Science - USA (2022-04-15)

decreasing CV. Sphingolipids populated the
top CV ranking positions, confirming that the
sphingolipid pathway (fig. S1F) is subjected to
high cell-to-cell variability (Fig. 2B).
To test whether the sphingolipid metabolic
pathway is coordinately modulated or if differ-

ent sphingolipid subsets are controlled in-

dependently in different cells, we created a

pairwise lipid-lipid correlation (PearsonÕsR)

matrix (Fig. 2C). Although phospholipid spe-

cies did not form biochemically meaningful

cliques, sphingolipids were clustered in groups

consisting of compounds bearing the same head

group (i.e., OH with Cers, hexose with HexCers,

trihexose with Gb3s, andN-acetyl-hexose-

trihexose with Gb4s) but with different Cer

backbones (mostly 34:1, 40:1, 42:1, and 42:2)

(fig. S1F). This suggests that specific enzymatic

Capolupoet al.,Science 376 , eabh1623 (2022) 15 April 2022 2 of 12

Fig. 1. Single-pixel MALDI-
MSI analysis on dHFs.
(A) Schematic drawing of
MALDI-MSI workflow.
Cells were fixed, matrix
was deposited, and MALDI-
MSI was performed by
rasterizing the laser across
a selected area. For each
spot, a mass spectrum was
collected and mass images
were obtained for each
ion by plottingm/zintensity
at the correspondingx
andycoordinates (left panel).
For peak identification,
total lipid extracts were
analyzed by ESI-LC/MS
(right panel). Lipids identi-
fied by ESI-LC/MS were
then compared with
the ones obtained by
MALDI-MSI. (B) Ion images
(50mm^2 /pixel; 354 ×
218 pixels) of selected lipids
recorded in positive-ion
mode. Insets show individual
cells images at higher mag-
nification. [PC(34:1)+Na]+,
phosphatidylcholine with
acyl chains consisting
of 34 carbon atoms and
one double bond complexed
with sodium; [SM(34:1)+Na]+,
SM with a backbone of
34 carbon atoms and
one double bond complexed
with sodium; [PC O-(36:4)
+H]+, phosphatidylcholine
plasmalogen with acyl
chains consisting of
36 carbon atoms and
four double bonds
complexed with hydrogen;
TIC, total ion current. Scale
bar, 500mm. (C) Images
displaying at each location
the PCA coordinate of each
pixel. PC1, PC5, and PC6
values are displayed using
a divergent color map;
positive coordinates are
shown in red and negative in
blue. Insets show individual
cell images at higher magnification. (D) Bar plots showing the contribution of the top 10 lipids with higher (red) and lower (blue) loadings for
each PC. Miniatures in the upper left corner show the entire distribution.

A B

C

min 0 Max

−0.15

0.0

0.15

−0.2

0.0

0.2

−0.2

0.0

0.2

D

10

45.

56

175

50

4.26486

1 124.54

237

10

84

.54922

SM 41:1SM 40:1SM 38:1SM

4 1:2

Cer

P 46

:6

SM

4 2:

1

SM

40:2

PC 32

:2

SM 36:

0

PA 34:

1

PC P-36

:5

PE O-40

:^6

Gb

4 40:1

Gb4 42:1Gb

4 34

:1

Gb

4 42:

2

0.2

0.2

-0.2

Loadings PC6

Loadings PC

5

Loadings PC1

0.1

-0.1

-0.2

13

44.64

233

10

35.517

86

1040

.544

19

12

69.5

9146

108

0.5

7622

Gb4 42:1Gb

4 4

2:2

Gb4

3 4:

1

Gb

4 4

0 :1

PA O - 3

7:0

PC 33

:2

Gb3 42:2Gb3

34:1

800.

55

65

2

PE O

-^4 0:

6

1 002.579

85

Gb3 40:

1

SM 36:0

1160.79

28

9

Gb3

42:

1

10

3 8.58432

1037

.5808

105

3.^5

5438

1213.58968 1 077.5

7352

69

7.526

23

117

3.5975

104

5.^5

617

5

CerP

36

:^3

CerP

46:6

PA

O

-37:0

PC 40

:6

Cer 3

4 :1

PC

32:

2

Cer 4

2 :1

PE

34

:1

PE 38:5PC

39

:^5

PE

3 8:2

PC

-O 3

4:1

PC #1 PC Coordinate

PC #5

PC #6 PC #6

Lipid Species

Min Max

Spectrum Peak Intensity

Normalized to TIC

[PC O-(36:4)+H]+

m/z 768.5917

[SM(34:1)+Na]+

m/z 725.5561

[PC(34:1)+Na]+

m/z 760.585

Matrix deposition

Laser

Intensity m/z

Pix

els

MALDI-MSI

Lipid #1 Lipid #2 Lipid #205

...

Optical Image

ESI-LC/MS

Lipid calling

Lipid #205

RT

Intensity

Lipid #2

PO

OOO

OOHON

O

PO

NHO

OOH

HO

N

O

Lipid #1

HOOOH

OHOH

ONH

HO O

TAG

Cer

GlcCer

PC

PS

PE

LBPA PGPAPI SM

PC #5

PC #1

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