13

C-Glutamine Qualitative Flux
Analysis of a Chondrosarcoma Cell
Line Using Agilent VistaFlux

Application Note
Metabolomics

Authors

Abstract

Hui Liu, Weige Qin, Hardik Shah,Interpretation of metabolomics data is often complicated by a lack of dynamic

Bhavapriya Vaitheesvaran,

information since signiﬁcant changes in ﬂux through a pathway can occur

Vladimir Yong, and Justin R. Cross

without corresponding changes in metabolite abundances. Stable isotope

Donald B. and Catherine C. Marron

tracing (qualitative ﬂux analysis) has tremendous potential to help address

Cancer Metabolism Center,

these situations, and allow a deeper understanding of biological systems. In

Memorial Sloan Kettering Cancer

qualitative ﬂux analysis, a stable isotope tracer (typically containing 13C, 15N, orCenter2

Lucia Salamanca-Cardona andKayvan R. Keshari
Department of Radiology andMolecular Pharmacology Program,
Memorial Sloan Kettering Cancer
CenterH) is introduced into the biological system, and results in changes in the isotopic

pattern (isotopologue distribution) of downstream metabolites. The approach isparticularly useful in cell culture models that are amenable to the introduction of
stable isotope tracers, and analysis of the resulting labeling pattern and kinetics oftracer incorporation provides insights into enzyme function, pathway dependence,and the effect of changes in gene expression or protein function.
Agilent MassHunter VistaFlux software is designed to be a comprehensive
workﬂow that helps scientists perform qualitative ﬂux analysis, and facilitates
the processing and visualization of high-resolution accurate mass TOF and
Q-TOF LC/MS data. Tools are provided for feature extraction, analysis of isotope
incorporation and isotopologue abundance, natural abundance correction,
statistical analysis, and data visualization on pathways.

This application note demonstrates
qualitative ﬂux analysis in achondrosarcoma cell line, using
13
C5-glutamine (U-13C-Gln) as the tracer.
Data were acquired on an Agilent LC/TOF
system, and MassHunter VistaFlux wasused for data analysis and interpretation.
The study demonstrates the effect ofan isocitrate dehydrogenase 2 (IDH2)
mutation on TCA cycle metabolism, inparticular the diversion of glutamine tothe production of 2-hydroxyglutarate(2-HG) by the mutant IDH2 isoform.

Introduction
Somatic IDH mutations have recently
been described in a broad range ofsolid and hematologic tumors, including
glioma, acute myeloid leukemia, andchondrosarcoma. Mutations in isocitrate
dehydrogenase enzymes (IDH1/2) occur
in the enzyme’s active site, and confera gain-of-function phenotype resulting
in an increased ability to produce themetabolite 2-hydroxyglutarate (2-HG).
Recent research has shown that 2-HG
accumulates to high levels in IDH mutant
cells due to saturation of the disposal
pathway, whereupon 2-HG becomes
a competitive inhibitor for a varietyof α-ketoglutarate (α-KG) dependent
dioxygenase enzymes including the TET
family of DNA demethylases, Jumonji Cfamily of histone demethylases, mRNA
demethylases FTO and ALKBH5, andprolyl hydroxylase (PHD) enzymes that
regulate hypoxia-inducible factors (HIFs)1.
These changes promote a repressive
chromatin environment that lock cells in
an undifferentiated state.
Chondrosarcomas, a type of sarcoma
arising from the chondrocytes that
deposit cartilage, exhibit high incidencesof IDH mutations. The availability oftumor-derived cell lines has madechondrosarcoma a useful model system
for understanding the consequences of
IDH mutations. An important componentof this has been the use of qualitativeﬂux analysis to show that 2-HG producedby the mutant enzyme is made fromglutamine through α-KG and the TCA
cycle.

Experimental
Cell culture and reagents
Human chondrosarcoma CS-1 cell line2,
was maintained in DMEM containing
10 % FBS, 25 mM glucose, 4 mM
L-glutamine, 100 units/mL penicillin, and
100 ug/mL streptomycin under 5 % CO2
in a 37 °C incubator. For metabolic tracing
experiments, 300,000 cells/well wereplated in standard 6-well culture plates
2 days before the start of the labeling
period, and triplicate wells were preparedfor each time point. The tracer-containing
medium was DMEM without glutamine,
supplemented with 10 % dialyzed FBS and
4 mM [U-13C]-L-glutamine (Isotec 605-166,
Sigma). At the start of the labeling
period, cells were washed once with
PBS, and transferred to tracer-containing
medium for 0.5, 1, 3, and 8 hours prior
to harvest. Since medium changes canalso perturb metabolite pools, the control(unlabeled) cells were harvested 8 hours
after changing into DMEM containing
10 % dialyzed FBS and 4 mM L-glutamine(without isotopic enrichment), as thecontrol cells were an exact match for the
8 hours U-13C-Gln time point.

Metabolite extraction
Culture medium was gently aspirated, andcells were extracted in 1 mL/well 80 %aqueous methanol (v/v). Plates wereincubated at –80 °C for >1 hour to aid
protein precipitation, then scraped. The
extract was transferred to microcentrifuge
tubes for clariﬁcation by centrifugation at
21,000 x g for 15 minutes at 4 °C. Then,the extract was transferred to clean
tubes, and evaporated to dryness in a
vacuum concentrator (Genevac).
Note that since changing the cell
culture medium and washes with
PBS can perturb rapidly-exchanging
metabolite pools, for this qualitativeﬂux experiment, cells were only washedwith PBS prior to addition of the stable
isotope-containing medium. At harvest,
the extraction solvent was added directly
after aspirating the medium without
additional washing to best preserve2

levels of intracellular metabolites and theassociated labeling patterns. However, if
such a procedure is followed, care must
be taken when interpreting results formetabolites also present at high levels in
the culture medium, since this approach
will result in a higher degree of residual
medium contamination in the cell extracts
than if a PBS wash had been included
immediately prior to harvesting the cells.

LC/MS analysis
Dried extracts were resuspended in
120 µL of water, and incubated on ice for
20 minutes, vortexing every 5 minutes.
Extracts were then centrifuged at
21,000 x g for 15 minutes at 4 °C toremove any residual particulate material.
A 100 μL volume was transferred to a
reduced-volume autosampler vial, and10 µL was removed from every sample
to assemble a pooled QC vial that was
injected six times at the start of theanalytical run, then regularly throughout
the analysis to ensure chromatographic
stability.
Data were acquired on an Agilent LC/MS
system consisting of:•Agilent 6230 time-of-ﬂight mass
spectrometer•

Agilent 1290 Inﬁnity Binary UHPLC
pump•

Agilent 1290 Inﬁnity thermostatted
column compartment•

Agilent 1290 Inﬁnity HTS
autosampler

An Agilent 1260 Inﬁnity isocratic pumpwith a 100:1 ﬂow splitter was used to
introduce the reference mass solution.
The LC was interfaced to the MS with adual electrospray ionization source, anddata were acquired in negative ion modeusing the 4 GHz high resolution mode at
1.5 spectra per second. Table 1 shows theanalytical method settings.
The analytical method was designed
for the metabolites of interest in thisexperiment. Because the TCA cycle
is composed of organic acids that arepoorly retained by reversed-phase
chromatography, ion-pair reversed-phase
chromatography was used. Negativemode electrospray ionization was
selected for best detection of the target
compounds.

Data analysis
Using Agilent MassHunter Pathways
to PCDL software, an Agilent Personal
Compound Database and Library ﬁle(.cdb) was created for the tricarboxylic
acid (TCA) cycle drawn from BioCyc. In
addition, lactate, pyruvate, aspartate,
glutamate, oxidized glutathione, and 2-HG
were appended to the custom PCDL using
Agilent METLIN PCDL as the source. Thiswas edited in Agilent PCDL Manager
software to remove metabolites notmeasured by this analytical method, andadd retention times for each compound.
The PCDL ﬁle was then used as a target
list for batch isotopologue extraction in
Agilent MassHunter Proﬁnder software(Table 2). The Proﬁnder batch results,
corrected for 13C natural abundance, were
exported to Omix Premium as a Proﬁnder
archive ﬁle (.PFA), which includes
sample group information, compound
identiﬁers, and isotopologue abundances.
Export of the complete dataset as acomma-separated value (.csv) ﬁle is also
supported.

Table 1. LC/MS analysis parameters.
Parameter

Value

LC conditions
ColumnWaters Cortecs C18+ 150 mm × 2.1 mm, 2.7 µm

Mobile phaseA) 5 mM N,N-dimethyloctylamine in water, pH 5.5
B) 5 mM N,N-dimethyloctylamine in 90 % methanol in water, pH 5.5

Flow rate

0.4 mL/min from 0–8 minutes, then 0.6 mL/min from 8.1–10 minutes

Gradient

10 %B at 0 minutes
100 %B at 8.0 minutes

Stop time

10 minutes

Post time

5 minutes

Column temperature

30 °C

Injection volume

15 μL

MS conditions
Ionization mode

Dual ESI

Ionization polarity

Negative

Gas temperature

250 °C

Drying gas

13 L/min

Nebulizer pressure

45 psiCapillary voltage

3,500 V

Octopole 1 RF voltage

600 V

Acquisition range

m/z 50–1,700

Reference masses

m/z 119.0363 and 980.01638

Table 2. Target metabolite list for isotopologue extraction.
CompoundFormulaCAS

RT (min)

L-Glutamate

C5H9NO4

56-86-0

2.26

L-Aspartic acid

C4H7NO4

56-84-8

2.29

L-Lactic acid

C3H6O379-33-4

2.93

Glutathione, oxidized

C20H32N6O12S227025-41-8

4.84

Succinic acid

C4H6O4110-15-6

5.05

2-Hydroxyglutarate

C5H8O52889-31-8

5.11

Fumaric acid

C4H4O4110-17-8

5.13

L-Malic acid

C4H6O597-67-6

5.13

Oxoglutaric acid (α-KG)

C5H6O5328-50-7

5.24

Guanosine diphosphate (GDP)

C10H15N5O11P2

146-91-8

5.83

Adenosine diphosphate (ADP)

C10H15N5O10P2

58-64-0

5.93

Aconitic acid

C6H6O6499-12-7

6.1

threo-Isocitric acid (not resolved from citric acid)

C6H8O76061-97-8

6.1

Citric acid (not resolved from threo-isocitric acid)

C6H8O7

77-92-9

6.1

Guanosine triphosphate (GTP)

C10H16N5O14P3

86-01-1

6.57

Adenosine triphosphate (ATP)C10H16N5O13P3

987-65-5

6.63

3

Results and Discussion
After isotopologue extraction in Proﬁnder,
results were reviewed and manuallycurated as necessary; only glutamate andα-KG needed to be manually adjusted formore consistent results. The isotopologue
display in Proﬁnder provides useful
insight into the incorporation of thestable label, as well as reproducibility of
replicates within a sample group. This canbe viewed as raw values or % correctedvalues, with and without an additionalcorrection for natural isotope abundance.
Figure 1 shows the isotopologue resultsfor 2-HG. Results are displayed as naturalabundance-corrected values groupedby time point. In this case, time points
demonstrate excellent consistency acrossthe biological replicates but, if necessary,
outlier samples could be excluded at
this stage. Initial inspection of the datashows that over time a decrease in 2-HG
M+0 is observed with a corresponding
increase in 2-HG M+5, revealing that all
ﬁve carbons of 2-HG are being derived
from the U-13C-Gln tracer, and that after
8 hours the majority of the 2-HG pool has
become labeled and is, therefore, derived
from glutamine.

Figure 1. Agilent MassHunter Proﬁnder isotopologue display of 2-HG.

4

Qualitative ﬂux analysis is most powerfulwhen used to follow the fate of a stablelabeled isotope tracer through the context
of biological pathways. To visualize
the Proﬁnder results in a pathways
context, a network was created in
Omix Premium from the human BioCyc
database. Additional metabolites ofinterest, not in the canonical TCA cycle(lactate, pyruvate, oxidized glutathione,
aspartate, and glutamine) weremanually added to the network. Some
metabolites such as ADP, GDP, ATP, and
GTP are ubiquitous, and the measured

abundance represents the total cellularconcentration. To monitor incorporation inthese compounds, metabolite nodes areshown separate from a speciﬁc pathway
with the same options for information
visualization. After creating the network,
Proﬁnder results were imported into
Omix Premium (Figure 2). In the program,the Component View on the left lists all
metabolites in the network, the main
window displays the network, and theData Manager window at the bottom
allows control of the imported Proﬁnder
results.

Figure 2. Omix Premium software displaying network and imported Agilent MassHunter Proﬁnder results.

5

Omix Premium offers graphical options
speciﬁcally designed for the visualizationand interpretation of qualitative ﬂux
data including the ability to change
properties of the metabolite node, andto add associated charts and graphics.
As networks become more complex,
a quick summary of the experimentcan be achieved displaying metabolite
abundance by coloring the node. In
Figure 2, the colored ﬁll intensity reﬂects
the abundance of the metabolites
measured (with grey-ﬁlled metabolite
nodes being compounds not measured inthis experiment).

The information shown on the metabolite
nodes is designed to indicate where themaximum fold changes are occurring
relative to samples designated as thecontrol group. Once this high level reviewof the data across the pathway has been
completed, a more detailed inspectionof the stable isotope incorporation ispossible. Figure 4 shows additional
information that can be displayed in
quilt plots and horizontal bar charts tothe side of each metabolite node. Theseplots summarize isotopologue abundance
and tracer incorporation, and expand onsummary information displayed on thenode. In this example, the time point
results are displayed on the y-axis of
the quilt plot, and the isotopologues
are shown on the x-axis. The ﬁll color
intensity indicates relative abundanceof tracer incorporation. The bar chart
provides an alternative view of the total
tracer incorporation as a proportion of
the total metabolite abundance (sum of
all isotopologues detected). These views
conﬁrm the result ﬁrst observed during
manual data inspection of the data in
Proﬁnder, namely that after 3 hours and8 hours of incubation with U-13C-Gln
there is signiﬁcant label incorporation of
the tracer into the M+5 isotopologue of
2-HG, demonstrating that glutamine is the
major substrate used to produce 2-HG in
IDH2-mutant CS-1 cells.

A

B

C

2-Hydroxyglutarate

2-Hydroxyglutarate

2-Hydroxyglutarate

Figure 3. 2-HG node ﬁlled as A) abundance of the metabolite relative to other detected metabolites, B)
percent stable label incorporation as a color gradient ,and C) split ﬁll with abundance on the left side andpercent incorporation on the right side.

Labeling incorporation
0

2E5 4E5 6E5 8E5 1E6 1.2E6 1.4E6

0
Hours

There are three options for the color of
the metabolite node, as shown in Figure 3
for 2-HG. The node ﬁll can be used toshow that this metabolite is present at ahigh level (intensity of color in Figure 3A),or that stable label incorporation is
occurring (Figure 3B). Figure 3C presents
a combined view, showing both pieces of
information.

0.5
1
3

+0 +1 +2 +3 +4 +5 +6

8
Labeling incorporation
UnlabeledFigure 4. Detailed information about U-13C-Gln tracer incorporation into citrate in the CS-1 cell line.

6Figure 5 shows another useful view for
inspecting detailed information about
the isotopologue incorporation. This plot
displays the isotopologue results for thedifferent groups (time points), with error
bars to indicate the standard deviation
observed for replicate measurements.
Similar to the isotopologue display in
Proﬁnder, this plot provides a numericalview of the raw data. In this example, theplot for oxidized glutathione clearly shows

Metabolite nodes can also be included
for compounds such as ADP, GDP, ATP,and GTP to show no incorporation orchange in cellular levels throughout theexperiment (Figure 6). This implies thatGln does not contribute signiﬁcantly
to biosynthetic pathways for thesemetabolites within an 8-hour time period.
However, including information about
the relative abundance of the di- andtri-phosphate nucleotide pools can stillbe helpful in interpreting cell health andviability.

that, over time, the cellular glutathionepool becomes labeled with ﬁrst one andthen two Gln-derived 5-carbon building
blocks. The node ﬁll also indicates thatoxidized glutathione is a highly abundant
metabolite; thus it takes time for asigniﬁcant proportion of the total cellularpool to incorporate one and then two
Gln-derived subunits.

Glutathione,
oxidized

0 hours
0.5 hours
1 hour3 hours
8 hours% Isotopologue abundances (corrected)
100
80
60
40
20
0
m+0

m+1

m+2

m+3

m+4

m+5

m+6

m+7

m+8

m+9

m+10

m+11

Figure 5. Glutathione isotopologue abundances as a bar chart showing the incorporation of glutamine tracer with time.

Labeling incorporation
5E50
1E6 1.5E6

0

ATP

0.5

Hours

Hours

0

ADP

Labeling incorporation
0 5E5 1E6 1.5E6 2E6 2.5E6 3E6

2E6

1
3

0.5
1
3

8

8
Labeling incorporation
UnlabeledLabeling incorporation
0 2E4 4E4 6E4 8E4 1E5 1.2E5

1.5E5

0

0

0.5

0.5

GTP

1

Hours

GDP

Hours

Labeling incorporation
5E4
0
1E5

Labeling incorporation
Unlabeled

1
3

3

8

8
Labeling incorporation
UnlabeledLabeling incorporation
UnlabeledFigure 6. Display of overall ﬂux for ubiquitous metabolites ADP, GDP, ATP, and GTP.

7

m+12

m+13

Combining these views, it becomes
possible to compare the isotopologue
distribution and labeling kinetics acrossthe network (Figure 7). For presentation
purposes, metabolite node ﬁlls canbe replaced with structural formulas
imported from BioCyc. Basic statisticalinformation can also be overlaid on thequilt plot. Figure 7 shows the resultsof Welch’s t-test for each isotopologue
relative to abundance in the control group(designated as the 0 hour time point
samples), with • indicating p < 0.05.

Inspection of these qualitative ﬂux
proﬁling results clearly reveals that the
U-13C-Gln tracer rapidly labels glutamate(Glu), somewhat faster than labeling of
the 2-HG pool. This is reﬂective of therelatively low activity of the mutant IDH2
enzyme for completing the reductive
reaction to produce 2-HG. It can also be
inferred that the pathway is likely direct,
since Glu, α-KG (not shown), and 2-HG all
retain all ﬁve 13C atoms from the U-13C-Glntracer. The results show that there isalso forward (oxidative) consumption

Figure 7. Summary of U-13C-Gln labeling of 2-HG and the TCA cycle metabolites.

8of U-13C-Gln in the TCA cycle, resulting
in rapid labeling of succinate, fumarate,
and malate predominantly as the M+4
isotopologue. Oxaloacetate (OAA) ispresent at low levels in most cells,
and is too unstable to reproducibility
survive extraction and analysis by
LC/MS. However, the OAA pool is inequilibrium with L-aspartic acid (Asp),
which is also rapidly labeled by the tracer,
predominantly as the M+4 isotopologue.

Later, M+3 labeling is seen in 2-HG,
succinate, fumarate, malate, andaspartate. This is the result of M+4labeled OAA combining with two
unenriched carbons contributed fromacetyl-CoA, and resulting in citrate
M+4. The M+3 isotopologue seen indownstream metabolites is the resultof decarboxylation by the wild type IDH
reaction, acting in the forward (oxidative)
direction. There is also evidence of
M+5 labeling in citrate, resulting fromreductive carboxylation catalyzed by thewild type IDH enzyme acting in reverse.
By 8 hours, more complex labeling
patterns are observed, resulting from:•

Further rounds of the TCA cycle•

Scrambling of labeling patterns
due to the symmetry in succinate,
fumarate, and citrate•

Potentially the action of malic
enzymeThese patterns could be investigatedfurther using alternative stable isotope
tracers, or with Gln labeled at speciﬁc
carbon positions. Finally, the summary
view makes it possible to assess therelative kinetics of metabolite labeling,
and it can be seen that Asp and Glulabeling are approaching steady stateby 8 hours of labeling. Longer time
points would be required for the othermetabolite pools to reach metabolicsteady state.

Conclusions

References

Using the Agilent MassHunter VistaFlux
workﬂow, qualitative ﬂux proﬁling was
performed in CS-1 chondrosarcoma cellswith U-13C-Gln as the metabolic tracer.
This analysis clearly demonstrates theactivity of the IDH2 mutant isoform toconvert Gln-derived α-KG into 2-HG.
Over 8 hours, the majority of the 2-HG
pool becomes labeled as the M+5
isotopologue in cells harboring an IDH
mutation. This increased production of
2-HG, driven by the neomorphic activityof the IDH2 R172S mutant enzyme,
overwhelms the endogenous 2-HG
dehydrogenases, allowing 2-HG toaccumulate to high levels. Then, 2-HG
becomes a competitive inhibitor for avariety of α-KG dependent dioxygenase
enzymes including TET family DNA
demethylases and Jumonji C family
histone demethylases, resulting in
epigenetic changes including DNA
hypermethylation, promoting an
undifferentiated chromatin environment.
The analysis also shows theconsequences of the U-13C-Gln tracer
entry into the canonical TCA cycle
pathway.

1. Losman, J. A.; Kaelin, W. G. Jr. Whata difference a hydroxyl makes: mutant
IDH, (R)-2-hydroxyglutarate, andcancer. Genes Dev. 2013, 27(8), 836-52.

Compared to manual data mining,
the VistaFlux workﬂow provides acomprehensive, automated, andaccelerated framework for the processingof stable isotope tracing data, including
isotopologue extraction and pathway
visualization of qualitative ﬂux proﬁling
data.

9

2. Susa, M.; et al. Alendronate inhibits
growth of high-grade chondrosarcoma
cells. Anticancer Res. 2009, 29,
1879–1888.

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic
procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2016
Published in the USA, May 6, 2016
5991-6810EN