Name: radiolabeling and quantification of cellular levels of phosphoinositides by high performance liquid chromatography-coupled flow scintillation
Uploaded: 2016-01-06T14:00:01.000+00:00
Duration: 10 min 52 s
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 the Discussion. Overall, the methodology to extract lipids, deacylate and extract the water-soluble Gro-InsPs from yeast is given and is illustrated in Figure 1.
1. Protocol for Labeling and Analysing PtdInsPs in Yeast
<ol>
	<li>Cell culture and radiolabeling
	<ol>
		<li>Grow a 20 ml liquid culture of the yeast strain SEY6210 at 30 &#176;C with constant shaking in complete synthetic media to mid-log phase or an optical density at 600 nm (OD600) of approximately 0.6-0.7. 
		Note: Do not use YPD media as this may affect 3H-myo-inositol incorporation. This culture size should provide sufficient material for 2-3 HPLC runs.</li>
		<li>Pellet a total of 10-14 OD of the yeast cells (note that 1 ml culture with an OD600 = 1 contains ~1x107 cells) by centrifugation at 800 x g for 5 min in a 12 ml round-bottom tube. Resuspend with 2 ml of inositol-free media (IFM) (see Table 1 for IFM composition).</li>
		<li>Repeat centrifugation and aspirate the media. Resuspend the pellet with 440 &#181;l of IFM and incubate at RT&#160;for 15 min.</li>
		<li>Add 60 &#181;l of 3H-myo-inositol (1 &#181;l = 1 &#181;Ci) to each sample and incubate at the growing temperature of 30 &#176;C for 1 to 3 hr with constant shaking. 
		Caution: 3H-myo-inositol is a radioactive material that should be handled after appropriate training. In addition, all consumables that make contact with 3H-containing material should be disposed appropriately and designated as radioactive waste. 
		Note: The growth temperature can be changed as required as long as all strains to be compared are grown at the same temperature.</li>
		<li>Transfer the cell suspension (500 &#181;l) to a microcentrifuge tube containing 500 &#181;l of 9% perchloric acid and 200 &#181;l of acid washed glass beads. Mix by inversion and incubate on ice for 5 min.</li>
		<li>Vortex the sample at top speed for 10 min and transfer the lysates to a new microcentrifuge tube using a gel-loading tip to avoid the glass beads.</li>
		<li>Pellet the sample at 12,000 x g for 10 min at 4 &#176;C and aspirate the supernatant.</li>
		<li>Resuspend the pellet by bath sonication in 1 ml of ice-cold 100 mM EDTA. Pellet again as before.</li>
	</ol>
	</li>
	<li>Deacylation and extraction
	<ol>
		<li>Freshly prepare 5.0 ml of the deacylation reagent by combining 2.3 ml of methanol, 1.3 ml of 40% methylamine, 0.55 ml of 1-butanol and 0.80 ml of water. Invert to mix.</li>
		<li>Aspirate the EDTA and sonicate the pellet in 50 &#181;l of water.</li>
		<li>Add 500 &#181;l of the deacylation reagent and sonicate to mix. Incubate at RT for 20 min.</li>
		<li>Heat lipids for 50 min at 53 &#176;C in a heat block. Dry the samples completely by vacuum centrifugation over 3 hr or O/N.
		<ol>
			<li>Ensure that a chemical trap is installed between the vacuum-centrifuge and the vacuum pump to prevent vapours from entering the air.</li>
		</ol>
		</li>
		<li>Resuspend the pellet with 300 &#181;l of water by bath sonication and incubate at RT for 20 min. Dry the samples by vacuum centrifugation for 3 hr or O/N. Repeat this step once more.</li>
		<li>Sonicate the pellet with 450 &#181;l of water. This is the aqueous phase during extraction.</li>
		<li>Freshly prepare 10 ml of the extraction reagent by adding 8.0 ml of 1-butanol, 1.6 ml of ethyl ether and 0.40 ml of ethyl formate. Invert to mix.</li>
		<li>Add 300 &#181;l of the extraction reagent to the aqueous phase. Vortex the mixture at top speed for 5 min and separate the layers by centrifugation at top speed (18,000 x g) for 2 min.</li>
		<li>Collect the bottom aqueous layer into a new microfuge tube while avoiding the top organic layer, the interface, and the pellet. Repeat extraction of the aqueous phase twice more with fresh extraction reagent.</li>
		<li>Completely dry the collected aqueous layer by vacuum centrifugation. Disperse the pellet in 50 &#181;l of water by bath sonication.</li>
		<li>Add 2 &#181;l of each sample to 4 ml of scintillation fluid in a 6 ml polyethylene scintillation vial. Determine the number of counts (in CPM) in each sample by liquid scintillation using an open window.</li>
		<li>Store the samples at -20 &#176;C until ready for HPLC.</li>
	</ol>
	</li>
	<li>Separation of&#160; 3H-glycero-inositides by HPLC
	<ol>
		<li>Prepare buffer A by filtering 1 L of ultrapure water with a bottle top 0.22 &#181;m vacuum filter, followed by degassing.</li>
		<li>Prepare buffer B by making a 1 L solution of 1 M ammonium phosphate dibasic (APS, MW 132.06) in water and adjust the pH to 3.8 with phosphoric acid. Filter the ammonium phosphate with a bottle top 0.22 &#181;m vacuum filter, followed by degassing.</li>
		<li>Assemble the injection vial by outfitting a 2 ml vial with a spring-loaded 250 &#181;l small volume insert. Load 10 million CPM and add water for a total volume of 55 &#181;l. Prepare a blank vial with 55 &#181;l of water.</li>
		<li>Cap the vials with screw caps outfitted with a PTFE/silicone septa (red side facing the inside of the vial). Place each vial in the auto-sampling tray of the HPLC, starting with the blank vial.</li>
		<li>Use an HPLC and associated software that controls buffer flow, degasses buffers and controls sample injection. Use an online flow scintillator and its associated software to regulate the scintillant flow rate and to monitor the 3H-decay signal.
		<ol>
			<li>Use a SAX liquid chromatography column with dimensions of 250 mm x 4.6 mm and containing 5 &#181;m silica resin in the HPLC. Outfit the column with a column guard to prevent injection of contaminants.</li>
			<li>Install a 3H-compatible 500 &#181;l flow cell in the flow scintillator. 
			NOTE: Fractionation can be done with an Agilent 1200 infinity series HPLC system controlled by Chemstation software or with other commercially available HPLC systems. Flow scintillation of the HPLC eluent can be done with a &#946;-RAM flow scintillator controlled by the Laura software or another commercially available flow scintillator or compatible software.</li>
		</ol>
		</li>
		<li>Initialize the quaternary pump at a flow rate of 1.0 ml/min with 100% buffer A.</li>
		<li>Set up an equilibration profile on the HPLC to control the gradient of Buffers A and B (call this &#34;Protocol A equilibration&#34;): 1% Buffer B for 5 min, 1-100% B for 5 min, 100% B for 5 min, 100-1% B for 5 min, 1% B for 20 min. Set the pressure limit at 400 bar, 1.0 ml/min flow rate, and 30 min run time.</li>
		<li>Set up an elution profile (Figure 2) on the HPLC to control the gradient of Buffers A and B (call this &#34;Protocol A&#34;): 1% B for 5 min, 1-20% B for 40 min, 20-100% B for 10 min, 100% B for 5 min, 100-1% B for 20 min, 1% B for 10 min. Set the pressure limit at 400 bar, 1.0 ml/min flow rate, and 90 min run time.</li>
		<li>Set up a detection protocol on the flow scintillator (call this &#34;Protocol A detection&#34;) to run for 60 min, with a scintillation fluid flow rate of 2.5 ml/min and an 8.57 sec dwell time.</li>
		<li>Program an automated injection sequence on the HPLC to begin with the water blank on &#34;Protocol A equilibration&#34;, followed by each radiolabelled sample on &#34;Protocol A&#34;. Initialize the sequence when ready.</li>
		<li>While the run is still on the equilibration protocol, create a batch on the flow scintillator to measure all of the samples with &#34;Protocol A detection.&#34; The injection of each new sample will initialize &#34;Protocol A&#34; on the HPLC while triggering the flow scintillator to begin &#34;Protocol A detection.&#34;</li>
		<li>At the completion of all runs, flush the HPLC, the column and flow scintillator with 100% Buffer A for 30 min at 1.0 ml/min flow rate.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Use Laura software or any other software that can quantify the chromatography spectra. The steps described in this section are depicted in Figure 3.</li>
		<li>Open the files by clicking &#34;File&#34;, &#34;Open&#34;, and select the raw data file for each sample.</li>
		<li>In the &#34;chromatograms&#34; tab, zoom in to stretch each of the lesser peaks (about 1000 counts [y-axis]) while retaining the time resolution. Zoom into each individual peak if necessary.</li>
		<li>Highlight each of the peaks for analysis using the &#34;Add ROI&#34; tool. Identify peaks by time of elution: Parental Gro-Ins at 10 min, Gro-Ins3P at 18 min, Gro-Ins4P at 20 min, Gro-Ins(3,5)P2 at 29 min and Gro-Ins(4,5)P2 at 32 min.</li>
		<li>In the &#34;regions tables&#34; tab, record the &#34;area (counts)&#34; of each peak. Note the &#34;start (mm:ss)&#34; time and &#34;end (mm:ss)&#34; time of each peak.</li>
		<li>For background subtraction, highlight a region adjacent to the peak spanning the same amount of time. Subtract the number of counts from the corresponding peak.</li>
		<li>Normalize the area of each peak against parental Gro-Ins (expressed as &#34;% of total PtdIns&#34;). Then, normalize each of the peaks in each experimental condition against the control condition (expressed as &#34;n-fold increase compared to control&#34;). 
		NOTE: Normalization of each peak can also be done against total counts but since the parental Gro-Ins is much more abundant than all other PtdInsPs the results tend to be similar.</li>
		<li>Export the data for the chromatographs by pressing &#34;File&#34;, &#34;Save as&#8230;&#34;, and choose the &#34;CSV (comma separated)&#34; file format. Plot the data on a spreadsheet and present as necessary.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

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.

<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.

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 ...

radiolabeling-quantification-cellular-levels-phosphoinositides-high

Research

JoVE Journal

Chemistry

10.4K Views.  Ryerson University.  Phosphoinositides signaleren lipiden waarvan de relatieve abundantie snel verandert in reactie op verschillende stimuli. Dit artikel beschrijft een methode om de overvloed aan phosphoinositides meten door metabolisch labelen van cellen met 3H-myo-inositol, gevolgd door extractie en deacylering. Geëxtraheerd glycero-inositiden omgezet worden vervolgens gescheiden door hoge-prestatie vloeistofchromatografie en gekwantificeerd door flow scintillatietell...

Video: 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.

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

Biochemistry

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.

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.

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.

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

Biology

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.

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.

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.

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.

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

Bioengineering

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.

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.

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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.
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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.
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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.
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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.
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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.
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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!
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Isotopic, Photoreactive and Bioorthogonal 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 ...

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

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