BRAIN GLYCEROPHOSPHOLIPIDS AND CHOLESTEROL IMAGING BY MASS SPECTROMETRY

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774.6

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BRAIN GLYCEROPHOSPHOLIPIDS AND

CHOLESTEROL IMAGING BY MASS SPECTROMETRY

C1

A. Héron ¹ *, V. Petit ² , A. Seyer ² , F. Benabdellah ² , D. Touboul ² , A. Brunelle ² , O. Laprévote ¹

1 Laboratoire de Chimie-Toxicologie Analytique et Cellulaire, EA 4463, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4 avenue de l’Observatoire 75006 Paris, France

2 Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse 91198 Gif-sur-Yvette, France

* Auteur correspondant/présentateur du poster anne.heron@parisdescartes.fr

m/z 798.8

PC 34:1+K

I : 0-200

MALDI-TOF Positive ion mode

PHOSPHATIDYLCHOLINES

I : 0-30

m/z 885.6

PI 38:4

I : 0-600

I : 0-80

m/z 774.7

PE (O-40:6)

I : 0-100

m/z 726.6

PE (O-36:2)

I : 0-200

MALDI-TOF Negative ion mode

PHOSPHATIDYLETHANOLAMINES

m/z 798.6

PC 34:1+K

Lipids are the most abundant biomolecules found in the brain,

following water, representing up to 50% of its dry weight. These

compounds are structural components of cell membranes, and

have various functions of precursors, biomessengers, and signal

transduction. Marked alterations in their composition have been

reported to occur during neurological disorders.

Until now, many studies have been interested in brain lipid

content. However, the methods used to identify lipids always

involve extraction prior to analysis, which destroys any

information relevant to tissue localization. Lipid staining in

tissues is possible with fluorescent or classic histological dyes

but is not specific. Rare are the specific molecular probes such

as immunohistochemical tools able to detect lipids in tissue.

Therefore, brain tissue lipidomic imaging is crucial to precise

anatomic localization of lipid species in cerebral structures.

In this study, we used two powerful mass spectrometry imaging

methods, MALDI-TOF/TOF ¹ (matrix-assisted laser desorption

/ionization) and TOF-SIMS ² (time-of-flight secondary ion mass

spectrometry), in order to detect and localize lipid species in rat

brain and human temporal cortex, both at regional and cellular

levels. The first method can perform in situ structural

identifications by MS/MS, while the second is able to localize

species with a spatial resolution of less than 1 µm ³ .

We focused on cholesterol and on the most quantitatively

relevant glycerophospholipids (PC, PE, PS, PI) described in

literature, their composition greatly altering neural membrane

stability, fluidity and permeability. The results showed differential

repartition of these lipids in cell bodies or dendrites of grey

matter, and in myelinated axons.

PHOSPHATIDYLSERINES

PHOSPHATIDYLINOSITOLS

cerebellum

striatum

TOF-SIMS

Positive ion mode

CHOLESTEROL

Species Localization and abundance

Grey

Matter

(GM)

White

Matter

(WM)

Structures

Cholesterol

[M+H-H

₂

O]

⁺

m/z 369.35

[M-H]

^-

m/z 385.35

38±2% of total lipids in rat brain cortex

⁶

14-16 mg/g brain ww

¹¹



PC

PC 34 :1

16 :0/18 :1

[M+K]

⁺

m/z 798.54

20-23 mg/g brain ww

^7,8,9,10

20±1% of total lipids in brain cortex

⁶

30-40% of PC in cortex

(PlsPC 34:0 :1-2% of PC)

^5,6



PE

PE 40 :6

(18 :0/22 :6)

[M-H]

^-

m/z 790.54

15-20 mg/g brain ww

^7,8,9,10

22±2% of total lipids in brain cortex

⁶

20-35% of PE in cortex

⁶



PE O-40:6

(p18 :0/22 :6)

[M-H]

^-

m/z 774.54

PE O-36 :2

(p18 :1/18 :1)

[M-H]

^-

m/z 726.54

10-20% of PE in cortex

^4,5,6

8-15% of PE in cortex

^4,5,6



PS

PS 40:6

(18:0/22:6)

[M-H]

^-

m/z 834.53

4-8 mg/g brain ww

^7,8,9,10

35-50% of PS in cortex

^5,6



PI

PI 38:4

(18:0/20:4)

[M-H]

^-

m/z 885.55

1-2 mg/g brain ww

^7,8,910

50-70% of PI in cortex

^5,6



Cholesterol represents 40% of total lipids in brain, Glycerophospholipids : 36%, Sphingolipids : 15%, Plasmalogens : 9.5%

⁶

Total amount of PL in rat brain = 42±1mg/g brain, wet weight

⁹

PC and PE represent 80% of total glycerophospholipids

HO

Rat brain sagittal sections Human brain sections :Temporal cortex 

Maldi imaging

WM

GM

Spatial resolution : 1 µm

m/z 1 to 2000 M/M  8000

Electron flood

gun

Time of flight

Detector

Reflectron

Liquid Metal Ion

Gun Bi

₃⁺

25 keV

Spectrum

4000 5000 6000 7000

0 20 40 60 80 100

m/z

% intensité

Fixation of the

inox plaque on

support

Matrix

deposit

Image

acquisition

MALDI-TOF/TOF TOF-SIMS

SAMPLES

 Thanks to Charles Duyckaerts GIE-Neuroceb

m/z 834.6

PS 40:6

I : 0-400

I : 0-20

Corpus callosum

Cortical Layer I

Str

Cx Hi

Thal Cb

Hy

Mes

Pons Bulb

TOF-SIMS

Negative ion mode

10000 µm Image 1

Image 7

2000 µm

1

2

3

4

5

6

7

M:385.38 [Cholesterol – H]^- mc:97 tc:3.504e+8 WM GM

I : 0-20

5 mm

5000 µm

2500 µm

m/z790.6

PE 40:6

I : 0-400

5 mm

1225 µm

5 mm

Region of interest

GM WM GM WM

5 mm

1225 µm

1225 µm 1225 µm

GM GM

m/z 726.6

m/z 774.7

5 mm

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0

200

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1400

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834.7

788.7

728.7

726.7

806.7

862.8

878.8

890.8

888.8 In te n si ty

m/z

Plasmalogens

WM WM

O

O O O P O

OH

O O

N

O

O O O P O

OH

O O

H2N

O O O P O

OH

O O

H₂N

O O O P O

OH

O O

H2N

O O O P O

OH

O O

HN

HO O

O

O O O P O

OH O

O HO

HO HO OH

OH

0 mc = maximal number

of counts in a pixel

Color scale for TOF-SIMS

imaging

tc = total number of counts

for the whole image

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