Basics of metabolite profiling and metabolic flux analysis

May 1st, 2020

Issam Ben-Sahra

Contact: issam.ben-sahra@northwestern.edu Phone : 312-503-5318

Membranes, Organelles & Metabolism

mailto:issam.ben-sahra@northwestern.edu

Cells respond to environment

Heat

Nutrients

Cold

Various external messages

Environmental conditions

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

Metabo-lome Metabolites

Proteins

Genes

External environmentAlters

AlterInitiate

Instructions to make

Alte

r

Metabolites are central in cell physiology

Genome

Proteome

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

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

Quantitative Metabolomics: Measurement of metabolite levels at one instant t

Steady-state Metabolomics

Know your textbook

Literature recommended

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

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

glutamine

glutamate

GLS

Valine

Methionine

methionineglutamate

glut

amin

e

valin

e

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

Metabolic flux

13C-Glucose

13C-F 1,6-BP

13C-pyruvate13C-lactate

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

-

-

-

-

-

-

++

+++ +

+

-

Carbon 12 Carbon 13-

-

-

-

-

-

++

+++ +

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.

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

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

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

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

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

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)

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

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

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

The software INCA can be used to perform MFA calculations.

Metabolic Flux Analysis (MFA)

Antoniewicz MR et al., Exp & Mol Med 2018

Acknowledgements

Eunus Ali Umakant Sahu

Lab members:

Metabolomics Developing Core Facility

Elodie Villa Brendan O’Hara

Peng GaoNav Chandel

“How to Conduct in vivo Metabolomics”

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