introduction to imaging metabolomics. · 7/20/2017 1 introduction to imaging metabolomics....

7/20/2017

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Introduction to Imaging Metabolomics.

7-20-2017Janusz Kabarowski, Dept. Microbiology, UAB.

0

1

2

3

4

5

6

-lo

g10

p

-8 -6 -4 -2 2 4 6 8

Inte

nsi

ty

Mass/Charge, Da

140 220 300 380 460 540 620 700 7800

500

1500

2500

3500

4500

5500

6500

7500

8500

9500

10500

782.5853

125.0021

184.0716

O

O

OO

O P

OH

HCH3

N CH3CH3

Matrix-Assisted Laser Desorption/Ionization (MALDI):Matrix molecules absorb laser light, enter an excited state, and collide with sample molecules, facilitating

charge transfer to create ions.

Mass Spectrometric Imaging for biomedical tissue analysisKamila Chughtai and Ron M.A. HeerenChem Rev. Vol.110(5): pp3237–3277, 2010.

MALDI-TOFinstrument

Conventional MALDI plate

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Vacuum sublimation is used to apply an even microscopically thin uniform layer of matrix

compound onto tissue sectionwithout the need for solvents.

Sublimation: the transition of a substance from solid togas phase without an intermediate liquid phase.

MALDI matrices for lipid imaging:

DHB: 2,5-dihydrobenzoic acid (+ve mode)

1,5-diaminonapthalene (-ve mode)

Cryosectioning onto Indium Tin Oxide (ITO) coated glass slides and scanning digital image of slide for

“teaching” FlexControl software on MALDI-TOF.

Minimum 2400dpicryosection image

Cryosectioning 10% gelatinembedded tissue block

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Vacuum sublimation apparatus for matrix application in MALDI Imaging.

Vacuummicro-valve Pirani vacuum

gaugeDigital vacuum

monitor

Vacuumsublimation

chambervacuumexhaust

Cold trap

Matrix deposition by vacuum sublimation.

Matrixcompound

Cold condensor unitof vacuum

sublimation chamber

ITO (indium-tin-oxide coated)slide with cryosections

140°C

0.05 Torr

5‐10°C

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Slides with matrix applied by vacuum sublimation.

Deposition of the matrixcompound is at the molecularlevel because gaseousmolecules recrystallize at therelatively cold surface of thetissue section attached to thecold condenser.

The uniformity of matrixdeposition onto the slideattached to the coldcondenser surface reflectsthe random Brownian motionof the released gaseousmatrix molecules.

Conventional MALDI plate MALDI plate for cryosections

Adapted MALDI plate holds slides forMALDI-IMS.

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Quantitative and SpatialAnalysis of Lipids Involved in

Acute Kidney Injury.

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SWATH (MS/MSALL) on 5600 Triple-TOF Mass Spectrometer

Normalizedkidney weight

Sham surgery or IR

8-10 weeksold C57Bl6/J

NephrectomyMALDI-Imaging MS

on a Bruker-TOF Mass Spectrometer

10% gelatincryosections

SWATH (Sequential Window Acquisition of all Theoretical Mass Spectra

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Pla

sma

crea

tin

ine

(mg

/dL

)

SHAM controlIR (0.5/6 hrs)

PAS

H&E 100m

Sham IR 0.5/6 hrs IR 0.5/24 hrs

IR (0.5/24 hrs)

*

*

*

0.9

1.0

1.1

1.2

1.3

Plasma creatinine and kidney histology in mice subjected to ischemia/reperfusion (IR) related

kidney injury at early and late time-points.

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PC O-38:1802.7_184.1

0.5

1.0

1.5

2.0

2.5

3.0

-15 -10 -5 0 5 10 15

-lo

g10

pLog2FC (6 hours)

PE O-40:4782.6_641.6

PE O-40:5780.6_639.6

GT2 26:0;2(LCB 18:0;2-H2O)900.8_284.3

PE O-42:3812.7_198.1

GD2 32:2;2(LCB 18:2;2-2H2O,LCB 18:1;3-3H2O)809.7_262.3

PC O-38:1802.7_184.1

2

4

6

8

10

12

-15 -10 -5 0 5 10 15

-lo

g10

p

Log2FC (24 hours)

PE O-40:5780.6_639.6

PE O-40:4782.6_641.6

PE O-42:3812.7_198.1

SWATH-MS on renal lipids following ischemia/reperfusion (IR)-related kidney injury

Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging. Rao S*, Walters KB*, Wilson L, Chen B, Bolisetty S, Graves D, Barnes S, Agarwal A, Kabarowski JH. Am J Physiol Renal Physiol, 310(10):F1136-47, 2016.

-15 -10 -5 0 5 10 15

Log2FC (6 hours)

1.5

2.0

2.5

3.0

0.5

1.0

PE O-40:4782.6_641.6

PC O-38:1802.7_184.1

PE O-42:3812.7_198.1

GD2 32:2;2(LCB 18:2;2-2H2O,LCB 18:1;3-3H2O)809.7_262.3

-lo

g10

p

-15 -10 -5 0 5 10 15

Log2FC (24 hours)

2

6

8

10

12

4

2

PC O-38:1802.7_184.1

PE O-42:3812.7_198.1

-lo

g10

p

SWATH-MS on renal lipids following ischemia/reperfusion (IR)-related kidney injury

Intensity >10

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100 180 260 340 420 500 580 660 740 820 9000

4080

120160200240280320360400440480520560600 255.2371

810.5516860.5161

861.5003

811.5511311.2301152.9984256.2399

862.4610

78.9614

18:0 acyl [M-H]-

283.2698

20:1 acyl [M-H]-

309.2493 774.5677

PC O-38:1 [M+AcO-]860.7307

Inte

nsi

ty [

a.u

.]

Mass/Charge, Da

Inte

nsi

ty [

a.u

.]

100 200 300 600 700 8000

100

200

300

400

500

600

700

800

184.0720phosphocholine

headgroup

PC O-38:1 [M+H]+

802.6843

125.0001

400 500

185.0752

Mass/Charge, Da

PC O-38:1 (O-18:0/20:1)

+ve mode

-ve mode

PC (184.0720)

20:1 (309.2493)

18:0 (283.2698)

O

OO

O P

OH

H NCH3

CH3

CH3

Plasma creatinine (IR 0.5/6h) (mg/dl)

PC

O-3

8:1

inte

ns

ity

(au

)

0

50

100

150

200

0 2 4 6 8 10

r=0.730p=0.0062

250

12

Sham (6h)IR (6h)


PE

O-4

0:4

inte

ns

ity

(au

)

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10

r=0.771p=0.0023

20

12


PE

O-4

0:5

inte

ns

ity

(au

)

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10

r=0.724p=0.0062

20

12


PE

O-4

2:3

inte

ns

ity

(au

)

0

20

40

0 2 4 6 8 10

r=0.717p=0.00872

12

60

80

100

120

140

160


GD

2 4

3:2

;2 in

ten

sit

y (a

u)

0

5

10

15

20

0 2 4 6 8 10

r=0.532p=0.0748 (ns)

25

12

Lipid changes correlate with extent of kidney injury.

PC O-18:0/20:1

CH3

O

OO

O P

OH

H N

CH3

CH3

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IR (6 h)

Sham (6 h)

802.7m/z [M+H+] 824.7m/z [M+Na+]

Inte

nsity

1mmSham (6 h)

Lotus LectinPECAM-1

IR (6 h)

Lotus LectinPECAM-1824.7m/z [M+Na+]

802.7m/z [M+H+]

Sham (6 h) 1mm

824.7m/z [M+Na+]

IR (6 h)

802.7m/z [M+H+]

IR (6 h)Sham (6 h)

824.678

0.0

0.5

1.0

1.5

2.0

2.5

100 200 300 400 500 600 700 800 900

Inte

nsi

ty [a

.u.]

(x1

04 )

Mass/Charge (m/z), Da

184.1 m/z phosphocholine

645.387

763.583

162.214

86.114

PC O-18:0/20:1 imaging in sham and IR kidneys.

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1-O-alkyl-glycerol-3-phosphate

Choline

Choline kinase

Phosphocholine

+

Fatty acyl coenzyme A

1-acyl DHAP synthase

1-acyl/alkyl DHAP reductase

Rate-limiting peroxisomal enzymes in ether lipid synthesis are most abundant in proximal tubules.

18:0 chain alcoholDihydroxyacetone phosphate (DHAP)

1-acyl-DHAP

1-O-alkyl-2-acyl-glycero-3-phosphate

1-alkyl-2-acyl-glycero-3-phosphocholine

+

…to plasmalogens via 1-alkyl-2-acyl-sn-glycerophosphocholine desaturase, methyltransferases and base-exchange enzymes.

1-alkyl-2-acyl-glycerol

1-alkyl-sn-glycerophosphate acyltransferase

CDP-choline

CTP:phosphocholinecytidylyltransferase

DHAP

acyltransferase

1-alkyl-2-acyl

glycerophosphate

phosphohydrolase

Other projects using SWATH lipidomicsand MALDI-IMS.

New therapeutic lipoprotein mimetic peptides (NIH) (Drs. Roger White and Anantharamaiah, UAB).

Eye lens lipid changes associated with aging (Dr. Steve Barnes, UAB).

Cosmic radiation effects on vascular inflammation and atherosclerosis risk (NASA).

NAD metabolites in kidney injury and inflammation (Drs. Samir Parikh and Anders Berg, Harvard).

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NAD m/z 664.5

Method development for targeted metabolite imaging projects.

1998 µL water + 2 µL TFA 4mg NAD

H2OTFA

H2OTFANAD

mix

2000 µL ACN

mixH2OTFANADACN

Final analyte solution is:50-50 Water-ACN

+ 0.1% TFA+ 1 mg/mL NAD

+ 10 mg/mL DHB

40mg DHB

mix H2OTFANADACNDHB

1998 µL water + 2 µL TFA

H2OTFA

mix

2000 µL ACN

mixH2OTFAACN

40mg DHB

H2OTFAACNDHB

Final diluent solution is:50-50 Water-ACN

+ 0.1% TFA+ 10 mg/mL DHB

mix

mix

100 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL

H2OTFANADACNDHB

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NAD m/z 664.5

Conventional MALDI-MS using a well plate with spotted standards of NAD + matrix solution

0

1000

2000

3000

Inte

ns.

[a.u

.]

0 100 200 300 400 500 600 700 800 900 m/z

Parent ion signal at 664.5 Da

NAD fragment at 542.3 Da

250125

500

62.5

1000

3.911.95

31.25

7.8115.62

0.50

0.12

1.0

0.25

524.06 Da 428.03 Da

314.54 Da (matrix) 542.37 Da (NAD fragment)505.33 Da (matrix)

MALDI-IMS using NAD standard spots and vacuum-sublimated matrix to simulate tissue

imaging method and data collection

1000ng NAD

542.37 Da

664.55 Da505.33 Da

314.54 Da

664.55 Da (protonated NAD)

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1000ng

500ng

250ng

125ng

62.5ng

31.25ng

15.6ng

7.8ng

8 intensity

3.9ng

9 intensity

1.95ng

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1.1 intensityNot visible on this y-scale

1ng

5.3 intensity

0.5ng

0.25ng

1.3 intensityNot visible

0.12ng

1.1 intensityNot visible

Kelly B. WaltersKiran B. Gupta

UAB, Dept of Microbiology

Stephen BarnesLandon Wilson

UAB, Targeted Metabolomics and Proteomics Lab

Anupam AgarwalSangeetha Rao

UAB, Dept of Medicine and UAB/UCSD O’Brien Center for Acute Kidney Injury

Research

Samir ParikhHarvard University

Rich DluhyUAB, Chair Dept of Chemistry

introduction to imaging metabolomics. · 7/20/2017 1 introduction to imaging metabolomics....

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