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.

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Research

JoVE Journal

Chemistry

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

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

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

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

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)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

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

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

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 of the materials were investigated. We used a one pot sol-gel approach to intercalate gold nano particles in the walls of mesoporous silica. To start with the synthesis, P123 was used as template to form micelles. Then TESPTS was used as a surface modification agent to intercalate gold nano particles. Following this process, TEOS was added in as a silica source which underwent a polymerization process in acid environment. After hydrothermal processing and calcination, the final product was acquired. Several techniques were utilized to characterize the porosity, morphology and structure of the gold intercalated mesoporous silica. The results showed a stable structure of mesoporous silica after gold intercalation. Through the oxidation of benzyl alcohol as a benchmark reaction, the GMS materials showed high selectivity and recyclability.

Here, we present a protocol with a sol-gel process to synthesize gold intercalated in the walls of mesoporous materials (GMS), which is confirmed to possess a mesoporous matrix with gold intercalated in the walls imparting great stability and recyclability.

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

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

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

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

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

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

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

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

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

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

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

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

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

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

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

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

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

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

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

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

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

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