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Basics of metabolite profiling and metabolic flux analysis
May 1st, 2020
Issam Ben-Sahra
Contact: [email protected] Phone : 312-503-5318
Membranes, Organelles & Metabolism
mailto:[email protected]
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Cells respond to environment
Heat
Nutrients
Cold
Various external messages
Environmental conditions
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Metabo-lome
Metabolome
Proteome
Genome
Res
pons
eR
espo
nse
Res
pons
eTime
External stimuli
Genome
Proteome
Signals (light, O2, food, etc.)
The Pyramid of Life
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Metabo-lome Metabolites
Proteins
Genes
External environmentAlters
AlterInitiate
Instructions to make
Alte
r
Metabolites are central in cell physiology
Genome
Proteome
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Metabolism
Me Me Ac Ac
Regulation of chromatin accessibility Gene expression
DNA
Histone octamer
Lactate
Zhang D, et al., Nature Oct 2019
Metabolites can directly control gene expression
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Metabolomics connects Proteome and Genome to Phenotype
• Metabolomics data provides insights into underlying biology
• Metabolomics data provides information behind the mechanisms by which genes function
• Multiple omics data pointing to the same biological pathways builds scientific hypotheses and bring us closer to translational science
Phenotype
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Quantitative Metabolomics: Measurement of metabolite levels at one instant t
Steady-state Metabolomics
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Know your textbook
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Literature recommended
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Steady-state Metabolomics
Gln
Glucose
pyruvate
citrate
isocitrate
Glu
AspAsn
Ser
α-KG
GlyATP UTP
UMPUDP
CTP
AMPADP
glycerol
lactate
NAD+
Arg Trp Pro
Tyr
His
dTTP
succinate
Fumarate
OAA
cAMPNADP NADPH
FolateTHF
DHF N-carb-asp
FADH2FAD creatine
Ala
Phe
Malate
Ac-CoA Lys
Met Cys Leu Ile Thr
CMP
dAMP
GMP
GTP
dTMPIMP
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Nutrient and the response of metabolism
Metabolic changes (unlabeled)
Time (min) Time (hours/days) Time (min)
Glucose added
Met
abol
ism
(e.g
., gl
ucos
e up
take
rate
) Glucose consumed
Fresh MediumGlucose 25mM
Time (hours/days)
MediumLow glucose
Steady-state period
The quantitative inflows and effluxes from each metabolite must be balanced
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glutamine
glutamate
GLS
Valine
Methionine
methionineglutamate
glut
amin
e
valin
e
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Glucose removal decreases flux though glycolysis but some glycolytic intermediates increase (e.g., PEP)
Metabolite levels Vs Metabolic flux
Metabolite level Flux
Adapted from Jang C, et al., Cell 2018
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Metabolic flux
13C-Glucose
13C-F 1,6-BP
13C-pyruvate13C-lactate
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C
C
CC
C
O
CH2OH
OH
HH
OHH
OHHO
HH C
C
CC
C
O
13CH2OH
OH
HH
OHH
OHHO
HH 13
13
1313
13
Glucose 13C6-GlucoseU-13C-Glucose
- Heavy isotope- Non radioactive- Similar physical properties
Isotopic tracer
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-
-
-
-
-
-
++
+++ +
+
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Carbon 12 Carbon 13-
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-
-
-
-
++
+++ +
Proton (+)
NeutronElectron(-)
Isotopic tracer
Carbon 13 has an extra neutron
Because the labeled atom has the same number of protons, it will behave in almost exactly the same way as its unlabeled counterpart and, will not interfere with the
reaction under investigation.
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Time (min) Time (hours) Time (hours)
13C
-enr
ichm
ent i
n m
etab
olite
s
Dynamic labeling
Steady-state labeling
13C-glycine/serine
Metabolite(e.g., IMP, AMP, GMP)
Isotopic tracing to study the activity of metabolic pathways in cells
lactate
Glyceraldehyde 3-P
G6-P
Time (min) Time (hours) Time (hours)
13C
-enr
ichm
ent i
n m
etab
olite
s
Dynamic labeling
Steady-state labeling13C-Glucose
Metabolite A(e.g., G6P, lactate) Metabolite B
(e.g., IMP, AMP, GMP)
pyruvate
ATP
ADP
ADP
Fructose 1,6-BP
ATP
glyc
olys
is
Ribose
Nucleotides(IMP, AMP, GMP)
ATP
ADP
U-13C-GlucoseIntensity of labeling glucose
lactate
Glyceraldehyde 3-P
G6-P
pyruvate
ATP
ADP
ADP
Fructose 1,6-BP
ATP
glyc
olys
is
Nucleotides(IMP, AMP, GMP)
Ribose
ATP
ADP
13C-glycine or 13C-serine
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glutamate(condition A)
glutamate(condition B)
13C5-glutamine
13C enrichment over time
met
abol
ite le
vels
Dynamic labeling data
Isotopic tracing: Consider the size of the unlabeled pool
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lactate
Glyceraldehyde 3-P
G6-P
pyruvate
ATP
ADP
ADP
Fructose 1,6-BP
ATP
glyc
olys
is
Ribose
Nucleotides(IMP, AMP, GMP)
ATP
ADP
U-13C-Glucose
ATP
TCA cycle
mitochondria
ETC
Isotopic tracing to study the activity of metabolic pathways
• Consider tracer uptake (e.g., glucose uptake) before performing the tracing
• Perform the flux in tracer-free medium (e.g., glucose-free medium)
• Timing of labeling matters:
For 13C-glucose:
- ~15-30 min of labeling enables to label glycolytic intermediates at the steady-state level
- 2-4h of labeling is required to label the TCA cycle
- 6-15h of labeling is required to label nucleotides
Recommendation for tracing experiments:
Intensity of labeling
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Acetyl-CoA
13C6-glucose
pyruvate
PCPDH n rounds (n≥2)
n rounds (n≥2)
n rounds (n≥2)
CitrateOAA
α-KG
TCA
glutamate
13C
≪≪
Representation of the metabolic tracing diagram
Lactate
12C
Asp
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α-ketoglutarate glutamate
Isotopic tracing: Impact on scientific research
Isot
opol
ogue
s
13C5-glutamine
M+1
M+2
M+3
M+4
M+5
α-KG
M+0
Adapted from Buescher JM et al., Curr Opin Biotechnol 2015
13C5-a-ketoglutarate (α-KG)
12C13C
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α-ketoglutarate glutamateM+0
M+1
M+2
M+3
M+4
M+5
Isotopic tracing: Impact on scientific research
815000
215000
35000
25000
95000
5615000
Fractional abundance α-KG (M+5) = 5615000
= 6800500
6800500~ 0.82
12C13C
Ion counts
Adapted from Buescher JM et al., Curr Opin Biotechnol 2015
α-KG
13C5-glutamine 13C5-a-ketoglutarate (α-KG)
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Isotopic tracing: Ben-Sahra labPRPP
PRA
GAR
FGAR
FGAM
AIR
CAIR
SAICAR
FAICAR
IMP
GART (E2)
PPAT
PFAS
GART (E1)
GART (E3)
PAICS (E1)
PAICS (E2)
ADSL
AICARATIC (E1)
ATIC (E2)
THFQ
E
Q
E
Fumarate
D
THF
Glycine
Formyl-THF
Tracer: 13C2-15N-glycine
CC
CCN
N
NC
De
novo
pur
ine
synt
hesi
s SK-MEL-28
Ali ES, Sahu U et al., Molecular Cell in press
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15.65 2.86
81.49
Untreated
27.10
10.9261.99
IL-4
citrate_13C1citrate_13C2citrate_13C3citrate_13C4
citrate_13C5
citrate_13C6
Unlabeled citrate Fan J et al, Nature 2014
Presentation of the tracing data
Chart Histogram
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Metabolic Flux Analysis (MFA)
13C metabolic flux analysis (MFA) is a mathematical approach for quantifying intracellular metabolic fluxes in cancer cells.
Antoniewicz MR et al., Exp & Mol Med 2018
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The software INCA can be used to perform MFA calculations.
Metabolic Flux Analysis (MFA)
Antoniewicz MR et al., Exp & Mol Med 2018
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Acknowledgements
Eunus Ali Umakant Sahu
Lab members:
Metabolomics Developing Core Facility
Elodie Villa Brendan O’Hara
Peng GaoNav Chandel
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“How to Conduct in vivo Metabolomics”
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