后，对于低丰度PtdInsPs，背景减除建议通过定义一个相邻的非峰面积与相同的"时标"作为关注（图3D）的峰值。 最后一个考虑涉及哺乳动物的样品，它们具有7 PtdInsP物种，其中包括三个单磷酸和三个双 - 磷酸化的物质的分析。不幸的是，它是难以完全解决PtdIns4P从PtdIns5P，和磷脂酰肌醇（3,5）P 2从磷脂酰肌醇（3,4）P 2的哺乳动物的样品中，产生连体双峰。有以前出版的多种方法提高这些峰的分辨率。通常，这涉及到修改洗脱的长度，磷酸铵缓冲液的起始浓度，速率和步骤由所述洗脱曲线在磷酸铵缓冲液浓度的变化和磷酸铵缓冲33,35-38的pH。也许，最成功的方法开发了单独的磷脂酰肌醇（4）P和磷脂酰肌醇（5）p是由Sarkes和Rameh 39，其中采用2个串联的SAX柱和在pH 6.0的磷酸铵缓冲最近描述。 与大多数的方法，用HPLC-Coupled流动闪烁量化PtdInsPs在细胞中都有优点和缺点相比其他可用的方法。例如，在线流量闪烁耦合到HPLC提供现场检测格罗-InsPs的但限制了闪烁计数时间，从而降低了该方法的灵敏度。或者，所述的HPLC洗脱剂可以是分数，收集用自动取样，而不是送入流动闪烁体。手动馏分随后可以通过离线液体闪烁计数器中读取的时间较长，从而提供更高的灵敏度-然而，这种方法是更费时和劳动强度大且降低分辨率取决于级分的数量收集38,40 。此外，全细胞的分析明确地测量一个特定PtdInsP的水平，但它不会告知更改到PtdInsP的亚细胞分布，虽然人们可以耦合流动闪烁到亚细胞分级分离法作为页上一页狡猾完成39。相比较而言，FP-基于PtdInsP结合结构域是有用的调查的PtdInsP物种在细胞中的位置和动态，但也结合因子比目标PtdInsP其他。另外，这里描述的方法通过消除酰基链，这大大增加PtdInsPs的分子多样性简化PtdInsP水平的分析。但是由于同样的原因，这也是一个主要缺点，因为脂质的脱酰导致了关于酰基链，的PtdInsP信令41,42一个被低估方面的结构信息的损失。如果这是极大的关注，那么液相色谱质谱（LC-MS）应采用14,17。然而，目前难以通过LC-MS 43以同时分析所有PtdInsPs物种。因此，相结合的方法是为了调查的位置，动力学，分子全部七个PtdInsPs物种的多样性和层次的最佳方法。

Phosphoinositides (PtdInsPs) are important signaling phospholipids that help regulate a variety of cellular functions, including signal transduction, membrane trafficking and gene expression, which then modulate higher-order cell behavior such as cell division, organelle identity and metabolic activity1-3. There are seven species of PtdInsPs that are derived from the phosphorylation of the 3, 4, and/or 5 positions of the inositol head group of phosphatidylinositol (PtdIns), the parent phospholipid. Importantly, the seven PtdInsPs are unequally distributed and the local concentration of each PtdInsP species can increase or decrease at specific subcellular sites where they bind to a distinct set of protein effectors, which together permits each PtdInsP to control the identity and function of its host membrane3,4. In addition, the levels of each PtdInsP need to be tightly controlled since this can significantly impact the signal intensityproduced by a PtdInsP. The localization and levels of each PtdInsP depends on the targeting and activity of numerous lipid kinases, phosphatases and phospholipases that mediate the synthesis and turnover of each PtdInsP3,4. Hence, misregulation of the PtdInsP regulatory machinery can perturb cell function, leading to diseases such as cancer and degenerative diseases2,5,6. To fully understand the roles and functions of PtdInsPs and their regulatory machinery, both microscopy-based and biochemical-based techniques have been developed to track and quantify PtdInsPs. 
In many cases, PtdInsPs bind to their protein effectors via a specific protein domain7-9. These protein modules often retain their proper fold and lipid recognition properties when expressed separately from the entire protein. This gave rise to PtdInsP probes by fusing a specific PtdInsP-binding protein domain to a fluorescent protein (FP) like green fluorescent protein (GFP) for the subcellular detection of PtdInsPs by microscopy. Indeed, many studies have used FP-fused PtdInsP-binding protein modules to identify the localization and dynamics of specific PtdInsP species by live-cell imaging1,10. For example, the Pleckstrin homology (PH) domain of phospholipase C &#948;1 (PLC&#948;1) fused to GFP specifically recognizes the phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] on the plasma membrane, whereas tandem copies of the FYVE domain of early endosome antigen 1 (EEA1) has been employed to track phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes10-13. Overall, microscopy-based techniques are great to visualize PtdInsP localization and dynamics, but there are several caveats including that PtdInsP-binding domains may also interact with additional factors other than the target PtdInsP species and that they cannot detect changes below cytosolic fluorescence of the FP-probes.
Biochemical techniques including thin-layer chromatography, mass spectrometry and radioisotope labeling can also be used to characterize and quantify the levels of each PtdInsP14-16. These methods require the isolation of lipids for the detection of cellular levels of PtdInsPs. Mass spectrometry can be used to characterize phospholipids from lipid extracts and is invaluable for determining the acyl chain composition of PtdInsPs14,17. However, mass spectrometry is mostly semi-quantitative and it remains difficult to resolve and concurrently quantify PtdInsP species of the same molecular weight14,17. In comparison, radioisotope labeling of PtdInsPs followed by high performance liquid chromatography (HPLC)-coupled flow scintillation is useful for the separation and concurrent quantification of all seven species of PtdInsPs18. The use of HPLC with a strong anion exchange (SAX) column achieves separation based on molecular weight, charge and shape, thus fractionating deacylated PtdInsPs (Gro-InsPs) even of the same molecular weight and charge. Coupling the HPLC eluent to a flow scintillator then generates radioactive-based signal peaks for each Gro-InsPs species relative to the original parent glycerol-inositol (Gro-Ins)18. This ultimately corresponds to relative levels of PtdInsPs in cells.
Radiolabeling of PtdInsPs and HPLC-coupled flow scintillation is a useful tool to investigate the regulation and function of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], a PtdInsP that only constitutes ~0.1-0.3% of PtdIns16,19,20. Synthesis of PtdIns(3,5)P2 is performed by the PtdIns3P 5-kinase called Fab1 in yeast and PIKfyve in mammals21. This reaction is counteracted by a PtdIns(3,5)P2 5-phosphatase called Fig4 in yeast or mFig4/Sac3 in mammals22-24. Interestingly, both PIKfyve/Fab1 and mFig4/Fig4/Sac3 exist in a single complex and are regulated by the scaffolding adaptor protein, Vac14 in yeast or mVac14/ArPIKfyve in mammals25,26. In VAC14-deleted yeast cells, Fab1 does not function efficiently leading to a 90% decrease in PtdIns(3,5)P2 levels27,28. On the other hand, Atg18 is a PtdIns(3,5)P2 effector protein that may control vacuolar fission 29. Atg18 is also a negative regulator of PtdIns(3,5)P2 since the deletion of its gene, ATG18, causes a 10 to 20-fold increase in PtdIns(3,5)P2 levels29,30. Overall, changes in the levels of PtdIns(3,5)P2 severely impacts the function of the yeast vacuole and the mammalian lysosomes, consequently affecting processes such as membrane trafficking, phagosome maturation, autophagy and ion transport 6,19,21,31. 
This article describes the process of radioisotope labeling of PtdInsPs with 3H-myo-inositol in yeast to detect the relative levels of PtdIns(3,5)P2 in wild-type, vac14&#916; and atg18&#916; yeast strains. Using this as an example, the resolving capabilities of HPLC for the separation of individual PtdInsPs as well as the sensitivity of flow scintillation for detection of trace amount of 3H-myo-inositol is shown. We also elaborate on how one might optimize the methodology for labeling and separating 3H-labelled PtdInsPs from mammalian cells, whose samples tend to be more complex since these cells possess all seven PtdInsP species.

<table border="0" cellpadding="0" cellspacing="0" width="1086">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">1-Butanol</td>
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			<td style="width:119px;">BC1800</td>
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			<td>APD001</td>
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			<td>B4501</td>
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			<td height="21" style="height:21px;">Copper(II) sulfate</td>
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			<td>451657</td>
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		<tr height="21">
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		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modification of Eagle&#39;s Medium</td>
			<td style="width:147px;">Life</td>
			<td>11995-065</td>
			<td>With 4.5 g/L glucose, 110 mg/L pyruate, L-glutamine</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modification of Eagle&#39;s Medium</td>
			<td style="width:147px;">MP biomedicals</td>
			<td style="width:119px;">0916429&#160;</td>
			<td>With 4.5 g/L glucose, without L-gluatmine, without inositol</td>
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			<td height="21" style="height:21px;">EDTA</td>
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			<td>EB0107</td>
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			<td>1/10/4700</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Ethyl formate</td>
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			<td>112682</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal bovine serum</td>
			<td style="width:147px;">Wisent</td>
			<td style="width:119px;">080-450</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, Dialyzed</td>
			<td style="width:147px;">Life</td>
			<td>26400044</td>
			<td>US origin</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:421px;">FlowLogic U</td>
			<td style="width:147px;">LabLogic Systems Ltd</td>
			<td style="width:119px;">SG-BXX-05</td>
			<td>Scintillation fluid for flow scintillation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Folic acid</td>
			<td>Biobasic</td>
			<td>FB0466</td>
			<td>USP grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer solution</td>
			<td style="width:147px;">Life</td>
			<td>15630080</td>
			<td>1 M solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Inositol, Myo-[2-3H(N)]</td>
			<td style="width:147px;">Perkin Elmer</td>
			<td>NET114005MC</td>
			<td>9:1 ethanol to water</td>
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			<td height="21" style="height:21px;">Insulin-Transferrin-Selenium-Ethanolamine</td>
			<td style="width:147px;">Life</td>
			<td>51500056</td>
			<td>100x solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Iron(III) chloride</td>
			<td>Sigma</td>
			<td>157740</td>
			<td>Reagent grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Laura - Chromatography data collection and analysis software</td>
			<td style="width:147px;">LabLogic Systems Ltd</td>
			<td style="width:119px;">Version 4.2.1.18</td>
			<td>Flow scintillator software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">L-glutamine</td>
			<td style="width:147px;">Sigma</td>
			<td>G7513</td>
			<td>200&#160;mM, solution, sterile-filtered, BioXtra, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
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			<td>M8266</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Manganese sulfate</td>
			<td>Biobasic</td>
			<td>MB0334</td>
			<td>Monohydrate, ACS grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Caledon labs</td>
			<td>6701-7-40</td>
			<td>HPLC Grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Methylamine solution</td>
			<td>Sigma</td>
			<td>426466</td>
			<td>40% (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monopotassium phosphate</td>
			<td>Biobasic</td>
			<td>PB0445</td>
			<td>Anhydrous, ACS grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinic acid</td>
			<td>Biobasic</td>
			<td>NB0660</td>
			<td>Reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">OpenLAB CSD ChemStation&#160;</td>
			<td style="width:147px;">Agilent</td>
			<td style="width:119px;">Rev. C.01.03&#160;</td>
			<td>HPLC software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-aminobenzoic acid (PABA)</td>
			<td>Bioshop</td>
			<td>PAB001.100</td>
			<td>Free acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:147px;">Sigma</td>
			<td>P4333</td>
			<td>100X, liquid, stabilized, sterile-filtered, cell culture tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perchloric acid</td>
			<td>Sigma</td>
			<td>244252</td>
			<td>ACS reagent, 70%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PhenoSpher SAX column</td>
			<td>Phenomenex</td>
			<td>00G-315-E0</td>
			<td>5&#181;m, 80&#197;, 250 x 4.6 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Phosphoric acid</td>
			<td style="width:147px;">Caledon labs</td>
			<td>1/29/8425</td>
			<td>Reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Biobasic</td>
			<td>PB0440</td>
			<td>ACS grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium iodide</td>
			<td>Biobasic</td>
			<td>PB0443</td>
			<td>ACS grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Pyridoxine hydrochloride</td>
			<td>Sigma</td>
			<td>P9755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Quaternary pump</td>
			<td style="width:147px;">Agilent</td>
			<td style="width:119px;">G1311C</td>
			<td style="width:399px;">Agilent 1260 infinity series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Riboflavin</td>
			<td>Bioshop</td>
			<td>RIB333.100</td>
			<td>USP grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Biobasic</td>
			<td>DB0483</td>
			<td>Biotech grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Sodium molybdate</td>
			<td>Sigma</td>
			<td>243655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermostatted Column Compartment</td>
			<td style="width:147px;">Agilent</td>
			<td style="width:119px;">G1316A</td>
			<td style="width:399px;">Agilent 1260 infinity series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Thiamine hydrochloride</td>
			<td>Sigma</td>
			<td>T4625</td>
			<td>Reagent grade; make solution of 0.02% (w/v), forms a suspension. mix and freeze aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Ultima Gold</td>
			<td style="width:147px;">Perkin Elmer</td>
			<td style="width:119px;">6013321</td>
			<td>Scintillation coctail for liquid scintillation counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zinc sulfate</td>
			<td>Biobasic</td>
			<td>ZB2906</td>
			<td>Heptahydrate, reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">&#946;-RAM 4</td>
			<td style="width:147px;">IN/US systems</td>
			<td style="width:119px;">Model 4</td>
			<td>Flow scintillator - 500 &#181;L flow cell; alternative Radiomatic Flow Scintillator Analyser by Perkin Elmer&#160;</td>
		</tr>
	</tbody>
</table>

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.

使用这种方法，酵母PtdInsPs进行代谢标记用 3 H- 肌醇 。标记后，磷脂沉淀，用高氯酸，接着脱酰磷脂和提取的水溶性格罗-InsPs（ 图1）。在这个阶段，重要的是要量化与所提取格罗-InsPs通过液体闪烁相关的总放射性信号，以确保有足够的信噪比为非常低丰度PtdInsPs像磷脂酰肌醇（3,5）P 2;总共5-10万的CPM应注射。由于酵母细胞表现出只有四个PtdInsPs，分辨率可以如所述（图2）中，使其与1小时洗脱法。 这里，野生型，atg18Δ和vac14Δ酵母突变体标记和处理，以分析磷脂酰肌醇的变化（3,5）P 2，第ê最丰富的酵母16,19,27的PtdInsPs的。如观察到的在图4A-C中，为产生3 H-相关信号峰值的所有三个菌株的洗脱曲线。亲格罗宏峰首先洗脱（8-9分钟; 在图3中示出，但没有在图4自格罗宏黯然失色磷酸化物种的信号），接着格罗-Ins3P（〜18分钟），GRO- Ins4P（约20分钟），格罗宏（3,5）P2（〜29分钟）和格罗宏（4,5）P2（〜32:00分），它们分别对应于酰化的磷脂酰肌醇磷脂， PtdIns3P，PtdIns4P，磷脂酰肌醇（3,5）P 2和磷脂酰肌醇（4,5）P2。有在4分钟和一个趋于紧跟在父格罗项（10分钟）的几个额外小峰;这些可能代表肌醇和非glycerated肌醇磷酸盐（图3）。洗脱模式是一致的特征，虽然采用迪菲时的确切时间可能有所不同租HPLC系统和/或一个新列。 与磷脂酰肌醇（3,5）P 2（图4，红色箭头）相关的峰经历野生型之间的最显着的变化，atg18Δ和vac14Δ酵母菌株。相对于野生型细胞（A）中，有一个非常大的格罗项（3,5）中的p atg18Δ细胞2机相关信号和在vac14Δ酵母细胞一个较小的峰。为了量化与每个峰相关的总放射性信号，所述计数的峰（图3）的区域下结合。另外，由于样品之间的绝对放射性信号变化时，亲本格罗宏物种用作由人反对正火（1也可以选择对正常化总计数）内部对照。通过这样做，数据表明，磷脂酰肌醇（3,5）P 2只占0.07％，在野生型酵母细胞磷脂酰肌醇，而磷脂酰肌醇（3,5）p 2对应于亲磷脂酰肌醇中atg18Δ细胞，或在磷脂酰肌醇戏剧性17倍的增加（3,5）P 2相对于野生型细胞（图4A，B和D）的1.2％。相比较而言，磷脂酰肌醇（3,5）P 2的水平仅为0.01％在VAC14的磷脂酰肌醇信号-deleted酵母细胞，在磷脂酰肌醇（3,5）P 2（图4C和D）一种 85％的损失。总体而言，这些测量是在与已发表 ​​的结果显示，磷脂酰肌醇（3,5）P 2为约0.1％在野生型细胞磷脂酰肌醇的协议，90％减少vac14 D和在atg18 D细胞10到20倍相对于野生型细胞27,29。 <img alt="图1" src="/files/ftp_upload/53529/53529fig1.jpg" /> 图1.脱酰化和提取的 3 H- 格罗-INSP。放射性标记的梨皮在高氯酸ð沉淀用的EDTA水溶液。这然后吸出，并加入去酰基化试剂和培养在53℃。脱酰化反应混合物，然后真空干燥，并用水洗涤两次，然后加入萃取试剂。这然后涡旋，离心，水相被收集。提取步骤重复三次总以除去有机和不溶性杂质。水溶性部分（蓝色），其中包括3 H-格罗-INSP，然后真空干燥和再水化60微升水中。放射性的量由液体闪烁（CPM）和整个样本数量相等加载到高效液相色谱法测定。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53529/53529fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53529/53529fig2.jpg" /> &lt;BR /&gt; 图2.梯度为 GRO-督察3 H-GRO-督察，分辨率洗脱铵磷酸盐缓冲液强阴离子交换层析取决于磷酸氢二铵，pH值3.8（缓冲液B）的应用程序。梯度的选择取决于格罗-INSP物种可用于分离混合物中。 议定书A用于酵母样本，其中具有只有四个PtdInsPs。每四个峰的分辨率足以在迅速增加（NH 4）2 HPO 4浓度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53529/53529fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53529/53529fig3.jpg" /> 图3.引导指令PtdInsP大量的数据分析，数据文件是在劳拉小号装直观地使用色谱仪（默认开启）oftware和评估。 "在缩放"工具使用（一），放大峰（B）。选择使用"添加投资回报率"工具（三）色谱明显的峰值。在"区域表"标签（d）中，从感兴趣的高亮区域的所有所得信息显示为一个单一的表（E）。导出的每个峰（F）的原始计数和规范各PtdInsP品种对父母的磷脂酰肌醇（g）或反对总计数。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53529/53529fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53529/53529fig4.jpg" /> 图4.磷脂酰肌醇（3,5）P 2 的水平在野生型和突变体酵母细胞。代表性的色谱示于提取物的流动闪烁ED 3 H-GRO-InsPs从野生型（A），atg18Δ（B），和vac14Δ（C）使用协议酵母株。从感兴趣相当于GRO宏区域的原始计数（3,5） P 2（箭头）中提取从色谱仪和背景减去。将所得的值进行比较，以野生型和表示为倍数变化在格罗项（3,5）P 2水平（D）。水平及变化格罗宏（3,5）P2被解释为显示水平和改变原来的磷脂酰肌醇（3,5）P2。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53529/53529fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><colgroup><col /></colgroup><tbody><tr height="24"><td height="24" style="height:24px;width:401px;"> 肌醇自由媒体（IFM） </td></tr><tr height="21"><td height="21" style="height:21px;"> 27.8毫硫酸铵</td></tr><tr height="21"><td height="21" style="height:21px;"> 2％葡萄糖</td></tr><tr height="21"><td height="21" style="height:21px;"> 0.01毫米磷酸二氢钾</td></tr><tr height="21"><td height="21" style="height:21px;"> 1X氨基酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 1X LPSM </td></tr><tr height="21"><td height="21" style="height:21px;"> 1X微量元素</td></tr><tr height="21"><td height="21" style="height:21px;"> 5X维生素</td></tr><tr height="24"><td height="24" style="height:24px;"> 10X磷酸和硫酸低介质（LPSM） </td></tr><tr height="21"><td height="21" style="height:21px;"> 9毫米氯化钙</td></tr><tr height="21"><td height="21" style="height:21px;"> 17毫米氯化钠</td></tr><tr height="21"><td height="21" style="height:21px;"> 67毫米氯化钾</td></tr><tr height="21"&gt; <td height="21" style="height:21px;"> 63毫米氯化镁</td></tr><tr height="24"><td height="24" style="height:24px;"> 1,000X微量元素 </td></tr><tr height="21"><td height="21" style="height:21px;"> 8毫硼酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 250μM硫酸铜</td></tr><tr height="21"><td height="21" style="height:21px;"> 602μMKI </td></tr><tr height="21"><td height="21" style="height:21px;"> 1.23毫米三氯化铁</td></tr><tr height="21"><td height="21" style="height:21px;"> 2.65毫摩尔硫酸锰</td></tr><tr height="21"><td height="21" style="height:21px;"> 971微米的钠钼酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 2.48毫摩尔硫酸锌</td></tr><tr height="24"><td height="24" style="height:24px;"> 500X维生素 </td></tr><tr height="21"><td height="21" style="height:21px;"> 4μM生物素</td></tr><tr高度="21"&gt; <td height="21" style="height:21px;"> 839μM钙泛酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 2.27μM叶酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 1.62毫米烟酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 729μM对氨基苯甲酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 973μMPryidoxine盐酸</td></tr><tr height="21"><td height="21" style="height:21px;"> 266微米的核黄素</td></tr><tr height="21"><td height="21" style="height:21px;"> 593μM硫胺盐酸</td></tr></tbody></table> 表1的组成酵母肌醇的培养基。酵母细胞被放射性标记与IFM为基础介质要与3 H- 肌醇补充。准备的100ml溶液IFM用该溶液的指示，过滤消毒用瓶盖0.22微米真空过滤和储存在室温。制备低磷酸盐和硫酸盐媒体（LPSM），使用指示的盐生成的100毫升的溶液，过滤消毒用瓶顶0.22微米真空过滤器，并储存在室温。溶解所指示的盐以制备微量元素的1L 1000倍的溶液。使用和/或储存分装于-20℃之前，调匀。化解表明维生素准备一个1升500倍的维生素解决方案。之前使用和储存分装于-20℃调匀。

Watch this Scientific Journal Video about Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation at JoVE.com

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

磷酸肌醇的信号脂类的相对丰度变化迅速响应各种刺激。本文通过代谢标记的细胞用 3 H- 肌醇 ，随后萃取和脱酰描述了一种用于测量磷酸肌醇的丰度的方法。萃取甘油基肌醇然后通过高效液相色谱法分离，并通过流式细胞闪烁定量。

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 ...

radiolabeling-quantification-cellular-levels-phosphoinositides-high

Nanyang Technological University

Research

JoVE Journal

Chemistry

10.3K 次观看.  Ryerson University. 磷酸肌醇的信号脂类的相对丰度变化迅速响应各种刺激。本文通过代谢标记的细胞用 3 H- 肌醇 ，随后萃取和脱酰描述了一种用于测量磷酸肌醇的丰度的方法。萃取甘油基肌醇然后通过高效液相色谱法分离，并通过流式细胞闪烁定量。 

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

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 ...

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 ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

柱后衍生用反应流高效液相色谱柱

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

高密度条码抗体芯片的水流模式的指导下制作

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

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 ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

简捷方法对硫甙的提取与分析用高压液相色谱法（HPLC）

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and non-porous supports (polymer, ceramic, metal, carbon, and graphene). We developed a novel crystallization technique, which is termed the ENACT approach: the electrophoretic nuclei assembly for the crystallization of highly intergrown thin films (ENACT). This approach allows for a high density of heterogeneous nucleation of MOFs on a chosen substrate via the electrophoretic deposition (EPD) directly from the precursor sol. The growth of well-packed MOF nuclei leads to a highly intergrown polycrystalline MOF film. We show that this simple approach can be used for the synthesis of thin, intergrown zeolite imidazole framework (ZIF)-7 and ZIF-8 films. The resulting 500 nm-thick ZIF-8 membranes show a considerably high H2 permeance (8.3 x 10-6 mol m-2 s-1 Pa-1) and ideal gas selectivities (7.3 for H2/CO2, 15.5 for H2/N2, 16.2 for H2/CH4, and 2655 for H2/C3H8). An attractive performance for C3H6/C3H8 separation is also achieved (a C3H6 permeance of 9.9 x 10-8 mol m-2 s-1 Pa-1 and a C3H6/C3H8 ideal selectivity of 31.6 at 25 &#176;C). Overall, the ENACT process, owing to its simplicity, can be extended to synthesize intergrown thin films of a wide range of nanoporous crystalline materials.

A simple, reproducible, and versatile approach for the synthesis of intergrown, polycrystalline metal-organic framework membranes on a wide range of unmodified porous and non-porous supports is presented.

超薄高性能金属-有机骨架膜的电泳结晶

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and ...