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 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.
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 '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 µM. 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.
1. Setting up the CE-ESI-MS system
2. Preparing buffer, capillary, and CE-MS system
3. Extraction of soluble inositol phosphates from mammalian cells
NOTE: HCT116NIH cells were a kind gift from Stephen Shears28. HCT116UCL cells were from Saiardi's Lab26.
4. Performing the CE-ESI-MS runs
5. Data analysis
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 µM 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 (µM) 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.
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 µM. 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. Please click here to view a larger version of this figure.
Figure 2: Representative InsP profile of HCT116UCL cells. (A) EIEs of the main inositol (pyro)phosphates in HCT116NIH and spiked SIL ISs 2 µM [13C6]1,5-(PP)2-InsP4 (1), 4 µM [13C6]5-PP-InsP5 (2), 4 µM [13C6]1-PP-InsP5 (3), 20 µM [13C6]InsP6 (4), and 20 µM [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 ± 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 ± SEM from three independent experiments. Please click here to view a larger version of this figure.
Figure 3: 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 ± SEM from three independent experiments. Please click here to view a larger version of this figure.
Figure 4: Inositol (pyro)phosphate levels in HCT116NIH cells, with NaF treatment. (A) EIEs of inositol (pyro)phosphate in 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 ± SEM from three independent experiments. Please click here to view a larger version of this figure.
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. Please click here to download this Table.
Table 2: Theoretical and experimental regression equation. Concentration of [13C6]5-PP-InsP5 [13C6]InsP6, and [13C6]Ins (1, 3, 4, 5, 6)P5 is 4 µM, 20 µM, and 20 µM, respectively. For regression equation, 5-PP-InsP5 concentration is at 0.04 µM, 0.1 µM, 0.2 µM, 0.4 µM, 1 µM, 2 µM, 4 µM, 8 µM, 16 µM, 24 µM. InsP6 and Ins (1, 3, 4, 5, 6)P5 concentration is at 0.2 µM, 0.5 µM, 1 µM, 2 µM, 5 µM, 10 µM, 20 µM, 40 µM, 80 µM, 120 µM. x is concentration, y is (Area InsP)12C/(Area InsP)13C. Please click here to download this Table.
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 °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.
The authors have nothing to disclose.
This project has received funding from the European Research Council (ERC) under the European Union'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.
Materials | |||
1.5 mL microcentrifuge tubes | Greiner Bio-One | 616201 | – |
15 cm tissue culture dishes | Thermo Fisher | 168381 | – |
2.0 mL microcentrifuge tubes | Greiner Bio-One | 623201 | – |
50 mL centrifuge tubes | Greiner Bio-One | 227261 | – |
96-well plates | Thermo Fisher | 260836 | for the DC protein assay |
CE fused silica capillary | CS Chromatographie | 105180 | 50 µM i.d. 360 µM o.d. |
Pipette tips | Starlab | I1054-0001, S1111-6701, S1113-1700, S1111-3700 | 10 mL, 1000 µL, 200 µL, 10 µL pipette tips |
Serological pipets | TPP | 94550, 94525, 94010, 94005 | 50 mL, 25, mL, 10 mL, 5 mL serological pipettes |
T75 flasks | TPP | 90076 | – |
Chemicals and Reagents | |||
NaOH | AppliChem | A6829,0500 | sodium hydroxide pellets for molecular biology, for preparation of cell lysis buffer |
0.25% trypsin-EDTA | Gibco | 25200056 | – |
Ammonium acetate | Thermo Fisher | 1677373 | HPLC grade |
BSA | Thermo Fisher | 23209 | albumin standard (2.0 mg/mL) for standard curve preparation |
DC protein assay | Biorad | 5000116 | DC protein assay reagents package |
DMEM | Gibco | 41966029 | high glucose, pyruvate |
FBS | Gibco | 10270106, 10500064 (heat inactivated) | 10270106 for HCT116UCL, 10500064 for HCT116NIH |
Isopropanol | Carl Roth | AE73.2 | 99.95% LC-MS grade |
NH4OH, 10% | Carl Roth | 6756.1 | for preparation of 3% NH4OH |
PBS | Gibco | 10010015 | – |
Perchloric acid, 70% | Carl Roth | 9216.1 | for preparation of 1 M perchloric acid |
SDS | SERVA | 20760.02 | for preparation of cell lysis buffer |
Sodium fluoride | Sigma Aldrich | S7920 | – |
TiO2 beads | GL Sciences | 5020-75000 | 5 µm particle size |
Trypan blue solution | Gibco | 15250061 | trypan blue stain (0.4%) |
Ultrapure (Type 1) water | Milli-Q | ZRQSVP3WW | model: Direct-Q 3 UV Water Purification System |
Equipment | |||
Analytical balance | Mettler Toledo | 30105893 | model: XPE26; for weighing of beads (5-6 mg per sample) |
Automated cell counter | Logos Biosystems | L40002 | model: LUNA-II Automated Cell Counter |
Benchtop centrifuge | Hettich | 1401 | model: UNIVERSAL 320 |
Benchtop centrifuge with cooling | VWR | 521-1647P | model: Microstar 17R |
CE system | Agilent | G7100A | – |
CE/MS Adapter Kit | Agilent | G1603A | – |
CE/MS Sprayer Kit | Agilent | G1607A | – |
Cell counting slides | Logos Biosystems | L12001 | LUNA Cell Counting Slides |
Centrifugal evaporator | Eppendorf | 5305000304 | model: Concentrator plus complete system |
ESI source | Agilent | AJS ESI | – |
Humidified incubator | Binder | 9040-0088 | model: CB E6.1, for cultivation of mammalian cells |
Ice box | – | – | should provide enough space for samples, dishes, etc. |
Isocratic LC system | Agilent | G7110B 1260 Iso Pump | model: Infinity II Quaternary system |
MSD | Agilent | G6495C | triple quadrupole |
Multiplate reader | Tecan | 30086375 | model: SPARK 10 M |
Pipette filler | Thermo Fisher | 10072332 | for serological pipettes |
Pipettes | Brand | 705884, 705880, 705878, 705872, 705870 | various pipettes |
Rotator | Labnet | H5500 | model: Mini LabRoller Rotator |
Shortix capillary column cutter | SGT | S0020 | – |
Test tube shaker (vortex mixer) | Carl Roth | HXH6.1 | model: Rotilabo-Mini Vortex |
Tilt table | Labnet | S0600 | model: EDURO MiniMix Nutating Mixer |
Water bath | Thermo Fisher | FSGPD05 | model: Isotemp GPD 05 |
Software | |||
MassHunter Workstation | Agilent | Version 10.1 | – |
MassHunter Workstation LC/MS Data Acquisition | Agilent | Version 10.1 | – |
MassHunter Workstation Optimizer | Agilent | Version 10.1 | – |
MassHunter Workstation Qualitative Analysis | Agilent | Version 10.0 | – |
QQQ Quantitaion Analysis | Agilent | Version 10.1 | – |