Imagerie biologique par spectrométrie de masse TOF-SIMS et ...ipnImagerie biologique par spectrométrie de masse TOF-SIMS et MALDI-TOF 1er Workshop Equipex ANDROMEDE. ... Focused

12 juin 2012

Alain BRUNELLE

Centre de recherche de GifInstitut de Chimie des Substances Naturelles

Gif-sur-YvetteFrance

[email protected]

Imagerie biologique par spectrométrie de masse TOF-SIMS et MALDI-TOF

1er Workshop Equipex ANDROMEDE

Bio-Imaging and Mass Spectrometry

1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 2

In vivo :

• X ray(absorption)

• PET (Positron Emission Tomography)

• MRI (Magnetic Resonance Imaging)

Ex vivo :

• Fluorescence (absorption/emission)

• Radio-labelling (emission)

• IR (absorption)

•AFM (Atomic Force Microscopy)…

All these techniques only target a few molecules, which have been selected a priori …whereas mass spectrometry allows the simultaneous analysis of a large number of moleculeswithout any a priori and with no labelling.

Focused beams of UV photons (MALDI) and/or cluster ions (SIMS) make possible to raster asurface

1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE

Mass Spectrometry ImagingMALDI-TOF versus TOF-SIMS imaging ?

MS Images Ion density maps 1 acquisition = one image for

each peak and one spectrumper pixel…

Huge amount of data

3

Section mincecongelée

TOF MS

4000 6000 8000 10000

Spectre de masse [m/z]

Images MS

Ions ouPhotons


TOF MS

4000 6000 8000 10000


Images MS

Ions ouPhotons

Frozen section MS images

Ions orphotons

Mass spectrum (m/z)

M. Stoeckli, P. Chaurand, D.E. Hallahan, R.M. Caprioli, Nat. Med. 2001, 7, 493-496.

Mass spectrometry ? Fast Simultaneous information from

many compounds Large mass range High sensitivity

Applications: Human health Screening of modified molecular

repartitions in tissues Diagnostic of pathologies Study of drug distribution in

biological tissue duringtreatments


Mass Spectrometry ImagingMALDI-TOF versus TOF-SIMS imaging ?

MS Images Ion density maps 1 acquisition = one image for

each peak and one spectrumper pixel…

Huge amount of data

4


TOF MS

4000 6000 8000 10000


Images MS

Ions ouPhotons


TOF MS

4000 6000 8000 10000


Images MS

Ions ouPhotons

Frozen section MS images

Ions orphotons

Mass spectrum (m/z)

M. Stoeckli, P. Chaurand, D.E. Hallahan, R.M. Caprioli, Nat. Med. 2001, 7, 493-496.

MALDI-TOF-TOF TOF-SIMS

Spatialresolution 50 µm 400 nm - 2 µm

SampleDehydrated,

Homogeneousmatrix coating

DehydratedNo fixationNo matrix

Mass range m/z > 200 m/z 1500

Compounds

Proteins,Peptides,Lipids,Drugs,

Metabolites,…

LipidsGlycosphingolipids

Cyclopeptides,Drugs,

Metabolites, Elements,…

Analyzer TOF-MS/MS TOF-MS

Lipids Lipids:

Various biological functions Major structural components of cell membranes Energy storage Antioxidants Cell signaling

Composition and distribution of lipids depend on: Cellular type Status of a group of cells Physiological conditions

Chemical imaging: anatomo-pathology lipophilic dyes

Poor specificity: all the lipids, or only a lipid family Mass spectrometry coupled to gas or liquid chromatography

Excellent for quantification and/or identification Preliminary extraction loss of the spatial information on tissue


Nile RedBlack Soudan

Excitation 450-500 nmEmission >528 nm

Excitation 515-560 nmEmission >590 nm

Apolar lipids Polar lipids


TOF-SIMS imaging

TOF-SIMS mass spectrometers equippedwith liquid metal ion sources (Ga+ ions) allowing lateral resolution << 1 µm

Widely used for surface analysis in polymerindustry, metallurgy, semi-conductor

Until 2002-2004, very few applications in organic mass spectrometry because of the poor efficiency of Ga+ ion sources for intact ion emission above 100-200 and separationbetween different scientific communities

Radical change with the advent of cluster ions sources since 2002

6

D. Touboul, F. Halgand, A. Brunelle, R. Kersting,E. Tallarek, B. Hagenhoff, O. Laprévote

Anal. Chem. 2004, 76, 1550-1559D. Touboul, O. Laprévote, A. Brunelle

Curr. Opin. Chem. Biol. 2011, 15, 725-732


Increase the secondary ion emission with clusters

The impact of a polyatomic projectile is the unique way to bombard a very small area

simultaneously with several atoms

Deposition of a highenergy density veryclose to the surface

Non Linear Effects

Ga1+ Projectile

Ions or Neutrals

Ions or Neutrals

Bi3+ Projectile

M.Benguerba, A. Brunelle, S. Della-Negra, J. Depauw,H. Joret, Y. Le Beyec, M.G. Blain, E.A. Schweikert,

G. Ben Assayag, P. SudraudNucl. Instrum. Methods. Phys. Res. B 1991, 62, 8-22

A. Brunelle, S. Della-Negra, J. Depauw, D. Jacquet,Y. Le Beyec, M. Pautrat, Phys. Rev. A 2001, 63, 022902, 1-10

N. Wehbe, M. Fallavier, S. Della-Negra,J. Depauw, A. Brunelle, H.H. Andersen,

Nucl. Instrum. Methods. Phys. Res. B 2010, 268, 2596–2602

S. Bouneau, A. Brunelle, S. Della-Negra,J. Depauw, D. Jacquet, Y. Le Beyec,M. Pautrat, M. Fallavier, J.C. Poizat,H.H. Andersen,Phys. Rev. B 2002, 65, 144106 1-10


Increase the secondary ion emission with clusters

The impact of a polyatomic projectile is the unique way to bombard a very small area

simultaneously with several atoms

Deposition of a highenergy density veryclose to the surface

Non Linear Effects

Ga1+ Projectile

Ions or Neutrals

Ions or Neutrals

Bi3+ Projectile

Bi3+Bi1+ Images recorded at the edge of the corpus callosum on a rat brainsection256x256 pixels, 256x256 µm2

Pixel size 1x1 µm2

Same fluences 1012 ions.cm-2

Acquisition time : 4 and 10 minutes

D. Touboul, F. Kollmer, E. Niehuis, A. Brunelle, O. Laprévote,J. Am. Soc. Mass Spectrom. 2005, 16, 1608-1618

How to read an ion image


Intensity bar

m/z value to which the

image corresponds

mc = max counts = numberof counts in the most

intense pixel

tc = total counts = total number of counts in the

image

1

mcImages of 256x256 pixels

= 65 536 pixels

= 65 536 mass spectra!!!

9

TOF-SIMS lipid imaging


D. Touboul, F. Kollmer, E. Niehuis, A. Brunelle, O. Laprévote, J. Am. Soc. Mass Spectrom. 2005, 16, 1608-1618


Sample preparation for tissue imaging

After dissection, fast freezing at -80°C Tissue is maintained with OCT glue 12-20 µm sections cut with a cryostat (-15 -20°C) Tissue sections are deposited directly onto a stainless steel plate,

a silicon wafer, or a glass plate Dehydrated under vacuum Fixed onto the standard sample holder

Tissue

Same method as for MALDI,with no matrix coating

MALDI Imaging


+ ++ + ++

Mixture between matrix and sample molecule(5000 for 1)

Co cristallization (solid sample)

Pulsed UV Laser (106 W.cm-2)

Desorption - Ionisation of sample molecules(molecular ions [M+H]+)

Analysis by mass spectrometry

Matrix Assisted Laser Desorption Ionization

a long work in methodology to obtain homogeneous and reproducible matrix

coatings, enabling analysis of the surface with a good sensistivity

HTX ImagingTM-Sprayer

MALDI Imaging: a long way since the beginning


"Image" of the m/z 7605 protein fragment from humanbuccal mucosa cells (cheek)

1997

• MALDI-TOF mass spectrometers with 2 kHz lasers• Data acquisition and processing software• Robots for matrix deposition• 50 µm resolution

Excellent method for in situ lipid analysis

Today

Rat brain sagittal sectionTM-Sprayer (HTX Imaging)

MALDI-TOF and TOF-SIMS Lipid imaging


H I J

K L M

(+)

N

D I : 0-250

m/z 283.1

F I : 0-1959

m/z 888.7I

E I : 0-200

m/z 303.2

(-)

A I : 0-40

m/z 780.7

B I : 0-38

m/z 796.8I

(+)

C I : 0-100

m/z 806.7

G

(-)

5 mm

O

Rat brainAdjacent sagittal sectionsMALDI pixel size: 50 µmSIMS pixel size: 87 µm

TOF-

SIM

SM

ALD

I-TO

F

F. Benabdellah, A. Seyer , L. Quinton, D. Touboul , A. Brunelle, O. Laprévote, Anal. Bioanal. Chem., 2010, 396, 151-162

Identification?


7 2 0 7 6 0 8 0 0 8 4 0 8 8 0 9 2 0 9 6 0 1 0 0 00

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

Inte

nsity

m /z

7 6 6 .6

7 2 8 .6

720 730 740 750 760 770 7800

20

40

60

80

766.6

Inte

nsity

m/z

728.6

723.6

m/z 728.6

Identification?


•Only glycerophospholipids•42 combinations:

•17 GPCho•9 GPSer•16 other

•Biological relevance:•even fatty acids

only (!) 19


Ion path in reflector mode

Laser path

MALDI TOF/TOF

4800 MALDI TOF/TOF Applied Biosystems

MALDI MS/MS: in-situ identification


0 100 200 300 400 500 600 7000

4000

8000

12000

16000

464.

3 (M

-C18

:1+H

2O)

446.

3 (M

-C18

:1)

436.

3 (M

-C20

:1+H

2O)

418.

3 (M

-C20

:1)

79.0

(PO

3- )97

.0 (H

2PO4- )

140

(pho

spho

etha

nola

min

e)

309.

3 (C

20:1

)

281.

2 (C

18:1

)

Inte

nsity

m/z

255.

2 (C

16:0

)

724 726 728 730 732 7340

40

80

120

Inte

nsity

728.56

PEDatabases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup

Brain is rich in plasmalogen phospholipids (1 acyl link and 1 alkenyl link)

No neutral loss of the fatty acid linked by the alkenyl function in MS/MS analysis.

C. D. Cerruti, F. Benabdellah, O. Laprévote,D. Touboul, A. Brunelle, Anal. Chem. 2012, 84, 2164-2171



0 100 200 300 400 500 600 7000

4000

8000

12000

16000

464.

3 (M

-C18

:1+H

2O)

446.

3 (M

-C18

:1)

436.

3 (M

-C20

:1+H

2O)

418.

3 (M

-C20

:1)

79.0

(PO

3- )

97.0

(H2P

O4- )

140

(pho

spho

etha

nola

min

e)

309.

3 (C

20:1

)

281.

2 (C

18:1

)

Inte

nsity

m/z

255.

2 (C

16:0

)

724 726 728 730 732 7340

40

80

120

Inte

nsity

728.56

PE 18:0p/18:1Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup






0 100 200 300 400 500 600 7000

4000

8000

12000

16000

464.

3 (M

-C18

:1+H

2O)

446.

3 (M

-C18

:1)

436.

3 (M

-C20

:1+H

2O)

418.

3 (M

-C20

:1)

79.0

(PO

3- )

97.0

(H2P

O4- )

140

(pho

spho

etha

nola

min

e)

309.

3 (C

20:1

)

281.

2 (C

18:1

)

Inte

nsity

m/z

255.

2 (C

16:0

)

724 726 728 730 732 7340

40

80

120

Inte

nsity

728.56

PE 16:0p/20:1Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup






0 100 200 300 400 500 600 7000

4000

8000

12000

16000

464.

3 (M

-C18

:1+H

2O)

446.

3 (M

-C18

:1)

436.

3 (M

-C20

:1+H

2O)

418.

3 (M

-C20

:1)

79.0

(PO

3- )

97.0

(H2P

O4- )

140

(pho

spho

etha

nola

min

e)

309.

3 (C

20:1

)

281.

2 (C

18:1

)

Inte

nsity

m/z

255.

2 (C

16:0

)PE 20:1p/16:0

724 726 728 730 732 7340

40

80

120

Inte

nsity

728.56

Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup






0 100 200 300 400 500 600 7000

4000

8000

12000

16000

464.

3 (M

-C18

:1+H

2O)

446.

3 (M

-C18

:1)

436.

3 (M

-C20

:1+H

2O)

418.

3 (M

-C20

:1)

79.0

(PO

3- )

97.0

(H2P

O4- )

140

(pho

spho

etha

nola

min

e)

309.

3 (C

20:1

)

281.

2 (C

18:1

)

Inte

nsity

m/z

255.

2 (C

16:0

)

PE 20:1p/16:0

724 726 728 730 732 7340

40

80

120

Inte

nsity

728.56

Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup



3 different isobaric lipid species, all from the same family, and all colocalized

PE 16:0p/20:1PE 18:0p/18:1


Non-alcoholic fatty liver disease(NAFLD)

Accumulation of lipids (mainly diacyl- and triacylglycerols) in hepatocytes,

The predominant risk factor for NAFLD appears to be insulin-resistance related to the metabolicsyndrome,

On stained sections, lipid vesicles are observed in fatty liver but are not observable in healthyliver,

NAFLD represents, in the absence of alcohol abuse, a wide spectrum of conditions ranging fromsimple steatosis (NAFL) to non-alcoholic steatohepatitis (NASH), that may progress to cirrhosisand end-stage liver disease.


1000 µm 1000 µm

Control liver Fatty liver

H&E staining (adjacent sections)

300 400 500 600 700 800 9000

1

2

3

4

5

6

Inte

nsity

per

uni

t are

a (c

ount

s x1

03 )

m/z

PC

DAG

Vit.E

Chol.

385.

33

369.34

339.

2931

3.28

523.47603.44

577.43551.43

430.36

758.55760.56

MAG

300 400 500 600 700 800 9000

5

10

15

20

Inte

nsity

per

uni

t are

a (c

ount

s x1

03 )

m/z

603.51

577.53

551.47

523.44

369.33

339.

28313.

27

Chol.

DAG

700 750 800 850 900 9500.0

0.1

0.2

0.3

0.4

0.5

853.69855.73857.74

758.45760.49

PC TAGMAG

Fatty liver - steatotic area

Control liver

Lipid profile of a fatty liver :Steatotic area, positive ion mode

Increase of the diacylglycerol signal in the steatotic area

Strong decrease of vitamin E signal Detection of triacylglycerols


TAG

DAG

MAG

Vit.E

D. Debois, M.P. Bralet, F. Le Naour, A. Brunelle, O. Laprévote, Anal. Chem. 2009, 81, 2823-2831

O

O

O

OP

O

OO

O

HOHO

HOOH

OH

X

X = H, Na, K, Ca...

O

HO


Fatty acids: O

OH

Sterols: Phospholipids:

α-TocopherolPhosphatidylcholine

Phosphatidylinositol

O

O

O

OP

O

O

O

ON

O

HN

OH

OP

O

OO

N

Sphingomyelin

HO Cholesterol

O

O

O

O

O O

Diglycerides: O

O

O

O

OH

Triglycerides:

Only mass spectrometry imaging makes possible to distinguish between the differentlocalizations among the same lipid species

Lipid profile of a fatty liver :Steatotic area, positive ion mode


100 µm

Fragments of TAGsFragments of TAGs and of other lipid species

In agreement with the diseasedescription

Cholesterol

16

0

179

0

7

0Sum of TAGs Sum of DAGs

19

0

50

0

79

0

64

0C30 DAGs C32 DAGs C34 DAGs C36 DAGs


Lipid profile of a fatty liver :Steatotic area, correlation between DAGs and FAs


19

0

50

0

79

0

64

0C30 DAGs C32 DAGs C34 DAGs C36 DAGs

17

0

67

0

39

0

41

0

36

0Myristic acid C14:0 Sum of C16 FAs Stearic acid C18:0 Oleic acid C18:1 Linoleic acid C18:2


Lipid profile of a fatty liver :DAGs C36


64

0C36 DAGs9

0

20

0

38

0

33

0

17

0C36:0 DAGsC36:1DAGsC36:2 DAGsC36:3 DAGsC36:4 DAGs

Composition of steatotic vesicles: Enrichment in cholesterol,

Lipids with longer acyl chains,

Enrichment in unsaturated lipids


Chronic glaucoma

Second cause of blindness in the world

Due to a trabecular failure

Induces an increase of the intra-ocular pressure (IOP) and a progressive optic neuropathy

Symptoms are insidious, only a progressive visual loss

Early diagnostic can be made only by systematic IOP measurements during ophthalmologic examination

Glaucoma treatments Eye drops like prostaglandins derivatives or beta

blockers predominantly used as first line therapy to reduce the IOP (800 000 people in France in 2004) Addition of a preservative is needed, usually benzalkonium chloride (BAK)

Laser Surgical resection of the trabecular

Many studies have suspected BAK to induce a long-term toxicity in eye


Reduced visual field of view

Benzalkonium chloride

Quaternary ammonium salt with variable length alkyl chains, two predominant forms C12 and C14

Surfactant, antiviral and antifungal depending on the alkyl chain length

Toxic preservative: Damage of tissues due to oxidative stress Apoptosis and necrosis depending on the

concentration used and the time of application

Dry eye, burning and/or tingling sensation


R=C8 to C18 but mainly C12 and C14

C12

C14

Very easy to analyze by MALDI-MS or SIMS

MALDI-ToF/ToF imaging of whole rabbit eye sections

BAK detected in: cornea, conjunctiva, iridocorneal angle AND in and near the optic nerve

Confirmation of results with a round-robin experiment in 2 laboratories:(second data not shown)

Matrix deposition: TM-Sprayer (HTX Imaging) SunCollect (SunChrome)

MALDI spectrometers: 4800 MALDI TOF/TOF (AB-Sciex) Autoflex Speed (Bruker Daltonics)

Processing software: TissueView (AB Sciex) FlexImaging (Bruker Daltonics)


2 near to the optic nerve

3 optic nerve

1 cornea

Optical

1

2

3

1

2

3

TOF-SIMS imaging: small areas of rabbit eye sections


Each image:m/z 332.36 (BAK C14)

mc: 28500 µm x 500 µm256 x 256 pixelsPixel size 2 µm

3.1011 ions.cm-2 (Bi3+)

Limbus

Fornix

Conjunctiva

Limbus

Fornix

Conjunctiva

TrabeculumTrabeculum

Acquisition of 5 x 4 adjacent images in the iridocorneal angle(total area 2.5 mm x 2.0 mm)

Optical view (x 1.25)

Iridocorneal angle

Anatomy of a human eye




Each image:m/z 332.36 (BAK C14)

mc: 28500 µm x 500 µm256 x 256 pixelsPixel size 2 µm

3.1011 ions.cm-2 (Bi3+)

Limbus

Fornix

Conjunctiva

Limbus

Fornix

Conjunctiva

TrabeculumTrabeculum

Acquisition of 5 x 4 adjacent images in the iridocorneal angle(total area 2.5 mm x 2.0 mm)

Optical view (x 1.25)



1. F

orni

x

TUNEL:Fluorescence methodshowing apoptotic cells

2. T

rabe

culu

m

Optical

3. C

orne

a

m/z 332.36 (BAK C14) mc : 57

m/z 304.32 (BAK C12) mc : 60

m/z 304.32 (BAK C12) Redm/z 332.36 (BAK C14) Green

out

in


Conclusion

TOF-SIMS and MALDI-TOF lipid imaging:

TOF-SIMS: micrometer resolution, no tissue preparation needed.

MALDI-TOF: in situ identifications of lipids.

For TOF-SIMS, many ions can also be evidenced by their co-localization with specific

fragments.

For both methods, the targeted compounds must be highly concentrated in well-defined

areas to be imaged.

Perspectives: data analysis, MS/MS, increase of TOF-SIMS sensitivity, neurodegenerative

diseases, liver diseases

36


Agence Nationale de la Recherche Scientifique (ANR)

European Union(FP6 STREP Program and COST)

Institut de Chimiedes Substances Naturelles

Centre National de la Recherche Scientifique

Documents

Imagerie biologique par spectrométrie de masse TOF-SIMS et ...ipnImagerie biologique par spectrométrie de masse TOF-SIMS et MALDI-TOF 1er Workshop Equipex ANDROMEDE. ... Focused