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）。 <p c...

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><tbody><tr><td>1-Butanol</td><td>Biobasic</td><td>BC1800</td><td>Reagent grade</td></tr><tr><td>Ammonium phosphate dibasic</td><td>Bioshop</td><td>APD001</td><td>ACS grade</td></tr><tr><td>Ammonium sulfate</td><td>Biobasic</td><td>ADB0060</td><td>Ultra Pure grade</td></tr><tr><td>Autosampler</td><td>Agilent</td><td>G1329B</td><td>Agilent 1260 infinity series</td></tr><tr><td>Biotin</td><td>Sigma</td><td>B4501</td><td></td></tr><tr><td>Boric acid</td><td>Biobasic</td><td>BB0044</td><td>Molecular biology grade</td></tr><tr><td>Calcium Chloride</td><td>Biobasic</td><td>CT1330</td><td>Ahydrous, industrial grade</td></tr><tr><td>Calcium pantothenate</td><td>Sigma</td><td>C8731</td><td></td></tr><tr><td>Copper(II) sulfate</td><td>Sigma</td><td>451657</td><td>Anhydrous</td></tr><tr><td>D-Glucose</td><td>Biobasic</td><td>GB0219</td><td>Anhydrous, biotech grade</td></tr><tr><td>Dulbecco&#039;s modification of Eagle&#039;s Medium</td><td>Life</td><td>11995-065</td><td>With 4.5 g/L glucose, 110 mg/L pyruate, L-glutamine</td></tr><tr><td>Dulbecco&#039;s modification of Eagle&#039;s Medium</td><td>MP biomedicals</td><td>0916429&amp;nbsp;</td><td>With 4.5 g/L glucose, without L-gluatmine, without inositol</td></tr><tr><td>EDTA</td><td>Biobasic</td><td>EB0107</td><td>Acid free, ultra pure grade</td></tr><tr><td>Ethyl ether</td><td>Caledon labs</td><td>1/10/4700</td><td>Anhydrous, reagent grade</td></tr><tr><td>Ethyl formate</td><td>Sigma</td><td>112682</td><td>Reagent grade</td></tr><tr><td>Fetal bovine serum</td><td>Wisent</td><td>080-450</td><td>US origin, premium quality, heat inactivated</td></tr><tr><td>Fetal Bovine Serum, Dialyzed</td><td>Life</td><td>26400044</td><td>US origin</td></tr><tr><td>FlowLogic U</td><td>LabLogic Systems Ltd</td><td>SG-BXX-05</td><td>Scintillation fluid for flow scintillation&amp;nbsp;</td></tr><tr><td>Folic acid</td><td>Biobasic</td><td>FB0466</td><td>USP grade</td></tr><tr><td>HEPES buffer solution</td><td>Life</td><td>15630080</td><td>1 M solution</td></tr><tr><td>Inositol, Myo-[2-3H(N)]</td><td>Perkin Elmer</td><td>NET114005MC</td><td>9:1 ethanol to water</td></tr><tr><td>Insulin-Transferrin-Selenium-Ethanolamine</td><td>Life</td><td>51500056</td><td>100x solution</td></tr><tr><td>Iron(III) chloride</td><td>Sigma</td><td>157740</td><td>Reagent grade</td></tr><tr><td>Laura - Chromatography data collection and analysis software</td><td>LabLogic Systems Ltd</td><td>Version 4.2.1.18</td><td>Flow scintillator software</td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G7513</td><td>200&amp;nbsp;mM, solution, sterile-filtered, BioXtra, suitable for cell culture</td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M8266</td><td>Anhydrous</td></tr><tr><td>Manganese sulfate</td><td>Biobasic</td><td>MB0334</td><td>Monohydrate, ACS grade</td></tr><tr><td>Methanol</td><td>Caledon labs</td><td>6701-7-40</td><td>HPLC Grade</td></tr><tr><td>Methylamine solution</td><td>Sigma</td><td>426466</td><td>40% (v/v)</td></tr><tr><td>Monopotassium phosphate</td><td>Biobasic</td><td>PB0445</td><td>Anhydrous, ACS grade</td></tr><tr><td>Nicotinic acid</td><td>Biobasic</td><td>NB0660</td><td>Reagent grade</td></tr><tr><td>OpenLAB CSD ChemStation&amp;nbsp;</td><td>Agilent</td><td>Rev. C.01.03&amp;nbsp;</td><td>HPLC software</td></tr><tr><td>p-aminobenzoic acid (PABA)</td><td>Bioshop</td><td>PAB001.100</td><td>Free acid</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td>100X, liquid, stabilized, sterile-filtered, cell culture tested</td></tr><tr><td>Perchloric acid</td><td>Sigma</td><td>244252</td><td>ACS reagent, 70%</td></tr><tr><td>PhenoSpher SAX column</td><td>Phenomenex</td><td>00G-315-E0</td><td>5 &amp;micro;m, 80 &amp;Aring;, 250 x 4.6 mm</td></tr><tr><td>Phosphoric acid</td><td>Caledon labs</td><td>1/29/8425</td><td>Reagent grade</td></tr><tr><td>Potassium Chloride</td><td>Biobasic</td><td>PB0440</td><td>ACS grade</td></tr><tr><td>Potassium iodide</td><td>Biobasic</td><td>PB0443</td><td>ACS grade</td></tr><tr><td>Pyridoxine hydrochloride</td><td>Sigma</td><td>P9755</td><td></td></tr><tr><td>Quaternary pump</td><td>Agilent</td><td>G1311C</td><td>Agilent 1260 infinity series</td></tr><tr><td>Riboflavin</td><td>Bioshop</td><td>RIB333.100</td><td>USP grade</td></tr><tr><td>Sodium Chloride</td><td>Biobasic</td><td>DB0483</td><td>Biotech grade</td></tr><tr><td>Sodium molybdate</td><td>Sigma</td><td>243655</td><td></td></tr><tr><td>Thermostatted Column Compartment</td><td>Agilent</td><td>G1316A</td><td>Agilent 1260 infinity series</td></tr><tr><td>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><td>Ultima Gold</td><td>Perkin Elmer</td><td>6013321</td><td>Scintillation coctail for liquid scintillation counting</td></tr><tr><td>Zinc sulfate</td><td>Biobasic</td><td>ZB2906</td><td>Heptahydrate, reagent grade</td></tr><tr><td>&amp;beta;-RAM 4</td><td>IN/US systems</td><td>Model 4</td><td>Flow scintillator - 500 &amp;micro;l flow cell; alternative Radiomatic Flow Scintillator Analyser by Perkin Elmer&amp;nbsp;</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?...

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

Universidad San Sebastian

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

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

科研

生物化学

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

使用SAX-HPLC从不同植物物种中提取和定量可溶性、放射性标记肌醇多磷酸盐

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

生物学

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

毛细管电泳电喷雾电离质谱法绝对定量肌醇焦磷酸盐

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

使用定量流式细胞术分析荧光标记蛋白与人工磷脂微泡的相互作用

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

生物工程

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

<div class="chapter_text" id="script_0">
Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
</div>
<div class="chapter_text" id="script_1">
Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
</div>
<div class="chapter_text" id="script_2">
The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
</div>
<div class="chapter_text" id="script_3">
In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
</div>

Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

代谢标记

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...