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

摘要

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。

摘要

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.

引言

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 activity^1-3. 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 membrane^3,4. 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 PtdInsP^3,4. Hence, misregulation of the PtdInsP regulatory machinery can perturb cell function, leading to diseases such as cancer and degenerative diseases^2,5,6. 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 domain^7-9. 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 imaging^1,10. For example, the Pleckstrin homology (PH) domain of phospholipase C δ1 (PLCδ1) 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 endosomes^10-13. 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 PtdInsP^14-16. 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 PtdInsPs^14,17. However, mass spectrometry is mostly semi-quantitative and it remains difficult to resolve and concurrently quantify PtdInsP species of the same molecular weight^14,17. 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 PtdIns^16,19,20. 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 mammals^22-24. 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 mammals^25,26. In VAC14-deleted yeast cells, Fab1 does not function efficiently leading to a 90% decrease in PtdIns(3,5)P₂ levels^27,28. 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 levels^29,30. 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, vac14Δ and atg18Δ 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.

研究方案

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

1. 细胞培养及放射性标记
   1. 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。
   2. 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）。
   3. 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。
   4. 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。
   5. 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。
   6. 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。
   7. 沉淀将样品在12000×g离心10分钟，在4℃，吸出上清液。
   8. 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。
2. 去酰基化和提取
3. 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。
4. 吸出EDTA和超声处理将沉淀于50μl水中。
5. 加入500μl脱酰试剂和超声处理混合。在室温下孵育20分钟。
6. 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。
   1. 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。
7. 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。
8. 超声处理以450微升水沉淀。这是提取期间水相。
9. 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。
10. 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。
11. 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。
12. 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。
13. 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。
14. 储存在-20℃的样本，直到准备用于HPLC。

分离^{3 H-}甘油基肌醇通过HPLC

1. 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。
2. 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。
3. 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。
4. 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。
5. 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。
   1. 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。
   2. 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。
6. 初始化四元泵以1.0毫升/分钟用100％缓冲液A的流速
7. 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。
8. 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。
9. 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。
10. 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。
11. 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 。"
12. 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。

数据分析

1. 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。
2. 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。
3. 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。
4. 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。
5. 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。
6. 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。
7. 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。
8. 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。

结果

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?...

讨论

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）。

材料

Name	Company	Catalog Number	Comments
1-Butanol	Biobasic	BC1800	Reagent grade
Ammonium phosphate dibasic	Bioshop	APD001	ACS grade
Ammonium sulfate	Biobasic	ADB0060	Ultra Pure grade
Autosampler	Agilent	G1329B	Agilent 1260 infinity series
Biotin	Sigma	B4501
Boric acid	Biobasic	BB0044	Molecular biology grade
Calcium Chloride	Biobasic	CT1330	Ahydrous, industrial grade
Calcium pantothenate	Sigma	C8731
Copper(II) sulfate	Sigma	451657	Anhydrous
D-Glucose	Biobasic	GB0219	Anhydrous, biotech grade
Dulbecco's modification of Eagle's Medium	Life	11995-065	With 4.5 g/L glucose, 110 mg/L pyruate, L-glutamine
Dulbecco's modification of Eagle's Medium	MP biomedicals	0916429	With 4.5 g/L glucose, without L-gluatmine, without inositol
EDTA	Biobasic	EB0107	Acid free, ultra pure grade
Ethyl ether	Caledon labs	1/10/4700	Anhydrous, reagent grade
Ethyl formate	Sigma	112682	Reagent grade
Fetal bovine serum	Wisent	080-450	US origin, premium quality, heat inactivated
Fetal Bovine Serum, Dialyzed	Life	26400044	US origin
FlowLogic U	LabLogic Systems Ltd	SG-BXX-05	Scintillation fluid for flow scintillation
Folic acid	Biobasic	FB0466	USP grade
HEPES buffer solution	Life	15630080	1 M solution
Inositol, Myo-[2-3H(N)]	Perkin Elmer	NET114005MC	9:1 ethanol to water
Insulin-Transferrin-Selenium-Ethanolamine	Life	51500056	100x solution
Iron(III) chloride	Sigma	157740	Reagent grade
Laura - Chromatography data collection and analysis software	LabLogic Systems Ltd	Version 4.2.1.18	Flow scintillator software
L-glutamine	Sigma	G7513	200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Magnesium Chloride	Sigma	M8266	Anhydrous
Manganese sulfate	Biobasic	MB0334	Monohydrate, ACS grade
Methanol	Caledon labs	6701-7-40	HPLC Grade
Methylamine solution	Sigma	426466	40% (v/v)
Monopotassium phosphate	Biobasic	PB0445	Anhydrous, ACS grade
Nicotinic acid	Biobasic	NB0660	Reagent grade
OpenLAB CSD ChemStation	Agilent	Rev. C.01.03	HPLC software
p-aminobenzoic acid (PABA)	Bioshop	PAB001.100	Free acid
Penicillin-Streptomycin	Sigma	P4333	100X, liquid, stabilized, sterile-filtered, cell culture tested
Perchloric acid	Sigma	244252	ACS reagent, 70%
PhenoSpher SAX column	Phenomenex	00G-315-E0	5 µm, 80 Å, 250 x 4.6 mm
Phosphoric acid	Caledon labs	1/29/8425	Reagent grade
Potassium Chloride	Biobasic	PB0440	ACS grade
Potassium iodide	Biobasic	PB0443	ACS grade
Pyridoxine hydrochloride	Sigma	P9755
Quaternary pump	Agilent	G1311C	Agilent 1260 infinity series
Riboflavin	Bioshop	RIB333.100	USP grade
Sodium Chloride	Biobasic	DB0483	Biotech grade
Sodium molybdate	Sigma	243655
Thermostatted Column Compartment	Agilent	G1316A	Agilent 1260 infinity series
Thiamine hydrochloride	Sigma	T4625	Reagent grade; make solution of 0.02% (w/v), forms a suspension. mix and freeze aliquots
Ultima Gold	Perkin Elmer	6013321	Scintillation coctail for liquid scintillation counting
Zinc sulfate	Biobasic	ZB2906	Heptahydrate, reagent grade
β-RAM 4	IN/US systems	Model 4	Flow scintillator - 500 µl flow cell; alternative Radiomatic Flow Scintillator Analyser by Perkin Elmer