Name: radiolabeling and quantification of cellular levels of phosphoinositides by high performance liquid chromatography-coupled flow scintillation
Uploaded: 2016-01-06T14:00:01
Description: 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

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.

Note: The text below describes in detail a method to measure PtdInsPs in yeast. It provides the experimental details for labeling yeast cells with 3H-myo-inositol, extracting and deacylating lipids and an HPLC-elution protocol to fractionate and quantify deacylated PtdInsPs. Please note that labeling, deacylation, resolution and quantification of PtdInsPs in mammalian cells require optimization and longer HPLC-elution profiles. These details can be found elsewhere, though we discuss some of aspects in...

This article details the experimental protocol required to quantify cellular levels of PtdnsPs by HPLC-coupled flow scintillation from yeast. The methodology enables the metabolic labeling of PtdInsPs with 3H-myo-inositol, followed by lipid processing and extraction of water-soluble 3H-Gro-InsPs, HPLC fractionation and analysis. Using this method, the relative levels of PtdInsPs in cells under various conditions can be quantified, as is shown for PtdIns(3,5)P2 in wild-type, v...

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.

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			<td height="21" style="height:21px;width:421px;">Ultima Gold</td>
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</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.

Using this method, yeast PtdInsPs were metabolically labelled with 3H-myo-inositol. After labeling, the phospholipids were precipitated with perchloric acid, followed by phospholipid deacylation and extraction of the water-soluble Gro-InsPs (Figure 1). At this stage, it is important to quantify the total radioactive signal associated with the extracted Gro-InsPs by liquid scintillation to ensure sufficient signal-to-noise ratio for the very low-abundan...

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

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

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

The overall goal of this procedure is to measure the amount of each phosphoinositide species in cells under various genetic and environmental conditions in order to understand phosphoinositide function and regulation. Phosphoinositide lipids control cell functions like migration, replication, and memory trafficking. This method identifies genetic and environmental conditions that impact phosphoinositides and increases our knowledge about their regulation and function.The main advantage of this technique is that it permits accurate, concurrent measurement of all phosphoinositides inside cells. Though this method can provide insight into the regulation of phosphoinositides in yeasts, it can also be applied to cultured mammalian cells. Individuals new to this method will struggle because of the many steps required for labeling, processing, and analysis.We recommend practicing without the radioactive label first. To begin, grow a 20 milliliter liquid culture of the yeast strain SEY6210, in complete synthetic medium at 30 degrees Celsius with constant shaking to mid-log phase. Next, transfer a total of 10 to 14 OD of the yeast cells to a 12 milliliter round bottom centrifuge tube and centrifuge at 800 times G for five minutes.Decant the medium. And re-suspend the pellet in 2 milliliters of inositol-free medium. Centrifuge the cells again.And then re-suspend the pellet in 440 microliters of inositol-free medium. Incubate the cells at room temperature for 15 minutes. After the incubation, add 60 microliters of tritium labeled myo-inositol and grow the cells for an additional one to three hours at 30 degrees Celsius with constant shaking.Transfer the cell suspension to a microcentrifuge tube containing 500 microliters of of 9%perchloric acid and 200 microliters of acid washed glass beads. Mix by repeatedly inverting the tube, and then incubate on ice for five minutes. Afterward, vortex the tube at maximum speed for 10 minutes.Then transfer the lysate to a new microcentrifuge tube using a gel loading tip to avoid aspirating the glass beads. Next, centrifuge the tube and discard the supernatant. Sonicate the pellet in one milliliter of ice cold 100 millimolar EDTA until it is completely dispersed, and centrifuge again.After centrifuging, aspirate the EDTA from the tube containing the radio-labeled pellet. Add 50 microliters of water and sonicate to re-suspend the pellet. Prepare fresh deacylation reagent according to the text protocol.Then add 500 microliters of the deacylation reagent to the tube, and sonicate to mix. Incubate the sample at room temperature for 20 minutes. Next, transfer the sample to a heating block at 53 degrees Celsius for 50 minutes.Afterward, place the sample in a vacuum centrifuge and allow it to dry for a minimum of three hours. Re-suspend the pellet by sonicating in 300 microliters of water, and incubate it at room temperature for 20 minutes. Afterwards, dry the sample completely in a vacuum centrifuge for a minimum of three hours.After drying, add 450 microliters of water to the tube and re-suspend the pellet by sonicating until it is completely dispersed. Prepare fresh extraction reagent according to the text protocol. Add 300 microliters of extraction reagent and vortex at maximum speed for five minutes.Centrifuge the tube at 18000 times G for two minutes, and carefully pipette the bottom aqueous layer into a new tube. Next, dry the final aqueous fraction by vacuum centrifugation for a minimum of three hours. Afterward, fully disperse the pellet by sonicating in 50 microliters of water, and store the sample at 20 degrees Celsius.Finally, determine the radioactivity of the sample by adding two microliters to four milliliters of scintillation fluid in a 6 milliliter polyethylene scintillation vial. Record the CPM of the vial using a liquid scintillation counter with an open window. To set up the HPLC system for separation of the tritium labeled glycero-inositides, first prepare buffers A and B, and chromatography column according to the text protocol.Next, load 10 million CPM of the sample into a two milliliter injection vial, fitted with a spring-loaded 250 microliter insert, and add water for a total volume of 55 microliters. Cap the vial with a screw cap outfitted with a PTFE silicone septum and place it into the auto-sampling tray of the HPLC system, along with a water blank in a similar vial. Using the software on the HPLC system, program an elution protocol.One percent buffer B for five minutes, one to 20 percent B for forty minutes, 20 to 100 percent B for ten minutes, 100%B for five minutes, 100 to one percent B for twenty minutes, and then one percent B for ten minutes. Set the pump flow rate at 1.0 milliliters per minute over 90 minutes, and the pressure limit at 400 bar. Follow this by setting up a detection protocol on the flow scintillator to run for 60 minutes, with the scintillation fluid flow rate of 2.5 per minute, and a dwell time of 8.57 seconds.Next, program an automated injection sequence on the HPLC to begin with the water blank running the equilibration protocol, followed by the radio-labeled sample on the elution protocol. Before the equilibration protocol is completed, create a batch sequence on the flow scintillator to measure all the samples using the detection protocol, which is triggered by the elution protocol. To analyze the HPLC data, first open the raw data file for each sample, using the chromatograph quantitation software.In the chromatograms tab, zoom in to stretch the lesser peaks while maintaining the time resolution. Use the add ROI tool to highlight each peak for analysis, and then identify the peaks based on their elution time. Use the regions table tab to locate and record the area of each peak.At the same time, note the start and end time of each peak. Next, perform a background subtraction for each peak by highlighting a region adjacent to the peak, spanning the same amount of time. Using the spreadsheet software, subtract the number of counts for that region from the counts of the corresponding peak.Normalize the area of each peak against the parental glycerated inositol peak, and record as percent of the parental phosphatidylinositol. Then normalize each of the peaks in each experimental condition against the control condition, and express as the end fold increase compared to the control. Export the files by clicking on File, and Save As, and choosing the CSV format.Finally, open the CSV file in a spreadsheet program to plot the data. The analysis of radio-labeled yeast phosphoinositides was performed by HPLC. Since yeast cells generate only four phosphoinositides, resolution can be achieved with a one hour double gradient elution method.The glycerated inositol peak alluding at eight to nine minutes overshadowed the signal from all the other phosphorylated species. Therefore, it is necessary to zoom in on the portion of the trace containing the other peaks before integrating the radioactive signals. Wild-type, ATG18 deleted, and VAC14 deleted yeast strains were assayed for changes in phosphatidylinositol 3, 5-bisphosphate using this HPLC method.The ATG18 deleted mutant was shown to produce the most phosphatidylinositol 3, 5-bisphosphate and the VAC14 deleted mutant produced the least. Once mastered, this technique can be done in four days if it is done properly. While attempting this procedure, it is important to remember to optimize growth and labeling conditions based on the specific yeast strain or cell types.Following this procedure, other methods, like GFP-fused biosensors, can be used to complement HPLC flow scintillation for detection of phosphoinositides. After watching this video, you should have a good understanding of how to radio label, extract and measure phosphoinositides by HPLC coupled flow scintillation. Don't forget that working with radioactivity and organic solvents can be extremely hazardous, and precautions such as wearing personal protective equipment and proper training should always be taken while performing this procedure.

Research

JoVE Journal

Chemistry

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (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 ...

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

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 ...

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 ...

Synthesis and Catalytic Performance of Gold Intercalated in the Walls of Mesoporous Silica

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties ...

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 ...

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 ...

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

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 ...

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 ...

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

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 ...

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 ...