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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</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 &#181;M i.d. 360 &#181;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 &#181;L, 200 &#181;L, 10 &#181;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>Chemicals and Reagents</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 HCT116UCL, 10500064 for HCT116NIH</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Carl Roth</td>
			<td>AE73.2</td>
			<td>99.95% LC-MS grade</td>
		</tr>
		<tr>
			<td>NH4OH, 10%</td>
			<td>Carl Roth</td>
			<td>6756.1</td>
			<td>for preparation of 3% NH4OH</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>TiO2 beads</td>
			<td>GL Sciences</td>
			<td>5020-75000</td>
			<td>5 &#181;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>Equipment</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>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>Software</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 ...

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Biochemistry

2.1K Views.  University of Freiburg. A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

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.

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

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 ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

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 ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...