This project has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (grant agreement no. 864246, to HJJ). DQ acknowledges the financial support from the Brigitte-Schlieben-Lange-Programm. AS is supported by the MRC program grant MR/T028904/1.

1. Setting up the CE-ESI-MS system
<ol>
	<li>Set up a CE-ESI-MS system consisting of a commercial CE system - and a triple quadrupole tandem mass spectrometer, equipped with an Agilent Jet Stream (AJS) electrospray ionization (ESI) source. A CE-ESI-MS sprayer kit and an isocratic LC (Liquid Chromatography) pump are requisite.</li>
	<li>Connect the sheath flow 1:100 splitter (included in the CE-MS sprayer kit) and the isocratic LC pump outlet.</li>
	<li>Ensure that the CE system inlet vial is at the same height as the sprayer tip of the mass analyzer.</li>
	<li>Utilize the MassHunter Workstation (Version 10.1) or comparable MS software to control the entire system, and for data acquisition and analysis.</li>
</ol>
2. Preparing buffer, capillary, and CE-MS system
<ol>
	<li>Prepare CE running buffer: Adjust the pH of 40 mM ammonium acetate to 9.0 with ammonium hydroxide. A 250 mL volumetric flask is recommended. Filter the 250 mL of buffer with 0.2 &#181;m pore-sized membrane filters. Ensure to use only ultra-pure deionized water and MS-grade reagents. 
	NOTE: This buffer can be kept at room temperature for 2-3 weeks or for several months in a fridge.</li>
	<li>Prepare sheath liquid: Mix 100 mL of ultra-pure water and 100 mL of LC-MS grade isopropanol in a 250 mL bottle. Change the sheath liquid at least once a week. Add mass reference into the sheath liquid when employing a high-resolution mass spectrometer.</li>
	<li>Install sheath liquid: Purge at 5 mL/min for 5 min and set the flow rate at 1 mL/min (10 &#181;L/min into the CE-MS sprayer). The pump pressure will be at ca. 180 bar. Ensure that the recycle tubing connects back into the sheath liquid bottle to reuse the solvent.</li>
	<li>Prepare capillary: Purchase a CE-MS capillary (50 &#181;m i.d. and 365 &#181;M o.d. with a length of 125 cm) with a UV detection window. The user can also obtain much cheaper bar-fused silica capillaries from specialized distributors. Cut a capillary with a length of 100 cm. Properly cut both capillary ends with a capillary column cutter with a rotating diamond blade and remove 2-3 cm of polyimide coating on both ends with a lighter. Clean the capillary surface with isopropanol.</li>
	<li>Install capillary: Match the capillary into the CE-MS cassette. Click on the Change Cassette button and install the cassette into the CE device. The inlet end of the capillary is around 2 mm lower than the electrode. Ensure that the inlet end is lower than the sample&#39;s surface during the injection process.</li>
	<li>Activate capillary: Prior to first use, flush the capillary with 1 M NaOH, followed by water for 10 min, and CE running buffer for 15 min.</li>
	<li>Insert the capillary end into the CE-MS sprayer: Gently put the capillary into the CE-MS sprayer and ensure that the capillary end protrudes approximately 0.1 mm out of the sprayer tip. Ensure to make precise adjustment of the capillary outlet end with a magnifying glass and the adjustment screw in the sprayer. Insert the sprayer back into the ion source and avoid touching the adjustment screw. The MS is on Standby mode when performing this operation.</li>
	<li>Check ESI spray: Check the stability of the ESI sprayer under full scan mode. The fluctuation of total ion electropherograms must be within 5%.</li>
	<li>Perform a test run with InsP standards: Employ a mixture of 2 &#181;M InsP3-InsP8 standards (adjusted by quantitative 31P NMR15,27) for test runs with an injection at 50 mbar for 10 s (10 nL). Set the detailed ESI and MS parameters as shown in Table 1. CE current is ca. 26 &#181;A. The peak width is around 0.4-0.5 min. Ensure that the signal-to-noise ratio reaches at least 400.</li>
</ol>
3. Extraction of soluble inositol phosphates from mammalian cells
NOTE: HCT116NIH cells were a kind gift from Stephen Shears28. HCT116UCL cells were from Saiardi&#39;s Lab26.
<ol>
	<li>Seeding cells
	<ol>
		<li>Culture HCT116NIH or HCT116UCL cells in T75 flasks at 37 &#176;C in a 5% high humidity CO2 atmosphere (further referred to as standard conditions) in Dulbecco&#39;s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).</li>
		<li>Wash the HCT116NIH and HCT116UCL stock cultures with phosphate-buffered saline (PBS) (5 mL) and incubate the cells with trypsin-ethylenediamine tetraacetic acid (EDTA) (3 mL, 0.25%) under standard conditions until they are completely detached. Quench trypsin activity by adding medium (7 mL), collect the cells into a centrifuge tube, and centrifuge (200 x g, 3 min).</li>
		<li>Remove the supernatant and resuspend the cells in medium (10 mL). Count the cells and determine the viability via trypan blue exclusion.</li>
		<li>Seed the cells (6 million HCT116 cells per assay) into a 150 mm dish and adjust 20 mL of cell culture medium in total. Premix the medium and the cells in a centrifuge tube prior to seeding to achieve an equal distribution of the cells in the dish. Prepare a parallel dish when normalization by cell number is required.</li>
		<li>Culture the cells under standard conditions for 72 h. The cells will reach about 80%-90% confluence.</li>
	</ol>
	</li>
	<li>Modulation of inositol phosphate levels with NaF and cell harvesting
	<ol>
		<li>NaF treatment: Add NaF (10 mM) 1 h before harvesting into the medium. Mix the medium by swirling the plate/pipetting and incubate the cells for 60 min under standard conditions.</li>
		<li>After the NaF treatment, remove the medium from the cells and place the cells on ice.</li>
		<li>Wash the cells twice with PBS (5 mL, 4 &#176;C) and remove the PBS completely from the dish.</li>
		<li>Add perchloric acid (PA) (1 mL, 1 M, 4 &#176;C). Ensure to cover the whole surface with PA (the cells will turn white as proteins precipitate). Incubate the cells for 10 min on a tilt table at 4 &#176;C.</li>
		<li>Collect PA into a centrifuge tube and remove the contaminating debris by centrifugation (17,000 x g, 5 min, 4 &#176;C). Add the supernatant to the prepared TiO2 beads for the pulldown of InsPs.</li>
		<li>Wash the post-extraction dish twice with PBS (5 mL, r.t.) for deacidification; remove PBS completely from the dish.</li>
		<li>Solubilize the proteins on the plate via the addition of cell lysis buffer (1.5 mL, r.t.; 0.1% sodium dodecyl sulfate [SDS] in 0.1 M NaOH). Incubate the dish for 15 min on a tilt table at r.t. Transfer the cell lysate into a centrifuge tube and centrifuge (17,000 x g, 5 min, 4 &#176;C). Store the supernatant at -80 &#176;C until the protein concentration is determined via the DC protein assay using bovine serum albumin as calibration standard (usually, one 150 mm dish contains around 10 mg of proteins).</li>
		<li>Determination of cell numbers from parallel dishes: Harvest the cells of the parallel dish via trypsin as described in step 3.1.2 (use 5 mL trypsin-EDTA for the 150 mm dish) and remove the medium. Resuspend the cell pellet in PBS (5 mL), mix properly, and count the cells. Perform this step right before harvest via direct quenching to obtain representative cell counts. Additionally, measure the volume of the cells with an appropriate method (e.g., with a multisizer machine).</li>
	</ol>
	</li>
	<li>TiO2 enrichment of inositol phosphates 
	NOTE: To avoid acidic decomposition of phosphorylated compounds, perform all the enrichment steps until elution on ice, and cool all reagents to 4 &#176;C. Keep the time for the extraction to a minimum (1.5-2 h). Perform all the extraction steps with 1 M PA.
	<ol>
		<li>Preparation of beads: Wash TiO2 beads (5 mg per sample) with ddH2O (1 mL) and centrifuge (3,500 x g, 1 min, 4 &#176;C). Remove ddH2O and wash the beads with PA (1 mL). Remove PA by centrifugation (3,500 x g, 1 min, 4 &#176;C). Resuspend the beads in PA (50 &#181;L per sample).</li>
		<li>Add the supernatant containing phosphorylated compounds (compare section 3.2, step 3.2.5) to the bead suspension, vortex, and then rotate the sample for 20 min at 4 &#176;C.</li>
		<li>Centrifuge the sample (3,500 x g, 1 min, 4 &#176;C) and discard the supernatant. Wash the beads with PA (500 &#181;L) and centrifuge (3,500 x g, 1 min, 4 &#176;C). Discard the supernatant and repeat the washing step.</li>
		<li>Add NH4OH (200 &#181;L, 3%) to the beads and resuspend. Rotate the sample for 5 min at r.t.</li>
		<li>Centrifuge the sample (3,500 x g, 1 min) and transfer the supernatant into a new centrifuge tube.</li>
		<li>Repeat elution steps 3.3.4 and 3.3.5 and combine the eluents. Discard the beads.</li>
		<li>Centrifuge the combined eluents (17,000 x g, 1 min, 4 &#176;C) to remove any insoluble residues.</li>
		<li>Completely dry the supernatant under vacuum evaporation (70 min, 60 &#176;C, V-AQ). Add ddH2O (50 &#181;L) to the dried extracts containing InsPs. Vortex to mix the sample until completely dissolved. Store the sample at -20 &#176;C until CE-ESI-MS analysis.</li>
	</ol>
	</li>
</ol>
4. Performing the CE-ESI-MS runs
<ol>
	<li>Prepare a mixture of internal standards containing 40 &#181;M [13C6]1,5-(PP)2-InsP4, 80 &#181;M [13C6]5-PP-InsP5, 80 &#181;M [13C6]1-PP-InsP5, 400 &#181;M [13C6]InsP6 and 400 &#181;M [13C6]Ins(1,3,4,5,6)P5. Determine the concentrations of SIL IS solutions by quantitative 31P and 1H NMR, with the aid of a certified reference standard, i.e., phosphoacetic acid. 
	NOTE: All above SIL internal standards (IS) with purities higher than 96% were synthesized and provided by Fiedler group15,27. Same as with nucleotides, these IS could carry many crystal water molecules and diverse counter ions. Instead of weighing the substance and calculating the concentration, concentration determination of 13C6 standard solutions by quantitative 31P NMR is recommended.</li>
	<li>Mix 10 &#181;L of the sample with 0.5 &#181;L of internal standards mixture in a CE sample vial. 2 &#181;M [13C6]1,5-(PP)2-InsP4, 4 &#181;M [13C6]5-PP-InsP5, 4 &#181;M [13C6]1-PP-InsP5, 20 &#181;M [13C6]InsP6, and 20 &#181;M [13C6]Ins(1,3,4,5,6)P5 are the final concentrations inside the samples.</li>
	<li>When using the replenishment system, click on the Change Bottle button, put the prepared 250 mL of CE running buffer into the electrolyte bottle, and click on Clean Tubes. Keep the replenishment needle in a water vial.</li>
	<li>Set ESI and MS parameters as shown in Table 1. Optimize the source parameters using a Source Optimizer with a mixture of inositol polyphosphate standards. Obtain the multiple reaction monitoring (MRM) settings using Masshunter Optimizer with all standards. Adjust the ESI and MS/MS settings for different instrumentation.</li>
	<li>Perform a run for the InsP extracts and check the result (Figure 2). Set a sequence when there are more samples.</li>
	<li>Let the MS be on Standby mode after measurements. Do not turn off the LC pump. The flow of sheath liquid protects the sprayer needle. Replace the CE-ESI-MS Sprayer with an LC-ESI-MS Sprayer when there is no sheath liquid supply.</li>
</ol>
5. Data analysis
<ol>
	<li>Open Quantitative Analysis (for QQQ) software, create a batch for all samples.</li>
	<li>Create a new method from the acquired MRM data. Set the [13C6]InsPs as internal standards - (ISTD). Check MRM compound setup, retention time setup, ISTD setup, concentration setup, and qualifier setup. Pass the validation and exit to apply the method to the current batch. Save the method.</li>
	<li>Check whether each peak in the batch is properly integrated; otherwise, manually integrate the peak.</li>
	<li>Export the results into a spreadsheet. Perform the quantitation of inositol (pyro)phosphates by comparing the analyte peak response with the respective peak response of SIL IS with known concentrations. Theoretical and experimental calibration curves are shown in Table 2 for 5-PP-InsP5, InsP6, and Ins(1,3,4,5,6)P5 with the linear range. 
	NOTE: The precondition applying theoretical calibration curve is the usage of a high-quality isotopic pattern of targeted analytes and with credible concentration. Evaluate the linear range. A calibration curve is essential for absolute quantitation when the full acquisition of the above-mentioned isotopic standards is impractical.</li>
	<li>With the measured concentration in the InsP extract solution and its volume, calculate the absolute amounts. Further, normalize the amount by cell counts or protein content. Calculate the cellular concentration based on cell counts and average cell volume of HCT116 (1.68 fL).</li>
</ol>

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The authors declare no competing interests.

Presented here is a practical and sensitive method for the quantitation of highly charged inositol pyrophosphates in mammalian cells. Combining this analysis approach with current state-of-the-art InsP extraction with perchloric acid followed by enrichment with TiO2, CE-ESI-MS analysis has unprecedented advantages. With regards to its throughput, sensitivity, stability, absolute quantitation, isomer identification, and matrix in-dependence, this method stands out compared to other approaches. This protocol is applicable to mammalian cells, but indeed this strategy succeeds in many different samples (e.g., yeast, plants, parasites, mouse tissues, etc.).
The applied extraction protocol fully recovers PP-InsPs and InsP6 from mammalian cell extracts16,19. It will also extract many other anionic metabolites, particularly phosphate-containing species, e.g., sugar phosphates and nucleotides. Evaluation of the recovery and decomposition for the user's analytes with this protocol would be necessary.
Generally, the CE-ESI-MS system runs smoothly and can accommodate around 200 samples every week using this protocol. Unlike HPLC, though, CE has been regarded as a method for experts and specialized persons for a long time, which restricted its market and limited its application. Thus, a CE-ESI-MS device is usually absent in analytical faculties. People who want to carry out CE-ESI-MS analysis probably lack CE experience and will spend more time troubleshooting. Here, the critical steps are highlighted. First and foremost is the quality of the capillary cut. The sensitivity and stability of ESI spray mostly rely on a first-class capillary cut. Secondly, the capillary outlet end should be exactly 0.1 mm out of the sprayer tip. The sprayer needle and the CE capillary should be in the axial direction. The quality of the ESI spray is critical for quantitation; technical runs should be performed to evaluate the repeatability. 
With the described protocol, the limit of quantitation (LOQ) for PP-InsPs is 40 nM with an injection at 50 mbar for 10 s (10 nL). There are several approaches to further increase the method sensitivity. Firstly, an injection at 100 mbar for 20 s (40 nL) will still result in a good peak shape and sufficient resolution for regioisomers 5-PP-InsP5 and 1-PP-InsP5. Secondly, InsP extracts can be dissolved in a smaller amount of water. Thirdly, the dwell time could be increased when using less MRM transitions for quantitation. In addition, a CE-MS ion source using ultra-low sheath liquid flow would significantly increase the sensitivity.
The CE running buffer with pH 9 provides the best resolution between InsP6-InsP8. When increasing pH to 9.7, the resolution among InsP3-InsP6 will significantly improve. Due to the excellent resolution, a shorter capillary length of 72 cm is recommended for further increasing the throughput. Besides, a higher CE cassette temperature at 40 &#176;C decreases the viscosity of the aqueous electrophoretic buffer and accelerates their movement under EOF. According to different research demands, modifications of this method can further facilitate InsPs and PP-InsPs analysis. Therefore, the described CE-ESI-MS protocols have the potential of opening novel research avenues into this multifaceted family of signaling molecules.

Since the initial discovery of myo-inositol pyrophosphates (PP-InsPs) in 19931,2, significant progress has been made to elucidate their biosynthesis, turnover, and functions3. Inositol pyrophosphates ubiquitously occur in eukaryotic cells4 and serve as metabolic signaling molecules critically involved in, e.g., phosphate homeostasis5,6, insulin sensitivity7, calcium oscillations8,9, vesicular trafficking10, apoptosis11, DNA repair12, immune signalling13, and others. The plethora of important processes under the control of inositol pyrophosphates calls for a deeper understanding of their cellular abundance, fluctuation, and localization.
Although InsPs and PP-InsPs attracted attention across disciplines, the analysis of their abundance is routinely performed using a method developed during the &#39;80s, consisting of labeling cells with tritiated inositol, resolving the extracted PP-InsPs by strong anion exchange chromatography Sax-HPLC with subsequent scintillation counting. Newer methods based on mass spectrometry still face significant challenges: inositol pyrophosphates with up to eight phosphate units harbor phosphate esters and anhydrides, leading to a significant negative charge and potential phosphate loss during ionization. There are four major types of PP-InsPs found in mammals (Figure 1): 1,5-(PP)2-InsP4 (or 1,5-InsP8), 5-PP-InsP5 (or 5-InsP7), 1-PP-InsP5 (or 1-InsP7), and 5-PP-Ins(1,3,4,6)P4 (or 5-PP-InsP4)3,14. The physiological levels of PP-InsPs are typically in the nano- to low-micromolar range, with 5-PP-InsP5 as the most abundant with cellular concentrations of 0.5 - 5 &#181;M. 1,5-(PP)2-InsP4 and 1-PP-InsP5 are believed to be up to around 10% of the 5-PP-InsP5 pool and remain difficult to trace in many cells15. 5-PP-InsP4 with a free OH group is even lower in abundance and usually only becomes detectable when phosphate hydrolases are inhibited with sodium fluoride (NaF)16.
The high charge density of PP-InsPs makes their separation difficult, and the occurrence of PP-InsP regioisomers further complicates these efforts. As a result, most experiments relied on quantitation by metabolic radioactive labeling of cells using [3H]-inositol, as background from the matrix is excluded and high sensitivity is achieved17,18. However, this method is costly, time-consuming, and does not allow to properly distinguish related PP-InsP regioisomers. Moreover, [3H]-inositol labeling does not account for endogenous inositol synthesis from glucose. A polyacrylamide gel electrophoresis (PAGE)-based method is a widely applied inexpensive alternative but limited in its sensitivity19,20,21,22. Other approaches avoiding radiolabeling have been published, including ion chromatography followed by post-column derivatization UV-detection23, hydrophilic interaction chromatography (HILIC)24, or weak anion exchange (WAX) coupled with mass spectrometry (MS)25. However, they are not (yet) on par with the classic [3H]-inositol SAX-HPLC protocol.
Recently, capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) was introduced as a transformative strategy for the analysis of InsPs and PP-InsPs metabolism, meeting all requirements discussed above16. Combined with current state-of-the-art InsP extraction by perchloric acid followed by enrichment with titanium dioxide beads26, CE-ESI-MS succeeded in every organism tested so far, from yeast to plants and mammals. Simultaneous profiling of InsPs and PP-InsPs, including all possible regioisomers, was easily achieved. Stable isotope-labeled (SIL) internal standards enabled a rapid and precise absolute quantitation, irrespective of matrix effects. Because MS can capture isotopic mass differences, CE-ESI-MS can also be applied to study compartmentalized cellular synthesis pathways of InsPs and PP-InsPs, e.g., by feeding cells with [13C6]-myo-inositol or [13C6]-D-glucose.
Described here is a detailed step-by-step protocol for the absolute quantitation of PP-InsPs and InsPs from mammalian cells by CE-ESI-MS. Apart from the major 5-PP-InsP5 isomer, 1,5-(PP)2-InsP4 and 1-PP-InsP5 are also quantified in this study, despite their lower abundance. Two HCT116 cell lines from different laboratories (NIH, UCL) are studied, and it is validated that HCT116UCL cells contain 7-fold higher levels of 1,5-(PP)2-InsP4 than found in HCT116NIH, while 5-PP-InsP5 concentrations are comparable. In addition, 1-PP-InsP5 synthesis in HCT116UCL is not significantly increased. Also, the increase of PP-InsP levels by blocking their dephosphorylation using sodium fluoride is studied quantitatively.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Materials&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Greiner Bio-One</td><td>616201</td><td>-</td></tr><tr><td>15 cm tissue culture dishes</td><td>Thermo Fisher</td><td>168381</td><td>-</td></tr><tr><td>2.0 mL microcentrifuge tubes</td><td>Greiner Bio-One</td><td>623201</td><td>-</td></tr><tr><td>50 mL centrifuge tubes</td><td>Greiner Bio-One</td><td>227261</td><td>-</td></tr><tr><td>96-well plates</td><td>Thermo Fisher</td><td>260836</td><td>for the DC protein assay</td></tr><tr><td>CE fused silica capillary</td><td>CS Chromatographie</td><td>105180</td><td>50 &amp;micro;M i.d. 360 &amp;micro;M o.d.</td></tr><tr><td>Pipette tips</td><td>Starlab</td><td>I1054-0001, S1111-6701, S1113-1700, S1111-3700</td><td>10 mL, 1000 &amp;micro;L, 200 &amp;micro;L, 10 &amp;micro;L pipette tips</td></tr><tr><td>Serological pipets</td><td>TPP</td><td>94550, 94525, 94010, 94005</td><td>50 mL, 25, mL, 10 mL, 5 mL serological pipettes</td></tr><tr><td>T75 flasks</td><td>TPP</td><td>90076</td><td>-</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Chemicals and Reagents&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>NaOH</td><td>AppliChem</td><td>A6829,0500</td><td>sodium hydroxide pellets for molecular biology, for preparation of cell lysis buffer</td></tr><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>25200056</td><td>-</td></tr><tr><td>Ammonium acetate</td><td>Thermo Fisher</td><td>1677373</td><td>HPLC grade</td></tr><tr><td>BSA</td><td>Thermo Fisher</td><td>23209</td><td>albumin standard (2.0 mg/mL) for standard curve preparation</td></tr><tr><td>DC protein assay</td><td>Biorad</td><td>5000116</td><td>DC protein assay reagents package</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41966029</td><td>high glucose, pyruvate</td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106, 10500064 (heat inactivated)</td><td>10270106 for HCT116&lt;sup&gt;UCL&lt;/sup&gt;, 10500064 for HCT116&lt;sup&gt;NIH&lt;/sup&gt;</td></tr><tr><td>Isopropanol</td><td>Carl Roth</td><td>AE73.2</td><td>99.95% LC-MS grade</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;OH, 10%</td><td>Carl Roth</td><td>6756.1</td><td>for preparation of 3% NH&lt;sub&gt;4&lt;/sub&gt;OH</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010015</td><td>-</td></tr><tr><td>Perchloric acid, 70%</td><td>Carl Roth</td><td>9216.1</td><td>for preparation of 1 M perchloric acid</td></tr><tr><td>SDS</td><td>SERVA</td><td>20760.02</td><td>for preparation of cell lysis buffer</td></tr><tr><td>Sodium fluoride</td><td>Sigma Aldrich</td><td>S7920</td><td>-</td></tr><tr><td>TiO&lt;sub&gt;2&lt;/sub&gt; beads</td><td>GL Sciences</td><td>5020-75000</td><td>5 &amp;micro;m particle size</td></tr><tr><td>Trypan blue solution</td><td>Gibco</td><td>15250061</td><td>trypan blue stain (0.4%)</td></tr><tr><td>Ultrapure (Type 1) water</td><td>Milli-Q</td><td>ZRQSVP3WW</td><td>model: Direct-Q 3 UV Water Purification System</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Equipment&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>30105893</td><td>model: XPE26; for weighing of beads (5-6 mg per sample)</td></tr><tr><td>Automated cell counter</td><td>Logos Biosystems</td><td>L40002</td><td>model: LUNA-II Automated Cell Counter</td></tr><tr><td>Benchtop centrifuge</td><td>Hettich</td><td>1401</td><td>model: UNIVERSAL 320</td></tr><tr><td>Benchtop centrifuge with cooling</td><td>VWR</td><td>521-1647P</td><td>model: Microstar 17R</td></tr><tr><td>CE system</td><td>Agilent</td><td>G7100A</td><td>-</td></tr><tr><td>CE/MS Adapter Kit</td><td>Agilent</td><td>G1603A</td><td>-</td></tr><tr><td>CE/MS Sprayer Kit</td><td>Agilent</td><td>G1607A</td><td>-</td></tr><tr><td>Cell counting slides</td><td>Logos Biosystems</td><td>L12001</td><td>LUNA Cell Counting Slides</td></tr><tr><td>Centrifugal evaporator</td><td>Eppendorf</td><td>5305000304</td><td>model: Concentrator plus complete system</td></tr><tr><td>ESI source</td><td>Agilent</td><td>AJS ESI</td><td>-</td></tr><tr><td>Super Support Film</td><td>Nisshin EM Co. Ltd, Tokyo</td><td>647</td><td></td></tr><tr><td>Humidified incubator</td><td>Binder</td><td>9040-0088</td><td>model: CB E6.1, for cultivation of mammalian cells</td></tr><tr><td>Ice box</td><td>-</td><td>-</td><td>should provide enough space for samples, dishes, etc.</td></tr><tr><td>Isocratic LC system</td><td>Agilent</td><td>G7110B 1260 Iso Pump</td><td>model: Infinity II Quaternary system</td></tr><tr><td>MSD</td><td>Agilent</td><td>G6495C</td><td>triple quadrupole</td></tr><tr><td>Multiplate reader</td><td>Tecan</td><td>30086375</td><td>model: SPARK 10 M</td></tr><tr><td>Pipette filler</td><td>Thermo Fisher</td><td>10072332</td><td>for serological pipettes</td></tr><tr><td>Pipettes</td><td>Brand</td><td>705884, 705880, 705878, 705872, 705870</td><td>various pipettes</td></tr><tr><td>Rotator</td><td>Labnet</td><td>H5500</td><td>model: Mini LabRoller Rotator</td></tr><tr><td>Shortix capillary column cutter</td><td>SGT</td><td>S0020</td><td>-</td></tr><tr><td>Test tube shaker (vortex mixer)</td><td>Carl Roth</td><td>HXH6.1</td><td>model: Rotilabo-Mini Vortex</td></tr><tr><td>Tilt table</td><td>Labnet</td><td>S0600</td><td>model: EDURO MiniMix Nutating Mixer</td></tr><tr><td>Water bath</td><td>Thermo Fisher</td><td>FSGPD05</td><td>model: Isotemp GPD 05</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Software&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>MassHunter Workstation</td><td>Agilent</td><td>Version 10.1</td><td>-</td></tr><tr><td>MassHunter Workstation LC/MS Data Acquisition</td><td>Agilent</td><td>Version 10.1</td><td>-</td></tr><tr><td>MassHunter Workstation Optimizer</td><td>Agilent</td><td>Version 10.1</td><td>-</td></tr><tr><td>MassHunter Workstation Qualitative Analysis</td><td>Agilent</td><td>Version 10.0</td><td>-</td></tr><tr><td>QQQ Quantitaion Analysis</td><td>Agilent</td><td>Version 10.1</td><td>-</td></tr></tbody></table>

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.

The results shown here aim to illustrate the potential of CE-ESI-MS analysis. The reported figures are descriptive of a technically flawless CE-ESI-MS run. Firstly, a mixture of inositol pyrophosphate standards (Figure 1) and a mammalian cell extract (Figure 2) are presented. Secondly, a comparison of two HCT116 cell lines (Figure 3) and NaF-treated HCT116 (Figure 4) cells are provided.
Extracted ion electropherograms (EIEs) of inositol (pyro)phosphate standards at a concentration of 2 &#181;M are shown in Figure 1. Metabolism of inositol pyrophosphates in mammals with their simplified structures is inserted. The four inositol pyrophosphates in mammals, 1,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, and 5-PP-Ins(1,3,4,6)P4 are well distinguished using the described method. A CE-ESI-MS run of HCT116UCL is depicted in Figure 2. With the aid of stable isotope-labeled (SIL) internal standards, an absolute quantitation can be readily achieved by comparing the signal response with the spiked-in SIL of known concentration. The integrated EIEs of the inositol phosphate from InsP5 to (PP)2-InsP4 and un-integrated EIEs of their isotopic patterns are displayed. RSDs of all analytes from six technical repeats are within 4%. With the measured concentration and the volume of extracts, the amount of analytes can be calculated. With the cell counts and cell volume, or protein content, absolute cellular concentration (&#181;M) or amount normalized by protein content (pmol/mg protein) are commonly the final outcomes of such an analysis.
According to an earlier study, two batches of diverged HCT116 cells have a variation of InsP8 levels, HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells29. With the CE-MS method, 1,5-(PP)2-InsP4 in HCT116NIH could be easily quantified (Figure 3), and HCT116UCL cells contain 7-fold higher levels of InsP8 than in HCT116NIH. In addition, significant accumulation of 1,5-(PP)2-InsP4 in HCT116UCL cells is paralleled by a significantly increased 1-PP-InsP5, which is now quantitatively shown in Figure 3.
PP-InsPs levels increase by inhibiting their dephosphorylation using sodium fluoride. CE-ESI-MS analysis of NaF-treated HCT116NIH cells demonstrated the -5-PP-InsP5 elevation along with a reduction in InsP6 and an appearance of 5-PP-Ins(1,3,4,6)P4 (Figure 4). Besides, the elevation of InsP8 levels is noticeable, while 1-PP-InsP5 decreases to some degree. 1-PP-InsP5 is not completely absent in NaF-treated HCT116NIH, but mostly either under the limit of detection or quantitation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62847/62847fig01.jpg" /> 
Figure 1: Typical extracted ion electropherograms (EIEs) of inositol (pyro)phosphate standards in CE-ESI-MS analysis using the described protocol. The concentration of each analyte is 2 &#181;M. Injected sample volume is ca. 10 nL with an injection at 50 mbar for 10 s. Inserts show the metabolism of inositol pyrophosphates in mammals. IPPK: inositol pentakisphosphate 2-kinase, IP6K: inositol hexakisphosphate kinase, PPIP5K: diphosphoinositol pentakisphosphate kinase, DIPP1: diphosphoinositolpolyphosphate phosphohydrolase 1. <a href="https://www.jove.com/files/ftp_upload/62847/62847fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62847/62847fig02.jpg" /> 
Figure 2: Representative InsP profile of HCT116UCL cells. (A) EIEs of the main inositol (pyro)phosphates in HCT116NIH and spiked SIL ISs 2 &#181;M [13C6]1,5-(PP)2-InsP4 (1), 4 &#181;M [13C6]5-PP-InsP5 (2), 4 &#181;M [13C6]1-PP-InsP5 (3), 20 &#181;M [13C6]InsP6 (4), and 20 &#181;M [13C6]Ins(1,3,4,5,6)P5 (5). Inserts show six technical repeats of InsP analysis by CE-ESI-MS, data are presented as means &#177; SD. (B) Cellular concentration of PP-InsPs and InsPs in human cell lines HCT116UCL and (C) PP-InsPs and InsPs amount normalized by protein content. Data are means &#177; SEM from three independent experiments. <a href="https://www.jove.com/files/ftp_upload/62847/62847fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62847/62847fig03.jpg" /> 
Figure 3:&#160;Variation in InsP8 levels between two diverged HCT116 cells. (A)EIEs of inositol pyrophosphate inHCT116UCL and HCT116NIH. InsP8 in HCT116UCL is markedly more abundant than in HCT116NIH. (B) Ratio of inositol pyrophosphate to InsP6 (%) in both HCT116 cells. HCT116UCL cells contain 7-fold higher levels of InsP8 as compared to in HCT116NIH, while the 5-PP-InsP5 levels are equal. Data are means &#177; SEM from three independent experiments. <a href="https://www.jove.com/files/ftp_upload/62847/62847fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62847/62847fig04.jpg" /> 
Figure 4: Inositol (pyro)phosphate levels in HCT116NIH cells, with NaF treatment. (A) EIEs of inositol (pyro)phosphate in&#160;HCT116NIH with sodium fluoride treatment (NaF, 10 mM). Levels of inositol pyrophosphate including 1,5-(PP)2-InsP4, 5-PP-InsP5, and 5-PP-Ins(1,3,4,6)P4 increase via blocking their dephosphorylation using NaF. (B) Inositol (pyro)phosphate levels (amounts are normalized by protein content) in untreated and NaF-treated HCT116NIH cells. Data are means &#177; SEM from three independent experiments. <a href="https://www.jove.com/files/ftp_upload/62847/62847fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: CE-ESI-MS parameter settings. Source parameter and iFunnel parameters are optimized by Source and iFunnel Optimizer. MSM parameter settings for inositol (pyro)phosphates are optimized by MassHunter Optimizer. <a href="https://www.jove.com/files/ftp_upload/62847/Table1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Theoretical and experimental regression equation. Concentration of&#160;[13C6]5-PP-InsP5 [13C6]InsP6, and [13C6]Ins (1, 3, 4, 5, 6)P5 is 4 &#181;M, 20 &#181;M, and 20 &#181;M, respectively. For regression equation, 5-PP-InsP5 concentration is at 0.04 &#181;M, 0.1 &#181;M, 0.2 &#181;M, 0.4 &#181;M, 1 &#181;M, 2 &#181;M, 4 &#181;M, 8 &#181;M, 16 &#181;M, 24 &#181;M. InsP6 and Ins (1, 3, 4, 5, 6)P5 concentration is at 0.2 &#181;M, 0.5 &#181;M, 1 &#181;M, 2 &#181;M, 5 &#181;M, 10 &#181;M, 20 &#181;M, 40 &#181;M, 80 &#181;M, 120 &#181;M. x is concentration, y is (Area InsP)12C/(Area InsP)13C. <a href="https://www.jove.com/files/ftp_upload/62847/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry at JoVE.com

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

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 ...

absolute-quantitation-inositol-pyrophosphates-capillary

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Biochemistry

2.2K Views.  University of Freiburg. Dit levert hoogwaardige resultaten op voor het inositolfosfaatmetabolisme in alle organismen die we tot nu toe hebben getest. Het zal ook inzicht geven in nieuwe cellulaire biosynthetische paden waarvan we ons momenteel bewust zijn. Analyse van inositol fosfaat metabolisme door CE-ESI-MS is sneller, zeer gevoelig, en in staat van het verstrekken van structuur informatie voor inositol fosfaat assemblages.Om te beginnen, zet een Capillaire Elektroforese Electrospray Ionization Mass Spect...

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

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.

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 ...

Chemistry

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 ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biology

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

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

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