Principles and Practice of Mass Isotopomeric …bioinformatics.cesb.uky.edu/pub/RCSIRM/WorkshopSymposium2018/... · 11/3/2015 · VDCC K ATP Canonical Insulin Secretion OXPHOS Insulin

Principles and Practice of Mass Isotopomeric MultiOrdinate Spectral

Analysis (MIMOSA) to Assess Metabolic Flux"

Richard G. Kibbey M.D., Ph.D. Associate Professor

Departments of Internal Medicine and Cellular & Molecular Physiology Yale University School of Medicine

What is wrong with my metabolism??!?

TCA rate RPM x10000

1 2

3 4 5 6

7

8 9

6.5

The KREBS Cycle

Glucose

Glucose

Pyruvate

TCAADPATP

K+

2+Ca

[Ca]2+ i

GLUT

VDCC

ATPK

Canonical Insulin Secretion

OXPHOS

InsulinRelease

SCS-GTP

cytoplasm

mitochondria

Canonical Insulin Secretion

CO2

NADH

NADPH

O2

GK

HHF6(GLUD1)

CongenitalHypoglycemia

MIMOSAwasdevelopedtoreconcileinconsistanciesinstableisotopeanalysis

HowtomeasureglucoseoxidaNonininsulinsecreNngcells?

[U-13C6]Glucose

Citrate

Glutamate

Acetyl-CoA

0 80 160 2400

20

40

60

80

100

Time (min)

13C

- En

richm

ent (

%)

PEP M+3 Glutamate M+2

?

Mitochondria

NNNN

N N

N

N

NPPPGluco

Kinase

PP

P PPPP

Aldolase

PyruvateCarboxy-lase

CitrateSynthase

Isocitratedehydro-genase

TransAminase

Fumarase

SuccinylCoA

Synthetase

OGDH

PPEPCK

PyruvateKinase

GlycolysisPyruvateDehydro-genase

Mitochondria

NNNN

N N

N

N

NPPP

PP

P PPPP

P

Glycolysis

GlucoKinase

Aldolase

Fumarase

PyruvateCarboxy-lase

PEPCK

PyruvateKinase

PyruvateDehydro-genase

CitrateSynthase

Isocitratedehydro-genase

TransAminase

OGDH

SuccinylCoA

Synthetase

Concentration-Based Flux Measurements

Concentration ≠ Flux

MetaboliteX

Isotope Enrichment-Based Flux Measurements

40% Enriched 25% Enriched

Enrichment ≠ Flux

Steadystateenrichments≠fluxProductEnrichmentdependsonprecursorenrichment

0

50

100

Time

13C

(%)

fA (V)

fB (V)0

50

100

Time

13C

(%)

fA (4V)

fB (4V)0

50

100

Time

13C

(%)

fA (V/2)

fB (V/2)

A BVV V

The Tyranny of reversible reactions…

Where to live?

Fate map ≠Flux map

TCA Jam:

Cycles are not Rings

MIMOSA Platform MIMOSA •  Mass •  Isotopomer •  Multi •  Ordinate •  Spectral •  Analysis

Resource

Integrated, Step-Wise, Mass-Isotopomeric FluxAnalysis of the TCA Cycle

Graphical Abstract

Highlightsd LC-MS/MS positional 13C-enrichment for steady-state and

dynamic flux analysis

d Intersecting metabolic fluxes are disentangled by

deciphering citrate isotopomers

d Comprehensive precursor/product positional 13C-label

transfer analysis

d Quantitative mitochondrial oxidative, anaplerotic, cycling,

and exchange rates

Authors

Tiago C. Alves, Rebecca L. Pongratz,

Xiaojian Zhao, ..., Gary W. Cline,

Graeme Mason, Richard G. Kibbey

[email protected]

In BriefQuantitative assessment of intracellular

metabolism requires measuring the

enzyme-to-enzyme flow of metabolites.

Mitochondria have multiple nodes where

metabolites intersect, scramble, and

diverge, complicating isotope labeling.

Alves et al. use LC-MS/MS to decipher

step-wise position-specific transfer of13C coming from glucose into subsequent

metabolites through glycolysis and

around the TCA cycle.

Alves et al., 2015, Cell Metabolism 22, 1–12November 3, 2015 ª2015 Elsevier Inc.http://dx.doi.org/10.1016/j.cmet.2015.08.021

Logues vs. Mers •  Isotopologues: Position Non-Specific

e.g., Citrate M+3 (20/64 potential combinations)

•  Isotopomer: Position Specific

�

Figure S4 – Characterization of citrate isotopomers. (A) Parent/daughter distribution of the

citrate fragments for M+2, M+3, M+4 and M+5. (B) Citrate isotopomers generated by reverse

ICDH.

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

A

B

�



ICDH.

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

A

B

�



ICDH.

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

A

B

8 species Oxidative Anaplerotic 3 families

Pyruvate Citrate

PDH 123645a

OAA

PCCS IDHTCA

h

c

j

g

f’

b’

Citrate

CitrateOAA

Glutamate

GlutamateOAA

IDHTCA

Glutamate

CS12345

b

f

i

d

e

k

21

34

OAA

IDH TCA IDH TCA TCACS IDH

Citrate Glutamate OAA

Citrate

Glutamate

OAAGlucose

Metabolism of glucose

Position from MS/MS data

1 2 3 4 5

4 5 1 2 3

+

Originated from Acetyl-CoA Detected by MS

Originated from OAA

M+0 M+1 M+2 M+3 M+4 M+5

146/41 147/41 147/42 148/41 148/42 148/43 149/41 149/42 149/43 150/42 150/43 151/43

aspartate

OAA

malate

citrate

α-ketoglutarate glutamate

succinate

AcCoA

pyruvateβ-oxidation

Φ

MIMOSA: Leveraging Position

Mapping Substrate Flows

Φ PAc

Φ AcCitΦ C

itGΦ GSΦ S

MΦ M

OΦ O

Cit'Φ O

P0.00

0.25

0.50

0.75

1.00

1.25* **

Glutamate(Succinate(

Malate(

OAA(

Aspartate((D)(

Acetyl7CoA(

Pyruvate(ΦPAc%

Citrate(

ΦAcCit%

ΦCitG%

ΦGS%

ΦSM%

ΦMOD%

ΦMO%

ΦOCit’%

ΦOP%

Alves et al. Cell Metabolism 2015

!

!

Supplemental Figure S2 related to Figure 3– Steady State (Φ) Analysis. Steady-state precursor-product relationships between the metabolic intermediates involved in the PC, PDH and TCA cycle reactions. The equations describing these relationships are shown in Supplemental Table S2. The (*) represents the relationship with the highest enrichments and therefore is used as a reference within a group of equivalent relationships. A red dashed line was drawn to facilitate that comparison. ! !

PAc PAcCit PAcCitG AcCit AcCitG0.0

0.5

1.0

1.5

Φ

0.990.79

0.42

0.80

0.42

CitG (1) CitG (2) CitG (3) CitG (4) CitG (5) CitG (6) CitG (7)0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.53 0.450.56

0.760.68

0.350.52

GS0.0

0.5

1.0

1.5

Φ

CitS(1) CitS(2) CitS(3) CitS(4)0.0

0.2

0.4

0.6

0.8

1.0

Φ0.53 0.47

0.59 0.54***

SM(1) SM(2) SM(3)0.0

0.5

1.0

1.5

Φ

1.080.82 1.00***

PO POD(1) POD(2) POM POC0.0

0.1

0.2

0.3

0.4

0.5

Φ

0.17

0.350.32

0.210.14

*** **

CitSM(1) CitSM(2) CitSM(3)0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.50 0.48 0.55

SMOD(1) SMOD(2) SMOD(3)0.0

0.5

1.0

1.5

Φ

1.281.03 1.21**

aspartate'(D)'

OAA'

malate'

citrate'

α�ketoglutarate''''''''''

glutamate'

succinate'

AcCoA'

pyruvate'β9oxida<on'

ΦPAc'

ΦPCit'

ΦAcCG'

ΦPAcCG'

ΦAcCit'aspartate'(D)'

OAA'

malate'

citrate'


glutamate'

succinate'

AcCoA'


ΦPOD192'

ΦPOM'ΦPOCit'

ΦPO'

aspartate'(D)'

OAA'

malate'

citrate'


glutamate'

succinate'

AcCoA'


ΦCitG'

ΦCitS'

ΦCitM'

aspartate'(D)'

OAA'

malate'

citrate'


glutamate'

succinate'

AcCoA'


ΦGS'

ΦSM'

ΦSMOD'

aspartate'(D)'

OAA'

malate'

citrate'


glutamate'

succinate'

AcCoA'


ΦDO' ΦDOCit'

ΦMOD' ΦMOCit'

ΦOCit'

ΦMO'

MO(1) MO(2) MO(3) MO(4)0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.87 0.890.80 0.85

DO(1) DO(2) DO(3) DO(4)0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.74 0.66 0.65 0.71

OC(1) OC(2) OC(3)0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.79 0.84 0.79

MOC(1) MOC(2) MOC(3)0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.690.75

0.63

DOC(1) DOC(2) DOC(3)0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.58 0.56 0.51

OP MP DP0.0

0.2

0.4

0.6

0.8

1.0

Φ

0.25 0.22 0.17

(PEP)

Φ CitG1* Citrate glutamate

Φ CitG2 Citrate glutamate





Φ POCit Citrate


Cita,d, f ,h,i, j∑ GlutC4,5+2∑

[U −13 C5 ]GlutamateCit h2+i4+ j

!

"#

$

%&

GlutC1,2,3+3∑Cit g+ h+ c

2+(i+ e)4

+ j!

"#

$

%&

Cit c2+e4+ g

!

"#

$

%& [1, 2,3−13 C3]Glutamate

Cit b+ f2

+h+ c4

+3(i+ e)4

!

"#

$

%& Glut

C1,2,3+2∑

Cit b+ f2

+3d4+32(192 / 68)

!

"#

$

%& Glut

C1,2,3+1∑

Cit( (b,c,d,e, f ,g,h, i, j, 32(192 / 68)))∑ Glut

C1,2,3+1∑ + GlutC1,2,3+2∑ + Glut

C1,2,3+3∑

0 1 20

1

2

VPC/VCS(Model Input)

V PC/V

CS

(Cal

cula

ted

Out

put)

Φ(5)

Φ(6)

= " + 3 "%&%'((*+)" + 3 *-./0%'( − " + 3 "%&%'((*+)

= " +3 "%&%'(" + 3 )*+,-%'(

= " +3 "%&%'((*+)" + 3 *-./0%'(

= " +3 "%&%'(" + 2 *+',%'(

Φ(2)

Φ(3) = " + 3 "%&%'( − " + 3 *+,,-.%'(" + 3 /01+2%'(

= " + 3 %&'()'*+ " + 5 %&'()'*" + 3 ,-(./)'*

Φ(1)

Φ(4)

Succinate

Pyruvate

Malate

Acetyl-CoAVPC

VME

VCSVPEPCK

M+3(PC)

M+3(Succinate)

Calculated vs. Actual Pyruvate Carboxylase

Isotopomers vs. Isotopologues


0 80 160 2400

20

40

60

80

100

APE

(%)

Time (min)PDH Label (Σ Cit a,f,i,d,h,j) PC Label (Σ Cit h,c)

PDH

PC

Change of Perspective

Pyruvate Oxidation Pyruvate Carboxylation

Fit to mathematical model: CWave

0 80 160 2400

10

20

30

40

50Cit a

Time (min)

APE

(%)

0 80 160 2400.0

0.5

1.0

1.5

2.0[1,3,6,4,5-13C5]Citrate

Time (min)

APE

(%)

0 80 160 2400

10

20

30

40

50

Time (min)

APE

(%)

Glutamate Total C45

0 80 160 2400

5

10

15

Time (min)

APE

(%)

[U-13C4]Succinate

0 80 160 2400

5

10

15

Time (min)

APE

(%)

Cit f

0 80 160 2400

1

2

3

4

5[U-13C6]Citrate

Time (min)

APE

(%)

0 80 160 2400

5

10

15

20

25

Time (min)

APE

(%)

Succinate M+2

0 80 160 2400

2

4

6

8[1,2,3,4,5-13C5]Citrate

Time (min)

APE

(%)

0 80 160 2400

20

40

60

80

Time (min)

APE

(%)

Cit a, f, i, d, h, j

0 80 160 2400

1

2

3

Time (min)

APE

(%)

[2,3,4-13C3]Succinate

0 80 160 2400

2

4

6

8[2,3,6,4,5-13C5]Citrate

Time (min)

APE

(%)

0 80 160 2400

10

20

30

Time (min)

APE

(%)

[4,5-13C2]Glutamate

0 80 160 2400

2

4

6

8

10

Time (min)

APE

(%)

[(1,2,3)(1,3,4)(1,2,4)-13C3]Succinate

0 80 160 2400

10

20

30

Time (min)

APE

(%)

Malate M+2

0 80 160 2400

5

10

15

Time (min)

APE

(%)

[1,2,3-13C3]Malate

0 80 160 2400

1

2

3

Time (min)

APE

(%)

[1,2,4-13C3]Malate

0 80 160 2400

2

4

6

8

10

Time (min)

APE

(%)

[U-13C4]Malate

0 80 160 2400

10

20

30

Time (min)

APE

(%)

OAA M+2

0 80 160 2400

5

10

15

Time (min)

APE

(%)

[1,2,3-13C3]OAA

0 80 160 2400

1

2

3

4

Time (min)

APE

(%)

[1,2,4-13C3]OAA

0 80 160 2400

2

4

6

8

10

Time (min)

APE

(%)

[U-13C4]OAA


Sensitivity to 0.5 mM change in glucose concentration Positionless analysis severely misses anaplerosis

!

!

!

!0 80 160 240

0

1

2

3

4[1,2,3-13C3]Citrate

Time (min)

AP

E (%

)

0 80 160 2400

1

2

3

4

Time (min)

AP

E (%

)

[1,2-13C2]Citrate

Isotopomer Fit Isotopologue Fit0.00

0.02

0.04

0.06

0.08

V Lip_ d

il (µ

M / µM

Tau

rine

/ min

)

VLip_dil / VCS = 20%

PDH CS PC0.0

0.1

0.2

0.3

0.4

µM

/ µ

M T

aurin

e / m

in

Isotopomer FitIsotopologue Fit

0 80 160 2400

2

4

6

8

Pyruvate M+2

Time (min)

APE

(%)

Isotopologue FitIsotopomer Fit

!!!!

0 80 160 2400

10

20

30

40

50Citrate M+2

Time (min)

APE

(%)

0 80 160 2400

1

2

3

4

5Citrate M+6

Time (min)

APE

(%)

0 80 160 2400

2

4

6

8Glutamate M+5

Time (min)

APE

(%)

0 80 160 2400

10

20

30Malate M+2

Time (min)

APE

(%)

0 80 160 2400

5

10

15Citrate M+3

Time (min)

APE

(%)

0 80 160 2400

10

20

30Glutamate M+2

Time (min)

APE

(%)

0 80 160 2400

5

10

15

20

25Succinate M+2

Time (min)

APE

(%)

0 80 160 2400

10

20

30Malate M+3

Time (min)

APE

(%)

0 80 160 2400

5

10

15

20Citrate M+4

Time (min)

APE

(%)

0 80 160 2400

5

10

15Glutamate M+3

Time (min)

APE

(%)

0 80 160 2400

5

10

15Succinate M+3

Time (min)

APE

(%)

0 80 160 2400

2

4

6

8

10Malate M+4

Time (min)A

PE (%

)

0 80 160 2400

5

10

15Citrate M+5

Time (min)

APE

(%)

0 80 160 2400

5

10

15Glutamate M+4

Time (min)

APE

(%)

0 80 160 2400

5

10

15Succinate M+4

Time (min)

APE

(%)

A

B C D E F

0.06 0.08 0.10 0.120

50

100

PC Flux (µM/µM Taurine/min)

Freq

uenc

y


SD0.009

SD0.005

G

Supplemental Figure S5 related to Figure 5 – Comparison between the use of isotopologue and isotopomer data in the CWave model for flux calculation. (A) Fit curves of the isotopologue target data for citrate, glutamate, succinate and malate from INS-1 cells incubated with 9mM [U-13C6]glucose. The red open circles correspond to the enrichment data measured. The black lines correspond to the enrichments fit by the model. (B) !!"# calculated using the isotopomer and isotopologue data. (C-D) Examples of isotopomer fits that results from unlabeled acetyl-CoA: (C) [1,2,3-13C3]citrate and (D) [1,2-13C2]citrate. (E) PDH, PC and CS fluxes calculated using isotopomer and isotopologue data. (F) Prediction of M+2 pyruvate enrichments using the isotopomer (red line) and isotopologue data (green line). The blue circles correspond to the measured data. (G) PC flux values calculated by CWave using the isotopomer (red line) and isotopologue data (green line) and their respective standard deviation of the distribution.

!

!

!

!0 80 160 240

0

1

2

3

4[1,2,3-13C3]Citrate

Time (min)

AP

E (%

)

0 80 160 2400

1

2

3

4

Time (min)

AP

E (%

)

[1,2-13C2]Citrate

Isotopomer Fit Isotopologue Fit0.00

0.02

0.04

0.06

0.08

V Lip_ d

il (µ

M / µM

Tau

rine

/ min

)

VLip_dil / VCS = 20%

PDH CS PC0.0

0.1

0.2

0.3

0.4

µM

/ µ

M T

aurin

e / m

in

Isotopomer FitIsotopologue Fit

0 80 160 2400

2

4

6

8

Pyruvate M+2

Time (min)

APE

(%)


!!!!

0 80 160 2400

10

20

30

40

50Citrate M+2

Time (min)

APE

(%)

0 80 160 2400

1

2

3

4

5Citrate M+6

Time (min)

APE

(%)

0 80 160 2400

2

4

6

8Glutamate M+5

Time (min)

APE

(%)

0 80 160 2400

10

20

30Malate M+2

Time (min)

APE

(%)

0 80 160 2400

5

10

15Citrate M+3

Time (min)

APE

(%)

0 80 160 2400

10

20

30Glutamate M+2

Time (min)

APE

(%)

0 80 160 2400

5

10

15

20

25Succinate M+2

Time (min)

APE

(%)

0 80 160 2400

10

20

30Malate M+3

Time (min)

APE

(%)

0 80 160 2400

5

10

15

20Citrate M+4

Time (min)

APE

(%)

0 80 160 2400

5

10

15Glutamate M+3

Time (min)

APE

(%)

0 80 160 2400

5

10

15Succinate M+3

Time (min)

APE

(%)

0 80 160 2400

2

4

6

8

10Malate M+4

Time (min)

APE

(%)

0 80 160 2400

5

10

15Citrate M+5

Time (min)

APE

(%)

0 80 160 2400

5

10

15Glutamate M+4

Time (min)

APE

(%)

0 80 160 2400

5

10

15Succinate M+4

Time (min)

APE

(%)

A

B C D E F

0.06 0.08 0.10 0.120

50

100

PC Flux (µM/µM Taurine/min)

Freq

uenc

y


SD0.009

SD0.005

G

Supplemental Figure S5 related to Figure 5 – Comparison between the use of isotopologue and isotopomer data in the CWave model for flux calculation. (A) Fit curves of the isotopologue target data for citrate, glutamate, succinate and malate from INS-1 cells incubated with 9mM [U-13C6]glucose. The red open circles correspond to the enrichment data measured. The black lines correspond to the enrichments fit by the model. (B) !!"# calculated using the isotopomer and isotopologue data. (C-D) Examples of isotopomer fits that results from unlabeled acetyl-CoA: (C) [1,2,3-13C3]citrate and (D) [1,2-13C2]citrate. (E) PDH, PC and CS fluxes calculated using isotopomer and isotopologue data. (F) Prediction of M+2 pyruvate enrichments using the isotopomer (red line) and isotopologue data (green line). The blue circles correspond to the measured data. (G) PC flux values calculated by CWave using the isotopomer (red line) and isotopologue data (green line) and their respective standard deviation of the distribution.

Succinate

MalateαKG

Pyruvate

PEP

OAA

Acetyl-CoA

Citrate

VPDH

VPC

VME

VPK

VCS

VICDH

GlutamateVX

Vβ-OxidaNon

VPEPCK

Glutamine

VGlutaminase

VLipogenesis

MIMOSAMassIsotopomerMul?-OrdinateSpectralAnalysis

[U-13C6]Glucose

Alvesetal,CellMetab2015

VSDH

Malonyl-CoA

Fa]yAcids

PyruvateCarboxylase(PC)isthemostresponsivefluxtochangesinglucoseconcentraNonsinINS-1

Alvesetal,CellMetab2015

G2.5 G5 G7 G90.0

0.1

0.2

0.3

Flux

es

(µM

/ µM

Tau

rine

/ min

)

VPDH

VCS

VPC

Vβ-Ox

VICDH

G2.5 G5 G7 G90

100

200

300

400

500

Insu

lin (n

g/hr

/mg

prot

ein)

P < 0.05

P < 0.001

0 1 2 3 40

20

40

60

80

100

Insulin (Normalized to G2.5)

Flux

es (N

orm

aliz

ed to

G2.

5)

VPDHR2 = 0.968

VPCR2 = 0.957

MIMOSA Workflow

Integration (MS/MS)

P/D Natural Abundance

Isotopomer Deconvolvement

Φ (Steady state) ν (dynamic)

http://elucidata.io/el-maven

Caveat Emptor •  Concentration ≠Flux •  Enrichment ≠Flux •  Fate map ≠Flux map •  Cycles ≠Rings •  Steady State ≠ Kinetic •  Exponential fit ≠ single point •  TCA turns more than once •  Reversible reactions matter •  Location, Location, Location •  MIMOSA: not just for brunch

MIMOSA:highsensiNvityofmassspectrometrywiththeposiNonspecificityof13C-labeling

SuccinateMalate

αKG

Pyruvate

PEP

OAA

Acetyl-CoA

Citrate

VCS

RawMassSpec

M+2M+3M+4M+5M+6 vs

PCLabel TCALabel

vs

PDHLabeledAcetyl-CoA UnlabeledAcetyl-CoA

1stturnTCA 2ndturnTCA

vs

Glucose

THANK YOU!! •  Abudukadier Abulizi •  Tiago Alves •  Rebecca Cardone •  Gary Cline •  Joelle Hillion •  Selin Isguven •  Sean Jesinkey •  Anila Madiraju •  Graeme Mason •  Rachel Perry •  Raaisa •  Doug Rothman •  Stephan Siebel •  Gerald Shulman •  Romana Stark •  Bei Wang •  OrLando Yarbarough •  Xiaojian Zhao •  Lingjun Ma

TCA rate RPM x10000

1 2

3 4 5 6

7

8 9

6.5

www.elucidata.io

Thank YouAbhishek Jha

[email protected]

Documents

Principles and Practice of Mass Isotopomeric …bioinformatics.cesb.uky.edu/pub/RCSIRM/WorkshopSymposium2018/... · 11/3/2015 · VDCC K ATP Canonical Insulin Secretion OXPHOS Insulin