Analysis of metabolically labeled inositol phosphate messengers by NMR

Article abstract of DOI:10.1016/bs.mie.2020.04.035

Inositol phosphates (InsPs) are an important group of eukaryotic messengers and mediate a wide range of processes. To elucidate the biological functions of these molecules, robust techniques to characterize inositol phosphate metabolism at the cellular level are highly sought after. This chapter provides a detailed protocol for the preparation of ¹³C-labeled myo-inositol, its use for metabolic labeling of mammalian and yeast cells, and the quantitative analysis of intracellular InsP pools from cell extracts using NMR spectroscopy.

Full text of DOI:10.1016/bs.mie.2020.04.035

CHAPTER TWO
Analysis of metabolically labeled
inositol phosphate messengers
by NMR
Robert Puschmanna,b,†, Robert K. Harmela,b,†, Dorothea Fiedlera,b,
aLeibniz-Forschungsinstitut fu€r Molekulare Pharmakologie, Berlin, Germany
bInstitut fu€r Chemie, Humboldt-Universit€at zu Berlin, Berlin, Germany
*Corresponding author: e-mail address: fiedler@fmp-berlin.de
*
Contents
1. Introduction
36
36
37
39
39
40
41
43
44
44
46
47
48
50
50
50
50
1.1 Inositol phosphate signaling molecules
1.2 Detection and quantification of endogenous inositol phosphates
2. Chemoenzymatic synthesis of [13C6]myo-inositol
2.1 General synthetic strategy
2.2 Materials
2.3 Protocol
2.4 Related techniques
3. Metabolic labeling
3.1 General approach
3.2 Materials
3.3 Protocol
3.4 Related techniques
4. Conclusion
Acknowledgments
Funding
References
Abstract
Inositol phosphates (InsPs) are an important group of eukaryotic messengers and
mediate a wide range of processes. To elucidate the biological functions of these mol-
ecules, robust techniques to characterize inositol phosphate metabolism at the cellular
level are highly sought after. This chapter provides a detailed protocol for the prepara-
tion of 13C-labeled myo-inositol, its use for metabolic labeling of mammalian and yeast
cells, and the quantitative analysis of intracellular InsP pools from cell extracts using NMR
spectroscopy.
†
R.P. and R.K.H. contributed equally.
#
Methods in Enzymology, Volume 641
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2020.04.035
2020 Elsevier Inc.
All rights reserved.
35

36
Robert Puschmann et al.
1. Introduction
1.1 Inositol phosphate signaling molecules
Inositol phosphates are a versatile class of small molecule messengers and are
key components in cellular decision-making processes. The inositol phos-
phates comprise two major groups: the lipid-anchored phosphatidyl inositol
phosphates (PtdInsPs), and the soluble inositol phosphates (InsPs) (Balla,
2013; Berridge, 2016; Irvine & Schell, 2001) (Fig. 1). Among the InsPs,
the most abundant species in most organisms and cell lines is InsP6
(app. 10–100μM in mammalian cells) and its high negative charge pro-
motes strong binding to di- and trivalent metal cations, as well as positively
charged amino acid side chains (Veiga et al., 2006; Wundenberg & Mayr,
2012). For example, InsP6 was proposed to act as a scaffolding molecule
that promotes HIV capsid maturation (Dick et al., 2018; Mallery et al.,
2018). Although InsP6 is already densely phosphorylated, inositol-based
molecules containing even seven (e.g., 5PP-InsP5/5-InsP7) or eight
(e.g., 1,5(PP)2-InsP4/InsP8) phosphate groups exist (Fig. 1). This unique
subgroup, termed the inositol pyrophosphates (PP-InsPs), harbors one or
two phosphoanhydride bonds and has been investigated more thoroughly
in recent years. Genetic perturbation of the kinases that synthesize
PP-InsPs (IP6Ks and PPIP5Ks) has linked these molecules to diverse
processes, including insulin signaling, tumor cell motility, apoptosis and
phosphate homeostasis (Auesukaree, Tochio, Shirakawa, Kaneko, &
Harashima, 2005; Bhandari et al., 2007; Chakraborty et al., 2010; Jadav
et al., 2016; Koldobskiy et al., 2010; Moritoh et al., 2016; Zhu et al.,
2019). Consequently, the enzymes involved in PP-InsP metabolism have
become intriguing targets for pharmacological intervention (Wormald,
Liao, Kimos, Barrow, & Wei, 2017).
Fig. 1 Examples of inositol phosphate messengers. The numbering of the carbon atoms
in the myo-inositol ring is indicated in the structure of InsP5.

Analysis of metabolically labeled inositol phosphate messengers
37
1.2 Detection and quantification of endogenous inositol
phosphates
Genetic studies have established a wide range of functions for InsPs and
PP-InsPs in a variety of cellular processes. However, to enable a detailed
understanding of the regulation, turnover and dynamic interconversion of
these messengers, and to connect specific messengers to defined down-
stream signaling events, the detection of endogenous InsPs levels remains
a crucial task. Due to the absence of UV-active moieties in these mole-
cules, metabolic labeling of cells with radioactive [3H]myo-inositol or
[32P]orthophosphate has been the most widely used technique in the field
(Menniti, Miller, Putney, & Shears, 1993; Stephens et al., 1993). The
resulting radioactively labeled InsPs can be isolated by perchloric acid
extraction of cell lysates and separated based on their charge using strong
anion exchange (SAX) HPLC. Subsequently, collected fractions are
analyzed by scintillation counting (Fig. 2A). The incorporation of radio-
active labels has contributed to many essential discoveries (Banfic,
Bedalov, York, & Visnjic, 2013; Gu et al., 2017; Menniti et al., 1993;
Stephens et al., 1993), but the requirement to work with radioactive
materials and dedicated equipment, and the limited compatibility with
Fig. 2 Analytical techniques to monitor endogenous InsPs and PP-InsPs from cell
extracts. (A) Metabolic labeling of cells with radioactive material and analysis of InsPs
and PP-InsPs by SAX-HPLC using scintillation counting. (B) Unlabeled InsPs and
PP-InsPs are enriched using TiO2 beads followed by PAGE separation and staining with
toluidine blue.

38
Robert Puschmann et al.
certain mammalian cell lines and tissues imposes limitations on its appli-
cability. In a subsequent approach, radioactive labeling could be circum-
vented by analyzing HPLC fractions using a metal-dependent dye
(Mayr, 1988). Even though this method allowed quantitative analysis
of InsPs, the prerequisite for non-standard HPLC equipment prevented
its wide use. More recently, an alternative method avoided the use of the
rather harsh HPLC conditions for separation (Wilson, Bulley, Pisani,
Irvine, & Saiardi, 2015). Here, InsPs and PP-InsPs were enriched from
acidified cell extracts by TiO2 beads, followed by separation via high
percentage polyacrylamide gel electrophoresis (PAGE) (Fig. 2B).
Unlabeled PP-InsPs and InsP6 were visualized using a cationic dye (tolu-
idine blue or DAPI) and were analyzed quantitatively by gel imaging.
This technique became widely appreciated because it omits radioactive
tracers and does not require advanced instrumentation. On the other
hand, a more elaborate sample preparation is necessary and the detection
is limited to only the most highly phosphorylated InsPs (Wilson
et al., 2015).
While all methods mentioned above have been instrumental to under-
stand InsP and PP-InsP function, they were only able to discriminate InsPs
based on their charge and lack a clear distinction of the structural isomers.
Furthermore, the necessity of various separation steps precluded direct mea-
surements in complex environments like cell extracts. Therefore, novel
approaches that discriminate InsPs and PP-InsPs based on their structure
and that generate reliable information with limited sample preparation were
highly needed. To tackle these challenges, we developed a new method to
measure InsP and PP-InsP levels using nuclear magnetic resonance (NMR)
spectroscopy (Harmel et al., 2019). In this approach, mammalian or yeast
cells are metabolically labeled with [13C6] myo-inositol and InsP and
PP-InsP levels can subsequently be quantified from acidic cell extracts,
without further separation steps (Fig. 3). NMR spectroscopy provides
structural information with atomic resolution, and the 13C-nuclei enable
targeted monitoring of low abundant InsP species within a highly complex
sample. In this chapter, a workflow for metabolic labeling of InsPs and
PP-InsPs is provided. Specifically, the chemoenzymatic synthesis of
[13C6]myo-inositol is covered, followed by a detailed protocol describ-
ing metabolic labeling of mammalian and yeast cells and the subsequent
analysis by NMR spectroscopy.

Analysis of metabolically labeled inositol phosphate messengers
39
Fig. 3 Metabolic labeling with [13C6]myo-inositol and analysis of InsPs and PP-InsPs by
NMR spectroscopy.
2. Chemoenzymatic synthesis of [13C6]myo-inositol
2.1 General synthetic strategy
[13C6]myo-Inositol can be synthesized from [13C6]glucose on a gram scale
using a three-step enzymatic synthesis, followed by a chemical purification
strategy. (Fig. 4) As a first step, [13C6]glucose is phosphorylated at the
6-position by hexokinase. To assure maximal conversion during this step,
an ATP regeneration system consisting of creatine kinase and phosphocre-
atine is applied. Next, the [13C6]glucose-6-phosphate is isomerized to [13C6]
myo-inositol-3-phosphate by inositol phosphate synthase. Inositol phos-
phate synthase is not commercially available but can be obtained in high
amounts from E. coli (Harmel et al., 2019). Using Mg2+ ions as a metal cofac-
tor, in place of commonly used paramagnetic Mn2+ ions, the reaction pro-
gress can be monitored by 13C NMR spectroscopy. During this step, full
conversion of the starting material is crucial for the overall synthetic
strategy, as residual [13C6]glucose-6-phosphate would contaminate the final
product with [13C6]glucose. Without any [13C6]glucose-6-phosphate
remaining, unspecific yet robust and commercially available alkaline phos-
phatase can be used to dephosphorylate [13C6]myo-inositol-3-phosphate to
yield [13C6]myo-inositol together with a large amount of salt- and buffer-
contaminants.
To isolate [13C6]myo-inositol from this mixture, the crude material is
derivatized with an excess of acetic anhydride in pyridine. The obtained
[13C6]myo-inositol hexakisacetate can then be purified by normal-phase

40
Robert Puschmann et al.
Fig. 4 Chemoenzymatic synthesis of [13C6]myo-inositol followed by derivatization and
purification. (A) Hexokinase, creatine kinase, ATP, MOPS pH 6.5, creatine phosphate,
DTT, MgCl2, H2O. (B) Inositol phosphate synthase, NAD+, Tris pH 8.0, NaCl, MgCl2,
H2O. (C) Alkaline phosphatase, glycine pH 9.8, ZnCl2, H2O. (D) Ac2O, pyridine.
(E) NaOMe, MeOH.
chromatography. Subsequent saponification and precipitation affords pure
[13C6]myo-inositol in 55% overall yield (with respect to [13C6]glucose).
2.2 Materials
Enzyme Stock Solutions: Hexokinase (Sigma-Aldrich, H4502): 100U/mL in
50mM citrate, pH 7, 10mM MgCl2, 1mg/mL BSA. Creatine kinase
(Sigma-Aldrich, C3755): 350U/mL in 200mM MOPS, pH 6.5, 20mM
MgCl2, 20mM DTT. Inositol phosphate synthase (Harmel et al., 2019):
4mg/mL in 20mM Tris, pH 7.4, 200mM NaCl, 1mM DTT. Alkaline
phosphatase (Sigma-Aldrich, P5521): 100U/mL, 10mM Tris, pH 8.5,
5mM MgCl2, 0.2mM ZnCl2, 50% (v/v) glycerol. Hexokinase and creatine
kinase stock solutions were kept at ꢀ20°C for several months, multiple
freeze-thaw cycles should be avoided. Alkaline phosphatase stock solution
was kept at 4 °C for several months.
Chemicals: NaOMe in MeOH, pyridine, MeOH, DCM, creatine phos-
phate, ATP, NAD+, glycine, DOWEX® 1ꢁ8 (Cl--form), silica TLC
plates, NH4OH, AcOH, NH4HCO3, (NH4)2CO3, silica gel, hexanes,
EtOAc, DOWEX 50WX8 (H+-form), MeCN, inositol phosphate
synthase, D-[13C6]glucose (Eurisotop CLM-1396-10, alternatively, differ-
ently labeled glucose can be used as well. [1-13C1]glucose yields [4-13C1]
myo-inositol, and [4,5-13C2]glucose yields [4-13C1]myo-inositol).

Analysis of metabolically labeled inositol phosphate messengers
41
2.3 Protocol
Day 1
1. Prepare a solution of D-[13C6]glucose (1.00g, 83mM), MOPS (100mM,
pH 6.5), creatine phosphate (87mM), ATP (1mM), DTT (20mM)
and MgCl2 (20mM) in 65mL (total reaction volume) deionized water.
2. Add hexokinase (1U/mL) and creatine kinase (1.75U/mL), mix gently
and incubate at 30°C overnight.
Day 2
3. Test conversion by TLC as explained in related techniques
(Section 2.4).
a. <90% conversion: add more creatine phosphate, hexokinase and
creatine kinase and incubate until full conversion is achieved.
b. >90% conversion: continue with the next step.
4. Prepare anion exchange column:
Add 50mL of DOWEX® 1ꢁ8 (Cl- form) to a fritted glass column
and wash with MeOH (ꢂ500mL) until the eluate becomes colorless,
followed by 500mL deionized water. Equilibrate the column with
300 mL 1 M NH4HCO3 and wash with deionized water until the
eluate reaches a pH < 8.
5. Dilute the reaction mixture with 320mL deionized water and load it
onto the column. Discard the flowthrough.
6. Wash the column with 300mL deionized water to remove unreacted
[13C6]glucose. Discard the flowthrough.
7. Elute [13C6]glucose-6-phosphate with 250–500mL of 0.1M
(NH4)2CO3 and lyophilize the combined eluates in a round-bottom
flask overnight.
Day 3
8. Dissolve the lyophilized solids in 50mL deionized water.
9. Start reaction by mixing dissolved solids, Tris (50mM, pH 8.0), NAD+
(0.5mM), NaCl (50mM), MgCl2 (2mM) and inositol-3-phosphate
synthase (40μg/mL) in 200mL (total volume) deionized water and
incubate at 85°C for 4h.
10. Combine 450μL of the reaction mixture and 50μL of D2O into an
NMR tube and control conversion by 13C NMR spectroscopy.
(Follow disappearance of the doublets at 92 and 96ppm)
a. incomplete conversion: add more IPS and incubate until full
conversion is achieved.
b. complete conversion: continue with the next step.

42
Robert Puschmann et al.
11. Cool the reaction mixture down to 35°C.
12. Add 15mL of 1M glycine pH 9.8, 75μL of 1M ZnCl2, fill up to
300mL with deionized water and adjust the pH to 9.8.
13. Add 1.5mL alkaline phosphatase stock solution and incubate overnight
at 35°C.
Day 4
14. Test conversion by TLC as explained in related techniques
(Section 2.4).
a. <90% conversion: add more alkaline phosphatase and incubate
until full conversion is achieved.
b. >90% conversion: continue with next step.
15. Concentrate the reaction solution in a 500mL round-bottom flask to
dryness using a rotary evaporator. Coevaporate with 30 mL pyridine
and resuspend the crude material in 160mL pyridine and 70mL acetic
anhydride.
16. Stir the solution under reflux overnight.
Day 5
17. Remove the pyridine by evaporation and re-dissolve the crude material
in 500mL DCM. Add 200 mL of 1M HCl and pass the suspension
through a glass filter.
18. Pour the filtrate into a separatory funnel and separate the organic phase
and aqueous phase. (Note: Phase separation is hard to see because of the
black solution, so use a flash light to find the separation layer.)
19. Extract the aqueous phase twice with 100mL DCM and discard
the aqueous phase. Wash the combined organic phases with 1M
NaHCO3, then brine.
20. Dry the organic phase with Na2SO4 and remove the solids by filtration.
21. Add Telos® 50–100mL to the filtrate and remove the DCM by rotary
evaporation.
22. Purify the immobilized product on a normal-phase silica column
(8ꢁ20cm) using a step-wise gradient elution (hexane:EtOAc,
10:1!5:1!1:1!1:2; 1.5L per step).
23. Analyze fractions by LC-MS (look for the Na adduct, m/z 461).
Combine and evaporate all fractions that contain product.
1
24. Analyze the product by NMR spectroscopy and HRMS: H NMR
(600MHz, CDCl3) δ 5.79–5.54 (m, 1.5H), 5.53–5.32 (m, 1.5H),
5.32–5.12 (m, 1.5H), 5.11–4.82 (m, 1.5H), 2.20 (s, 3H), 2.01
(s, 3H), 2.01 (s, 6H), 2.00 (s, 6H). 13C NMR (151MHz, CDCl3) δ

Analysis of metabolically labeled inositol phosphate messengers
43
169.94 (s), 169.81 (s), 169.56 (s), 71.68–70.65 (m), 70.11–
69.15 (m), 69.11–67.90 (m), 20.89 (s), 20.69 (s), 20.60 (s). HRMS
13
12
(ESI/Orbitrap) m/z: [M+K]+ calcd. For C C6H24KO12 477.1101;
Found 477.1091.
Day 6
25. Dissolve [13C6]myo-inositol hexakisacetate (1.20g, 2.7mmol) in meth-
anol (249mL) and add 5.4M NaOMe in MeOH (3.35mL,
18.1mmol). Stir for 2h.
26. Neutralize the resulting suspension by addition of DOWEX 50WX8
(H+ form).
27. Pass the suspension through a glass filter and wash the residue with
methanol (100mL) and water (100mL) to dissolve all precipitates.
Concentrate the filtrate to dryness with a rotary evaporator.
28. Re-dissolve the solids using minimal amount of water and precipi-
tate the [13C6]myo-inositol by addition of a large excess of cold
MeCN.
29. Collect the white precipitate with a glass filter, wash the residue with
cold MeCN and re-dissolve in water. Lyophilize [13C6]myo-inositol
overnight.
Day 7
1
30. Analyze the product by NMR spectroscopy and HRMS: H NMR
(600MHz, D2O, pD 7.0) δ 4.29–4.14 (m, 0.5H), 4.03–3.90
(m, 0.5H), 3.82–3.60 (m, 2H), 3.59–3.34 (m, 2.5H), 3.25–3.12
(m, 0.5H). 13C NMR (151MHz, D2O, pD 7.0) δ 74.96–73.80 (m),
72.86–71.71 (m), 71.60–70.50 (m). HRMS (ESI/Orbitrap) m/z:
[M–H]ꢀ calcd for 13C6H11O6 185.0762; Found 185.0814.
31. Prepare 50mM stock solutions in milli-Q water for metabolic labeling
experiments.
2.4 Related techniques
Thin layer chromatography (TLC)
1. Spot a drop of the reaction mixture on a silica normal-phase TLC plate.
2. Spot reference compounds (glucose, glucose-6-phosphate, inositol) on
the TLC plate.
3. Dry TLC plate gently with a heat gun or under vacuum.
4. Run the TLC plate with MeOH:H2O:NH4OH:AcOH, 50:30:15:5
until the solvent front is close to the top of the TLC plate.

44
Robert Puschmann et al.
5. Dry the TLC with a heat gun and heat until yellow/brown dots appear
(for glucose and glucose-6-phosphate) or stain the plate with KMnO4
(for inositol).
3. Metabolic labeling
3.1 General approach
Metabolic labeling with [13C6]myo-inositol is conducted analogously to
labeling with [3H]myo-inositol (Azevedo & Saiardi, 2006). Both approaches
are applicable to cell lines that are able to import exogenous myo-inositol,
and importantly [13C6]myo-inositol is non-toxic. The cells of interest are
grown in synthetic medium that is devoid of regular 12C-myo-inositol
and instead supplemented with [13C6]myo-inositol. Steady state labeling is
usually achieved after at least seven doublings. The labeled cells are sub-
sequently harvested and the inositol phosphates are extracted using
perchloric acid. In our hands, the PP-InsPs are stable during this procedure
despite the low pH, as long as all solutions are kept on ice and the samples are
processed in a timely manner. The acidic extracts are neutralized with KOH
to assure pH stability during the following lyophilization step. Although
significant amounts of KClO4 precipitate out of solution during the neu-
tralization, it is important to reduce the salt concentration further to enable
high quality NMR measurements. Therefore, the lyophilized residue is
resuspended in a small volume of D2O and incubated on ice to lower the
salt content as much as possible.
Depending on the biological question to be addressed, different types
of NMR measurements can be employed. Only a small spectral window
to cover the InsP and PP-InsP resonances is defined (spectral width in the
indirect dimension(F1): 40ppm), when the focus lies exclusively on
the highly phosphorylated inositols. In an initial measurement, a high
number of points in the F1 dimension (number of FIDs: 512) with a
low number of scans (number of scans: 40) should be recorded to observe
splitting of the 13C-nuclei. The triplet pattern is characteristic for peaks
that correspond to [13C6]myo-inositol derived compounds. Once the
characteristic peaks are assigned, their signal intensities can be increased
in a second measurement with lower resolution (number of FIDs: 128)
and a higher number of scans (number of scans: >160) (for representative
spectra see Fig. 5).

Analysis of metabolically labeled inositol phosphate messengers
45
Fig. 5 NMR-spectra of extracts from (A) HCT116, (B) HT29, and (C) S. pombe. The spectra
were recorded at 310K and pH* 6.0. The red and black arrows indicate the InsP5 and
InsP6 peaks, respectively.
In order to unambiguously assign the observed peaks to a certain InsP
species, a synthetic standard should be used as a reference (for synthesis of
the different standards please refer to (Puschmann, Harmel, & Fiedler,
2019)). For preliminary assignments, a table of characteristic chemical shifts
is included in this protocol (Table 1). In case the InsPs and PP-InsPs signals
are obstructed by other, high-intensity peaks, a TiO2 enrichment can sim-
plify the spectrum by retaining only InsPs and PP-InsPs in the sample
(Wilson et al., 2015).

46
Robert Puschmann et al.
Table 1 Reference peaks for myo-inositol, InsP5, InsP6, and PP-InsP5.
Compound H1/C1 H2/C2 H3/C3 H4/C4 H5/C5
H6/C6
myo-inositol 3.70/74.0 4.23/75.1 3.70/74.0 3.79/75.3 3.45/77.3 3.79/75.3
InsP5
InsP6
4.34/77.1 4.57/73.6 4.34/77.1 4.62/78.9 4.35/80.3 4.62/78.9
4.37/76.2 5.11/72.9 4.37/76.2 4.62/78.9 4.35/80.3 4.62/78.9
5PP-InsP5 4.23/75.1 5.07/78.1 4.23/75.1 4.69/78.9 4.45/80.3 4.69/78.9
1
The chemical shifts are given as H (ppm)/13C (ppm) at 310K and pH* 6.0.
NMR spectroscopy is a quantitative method and the spectra can be used
to determine the absolute concentration of the compounds (with respect to
packed cell volume). The peaks of the C2-position of myo-inositol, InsP5,
InsP6, 1PP-InsP5, 5PP-InsP5 and 1,5(PP)2-InsP4 are distinctive and can
be used for quantification of these compounds, relative to an internal stan-
dard (tetramethyl phosphonium bromide). The ratio of the signal
intensities of the internal standard and the C2-positions can be used for
quantification, when referenced to a corresponding calibration curve
(for each compound an individual curve is required (Harmel et al., 2019)).
3.2 Materials
Cell Culture: [13C6]myo-inositol (see Section 2.3), DPBS (gibco 14190-094),
TrypLE express (gibco 12605-010), 100ꢁ glutamine (gibco 25030-024),
Pen-Strep (gibco 15140-122), cell culture dish 150mmꢁ25mm (corning
430599), vacuum filtration unit 0.2μm, FBS dialyzed (Gibco 26400-044),
PCV Packed Cell Volume Tube (TPP 87005), DMEM (gibco custom
formulation of DMEM powder without glucose, without myo-inositol,
without glutamine, without serine, without sodium bicarbonate, without
sodium phosphate, without pyruvate. All missing components, plus
10mM HEPES, were added back in, substituting 50μM [13C6]myo-
inositol for regular myo-inositol and the pH was adjusted to 7.4. The
medium was sterile-filtered).
Sample preparation: D2O (Deutero 00506), D2O ampule (Eurisotop
D215T), 5mm NMR tubes (Deutero Boro500-5-7).
NMR Spectrometer: Bruker Avance III NMR spectrometer
(1H-frequency: 600.13MHz), cryogenically cooled 5mm QCI-triple reso-
nance probe equipped with a one-axis self-shielded gradient, TopSpin
Acquisition Version 3.5-pl6.

Analysis of metabolically labeled inositol phosphate messengers
47
3.3 Protocol
1. Grow the mammalian cell line of choice up to 80–90% confluency
in a 15 cm dish in DMEM (+ 50 μM myo-inositol (unlabeled), 10%
DFBS, + 1 ꢁ Pen-Strep, + 1 ꢁ Gln) and subsequently dilute the cells
1:10 in a 15cm dish in DMEM (+50μM [13C6]myo-inositol, +10%
DFBS, +1ꢁ Pen-Strep, +1ꢁ Gln). Note: cells in medium containing
DFBS grow slower than when supplemented with regular FBS.
2. Split the cells at 80–90% confluency with a 1:10 dilution and distribute
them in the appropriate number of 15cm dishes. Depending on the InsP
and PP-InsP concentrations in the cell line of interest, up to 10 dishes
might be necessary to allow for reliable quantification. Continue the
growth in DMEM (+50μM [13C6]myo-inositol, +10% DFBS, +1ꢁ
Pen-Strep, +1ꢁ Gln).
3. Once the cells reach 80–90% confluency, they are washed with DPBS
and trypsinized. Combine the cells, wash with DPBS, and determine
the packed cell volume of the cell suspension (this is only required if
cellular concentrations of InsPs and PP-InsPs will be measured). At this
point, the cell number can be determined as well.
4. Steps 4 through 7 are performed at 4°C! Centrifuge the cell suspension
and lyse the cell pellet by adding 5mL lysis solution (1M HClO4,
6mM EDTA). Vortex the suspension repeatedly over the course of
5min and centrifuge at 20,000 g for 10min to remove all cell debris.
5. Transfer the supernatant into a fresh tube and adjust the pH of the solu-
tion to 6.0 by adding 2M KOH (in our hands KOH is superior to
K2CO3 as the pH of the solution is more stable during lyophilization).
Incubate the neutralized solution on ice for half an hour to ensure pre-
cipitation of as much KClO4 as possible, and centrifuge the solution.
Transfer the supernatant into a fresh tube and lyophilize the sample.
6. The next day, dissolve the sample in 600μL D2O and incubate on ice
for 30min to allow more KClO4 to precipitate. This step is crucial, as
a high ionic strength is problematic for the NMR measurements.
Remove the precipitate by centrifugation.
7. Optional step: This step is only necessary if weaker signals close to the
HDO solvent peak need to be observed. Lyophilize the supernatant
and dissolve the residue in 600μL D2O from an ampule.
8. Transfer the solution into an NMR tube and record an NMR spectrum
(Fig. 5, Table 1) at 310 K: HMQC with a BIRD pulse for suppression of
signals from protons not bound to 13C nuclei. The spectra are recorded

48
Robert Puschmann et al.
using 1024 points, a spectral width of 10,000 Hz (ꢂ 16.7 ppm) and
a center of the spectrum at 4.7 ppm in the F2 dimension. In the F2
dimension spectra are recorded as follows:
- For an overview spectrum: 128 points, a spectral width of 15,000Hz
(ꢂ100ppm), a center of the spectrum at 48ppm and 20 scans.
- For high resolution in the inositol region: 256 points, a spectral width of
6000Hz (ꢂ40ppm), a center of the spectrum at 70ppm and 40 scans.
- For higher sensitivity in the inositol region: 64 points, a spectral width
of 6000Hz (ꢂ40ppm), a center of the spectrum at 70ppm and 200 scans.
3.4 Related techniques
Labeling of Schizosaccharomyces pombe
Bead beater (Precellys Evolution), glass beads.
Prepare a 50ꢁ salt stock (52.5mg/mL MgCl2 ꢁ6 H2O, 0.735mg/mL
CaCl2 ꢁ2 H2O, 50mg/mL KCl, 2mg/mL Na2SO4) and a 20ꢁ glucose
stock (400mg/mL glucose). Sterilize by autoclaving and store at 4°C.
Prepare a 1000ꢁ vitamin stock (w/o myo-inositol) (1mg/mL sodium
pantothenic acid, 10mg/mL nicotinic acid, 10mg/mL biotin), a
10,000ꢁ mineral stock (5mg/mL H3BO3, 4mg/mL MnSO4,
4mg/mL ZnSO4 ꢁ7 H2O, 2mg/mL FeCl3 ꢁ6 H2O, 0.4mg/mL
MoO3, 1mg/mL KI, 0.4mg/mL CuSO4 ꢁ5 H2O, 10mg/mL citric
acid), a 100ꢁ amino acid stock (7.5mg/mL lysineꢃHCl, 7.5mg/mL
arginineꢃHCl, 7.5mg/mL leucine), a 100ꢁ histidine (7.5mg/mL
histidine), a 500ꢁ [13C6]myo-inositol stock (9.3mg/mL [13C6]myo-
inositol), a 25ꢁ uracil stock (1.875mg/mL uracil), and a 10ꢁ adenine
stock (0.75mg/mL adenine). Sterilize by filtration and store at 4°C.
Minimal medium (MM; for 1L): 2.2mg/mL Na2HPO4, 3mg/mL
potassium phthalate monobasic, 1mg/mL glutamic acid, 20mL 50ꢁ salt
stock, 100μL 10,000ꢁ mineral stock, 767mL milliQ water. Mix, auto-
clave and store at 4°C. Add 100mL 10ꢁ adenine stock (depends on
nutritional marker), 40mL 25ꢁ uracil stock (depends on nutritional
marker), 50mL 20ꢁ glucose stock, 10mL 100ꢁ histidine stock (depends
on nutritional marker), 1mL 1000ꢁ vitamin stock, 10mL amino acid
stock, 2mL [13C6]myo-inositol or [12C6]myo-inositol.
1. Pick an individual colony of S. pombe from an agar plate and inocu-
late into 50mL MM+[13C6]myo-inositol and grow for 2 days (or until
OD>7).

Analysis of metabolically labeled inositol phosphate messengers
49
2. Use the prepared culture to inoculate in 2L MM+[13C6]myo-inositol
at OD 0.05 and grow the culture overnight until it reaches OD 1.4.
3. Harvest the cells by centrifugation at 4°C and wash them with ice
cold water. Combine all pellets into one tube.
4. Resuspend the cells in a small volume of ice cold water and determine
the optical density (OD600).
5. Aliquot 100 ODs of cells into 1.5mL screw cap tubes and centrifuge.
6. Resuspended the pellet in 600μL 1M HClO4, 3mM EDTA and add
glass beads to the meniscus of the suspension. Lyse the cells by bead
beating (2ꢁ20s, 6500rpm, 1min rest on ice in-between cycles).
7. Pierce the bottom of the tubes and collect the cell extract by shortly
centrifuging into another tube (30s, 500 g). The debris and the pre-
cipitated protein is separated by centrifugation at 20,000ꢁ g for
30min at 4°C.
8. Continue with Section 3.3, step 5.
TiO2-enrichment
TiO2 beads (GL Sciences Inc. 5020-75000), 1M HClO4, 10% NH4OH
1. Wash and equilibrate the TiO2 beads (5mg per 15cm dish) with 500μL
water followed by 500μL 1M HClO4 (centrifuge for 1min at 3500 g).
2. Add the acidic supernatant from Section 3.3 step 4 to the beads, briefly
vortex and rotate the samples for 15min at 4°C. If the sample is already
neutralized, mix it in a 1:1 ratio with 1M HClO4.
3. Pellet the beads by centrifugation (1min, 3500g) and wash them with
500μL ice cold 1M HClO4. Briefly vortex and repeat the wash. The
supernatant can be kept to check if all inositol pyrophosphates were
removed.
4. To elute, add 250μL 10% NH4OH to the pelleted beads, vortex and
rotate for 5min at 4°C. Pellet the beads and transfer the supernatant
(contains the InsPs) to a new tube. Repeat the elution and combine
the supernatants to ensure full recovery.
5. Filter the combined eluates through a syringe filter to ensure complete
removal of the TiO2 beads and lyophilize the sample (we recommend
lyophilization but alternatively, vacuum concentration using a speed-
vac can be implemented).
6. Dissolve the residue in D2O and check the pH*. Adjust with DCl or
NaOD to pH* 6.0 as necessary.

50
Robert Puschmann et al.
4. Conclusion
The analysis and quantification of InsPs and PP-InsPs in complex
samples constitutes a challenging task, since these molecules lack a handle
that would facilitate their detection. Most commonly, researchers rely on
specific separation techniques, radiolabeling methods, or both, to be able
to distinguish and quantify the structurally closely related InsPs and
PP-InsPs. Here, we provide a detailed workflow for an alternative approach,
in which metabolic labeling of cells with [13C6]myo-inositol is followed by
NMR analysis. In analogy to previous radiolabeling experiments, eukaryotic
cell are exposed to [13C6]myo-inositol, but the benign 13C-labeled com-
pound does not require specialized equipment for handling radioactive
materials, nor is cell viability affected negatively. The cell extracts are
analyzed directly by NMR spectroscopy, and cellular InsPs and PP-InsPs
can be quantified within these complex mixtures. In addition to provid-
ing information on common InsP species, the labeling strategy with
[13C6]myo-inositol has also the potential to identify hitherto uncharacterized
myo-inositol derived metabolites (Harmel et al., 2019). As more research
groups will utilize the method described here in a variety of cell lines and
organisms, we are certain to uncover novel components and regulatory
factors in inositol metabolism.
Acknowledgments
The authors thank Dr. Peter Schmieder for his support with NMR measurements and
analysis.
Funding
R.K.H. and R.P. gratefully acknowledge funding from the Leibniz-Gemeinschaft
(SAW-2017-FMP-1).
References
Auesukaree, C., Tochio, H., Shirakawa, M., Kaneko, Y., & Harashima, S. (2005). Plc1p,
Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential
for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.
The Journal of Biological Chemistry, 280, 25127–25133.
Azevedo, C., & Saiardi, A. (2006). Extraction and analysis of soluble inositol polyphosphates
from yeast. Nature Protocols, 1, 2416–2422.
Balla, T. (2013). Phosphoinositides: Tiny lipids with giant impact on cell regulation.
Physiological Reviews, 93, 1019–1137.

Analysis of metabolically labeled inositol phosphate messengers
51
Banfic, H., Bedalov, A., York, J. D., & Visnjic, D. (2013). Inositol pyrophosphates modulate
S phase progression after pheromone-induced arrest in saccharomyces cerevisiae.
The Journal of Biological Chemistry, 288, 1717–1725.
Berridge, M. J. (2016). The inositol trisphosphate/calcium signaling pathway in health and
disease. Physiological Reviews, 96, 1261–1296.
Bhandari, R., Saiardi, A., Ahmadibeni, Y., Snowman, A. M., Resnick, A. C.,
Kristiansen, T. Z., et al. (2007). Protein pyrophosphorylation by inositol pyrophosphates
is a posttranslational event. Proceedings of the National Academy of Sciences of the United States
of America, 104, 15305–15310.
Chakraborty, A., Koldobskiy, M. A., Bello, N. T., Maxwell, M., Potter, J. J., Juluri, K. R.,
et al. (2010). Inositol pyrophosphates inhibit akt signaling, thereby regulating insulin sen-
sitivity and weight gain. Cell, 143, 897–910.
Dick, R. A., Zadrozny, K. K., Xu, C., Schur, F. K. M., Lyddon, T. D., Ricana, C. L., et al.
(2018). Inositol phosphates are assembly co-factors for HIV-1. Nature, 560, 509–512.
Gu, C., Nguyen, H.-N., Hofer, A., Jessen, H. J., Dai, X., Wang, H., et al. (2017). The
significance of the bifunctional kinase/phosphatase activities of diphosphoinositol
pentakisphosphate kinases (PPIP5Ks) for coupling inositol pyrophosphate cell signaling
to cellular phosphate homeostasis. The Journal of Biological Chemistry, 292, 4544–4555.
Harmel, R. K., Puschmann, R., Nguyen Trung, M., Saiardi, A., Schmieder, P., &
Fiedler, D. (2019). Harnessing 13 C-labeled myo -inositol to interrogate inositol phos-
phate messengers by NMR. Chemical Science, 10, 5267–5274.
Irvine, R. F., & Schell, M. J. (2001). Back in the water: The return of the inositol phosphates.
Nature Reviews. Molecular Cell Biology, 2, 327–338.
Jadav, R. S., Kumar, D., Buwa, N., Ganguli, S., Thampatty, S. R., Balasubramanian, N.,
et al. (2016). Deletion of inositol hexakisphosphate kinase 1 (IP6K1) reduces cell migra-
tion and invasion, conferring protection from aerodigestive tract carcinoma in mice.
Cellular Signalling, 28, 1124–1136.
Koldobskiy, M. A., Chakraborty, A., Werner, J. K., Snowman, A. M., Juluri, K. R.,
Vandiver, M. S., et al. (2010). p53-mediated apoptosis requires inositol hexakisphosphate
kinase-2. Proceedings of the National Academy of Sciences of the United States of America, 107,
20947–20951.
Mallery, D. L., Ma´rquez, C. L., McEwan, W. A., Dickson, C. F., Jacques, D. A.,
Anandapadamanaban, M., et al. (2018). IP6 is an HIV pocket factor that prevents capsid
collapse and promotes DNA synthesis. eLife, 7, e35335.
Mayr, G. W. (1988). A novel metal-dye detection system permits picomolar-range h.p.l.c.
analysis of inositol polyphosphates from non-radioactively labelled cell or tissue speci-
mens. The Biochemical Journal, 254, 585–591.
Menniti, F. S., Miller, R. N., Putney, J. W., Jr., & Shears, S. B. (1993). Turnover of inositol
polyphosphate pyrophosphates in pancreatoma cells. The Journal of Biological Chemistry,
268, 3850–3856.
Moritoh, Y., Oka, M., Yasuhara, Y., Hozumi, H., Iwachidow, K., Fuse, H., et al. (2016).
Inositol hexakisphosphate kinase 3 regulates metabolism and lifespan in mice. Scientific
Reports, 6, 32072.
Puschmann, R., Harmel, R. K., & Fiedler, D. (2019). Scalable chemoenzymatic synthesis of
inositol pyrophosphates. Biochemistry, 58, 3927–3932.
Stephens, L., Radenberg, T., Thiel, U., Vogel, G., Khoo, K. H., Dell, A., et al. (1993). The
detection, purification, structural characterization, and metabolism of diphosphoinositol
pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s). The Journal of
Biological Chemistry, 268, 4009–4015.
Veiga, N., Torres, J., Dominguez, S., Mederos, A., Irvine, R. F., Diaz, A., et al. (2006). The
behaviour of myo-inositol hexakisphosphate in the presence of magnesium(II) and
calcium(II): Protein-free soluble InsP6 is limited to 49 micro M under cytosolic/nuclear
conditions. Journal of Inorganic Biochemistry, 100, 1800–1810.

52
Robert Puschmann et al.
Wilson, M. S. C., Bulley, S. J., Pisani, F., Irvine, R. F., & Saiardi, A. (2015). A novel method
for the purification of inositol phosphates from biological samples reveals that no phytate
is present in human plasma or urine. Open Biology, 5, 150014.
Wormald, M., Liao, G., Kimos, M., Barrow, J., & Wei, H. (2017). Development of a
homogenous high-throughput assay for inositol hexakisphosphate kinase 1 activity.
PLoS One, 12, e0188852.
Wundenberg, T.,
& Mayr, G. W. (2012). Synthesis and biological actions of
diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis.
Biological Chemistry, 393, 979–998.
Zhu, J., Lau, K., Harmel, R. K., Puschmann, R., Broger, L., Dutta, A. K., et al. (2019). Two
bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate
homeostasis. Elife, 8, e43582.