Mass-Spectrometric Analysis of
Lipids (Lipidomics)
1. Identification
2. Quantification
3. Metabolism
Why to do lipidomics?
Biology: Functions of different lipids?
Medicine: Diagnostics and Therapy
Industry: Healthier food, Quality control
GlyceroPhospholipids
H
H
H
H
O
P
O
O
H2C
H
C
O
O
CH2
H
H
O
N
H
O
H
H
H
H
H
H
>10 classes (PC, PE, PS, PI, PA etc)
O
- Each class consists of numerous species
with different fatty acid combinations
(>20 different fatty acids)
=> Thousands of different molecular
species possible
Phosphatidylcholine (PC)
Neutral Glycerolipids
- Triacylglycerols (TG)
- Diacylglycerols (DG)
- Monoacylglycerols (MG)
-Each class consists of numerous
species due to different fatty acid
combinations
=> Hundreads of different
molecular species
TG
DG
Sphingolipids
- Ceramides
- Neutral Glycosphingolipids
- Acidic Glycosphingolipids
-Each class consists of numerous
species due to different fatty acid
=> Hundreads of different
molecular species
Lactosylceramide Ganglioside Sulfatide
The complete lipidome of no cell or
tissue has ever been determined
...because of technical limitations
Advantages of MS analysis
Sensitivity
>1000-fold higher than with conventional methods
Resolution
- Allows quantification of hundreds of lipid species
Speed
-100 times faster
Can be automated
- High troughput possible
Ionization methods used in lipid MS
Electrospray (ESI)
– Does not cause fragmentation
– Can be easily automated
– Compatible with on-line LC
Matrix-assisted laser desorption (MALDI)
– Less used thus far
– Suppression by PC/SM > All lipids not detected
– On-line LC separation not feasible
Electrospray ionization
Competition for charge => Suppression effects!
MS spectrum of cellular lipid extract = a Mess!
Intens.
PE 18:0/18:2
PS 18:0/18:1
PE 18:0/20:4
6000
PE 18:0/22:5
4000
PE 18:0/20:4
PE 16:0/18:1
LBPA 18:1/18:1
2000
0
PI 18:0/20:3
PI !8:0/18:2
PA 18:0/18:2
700
725
750
775
800
825
850
875
m/z
Scanning
MS1
Collision cell
MS2
Detector
How to improve selectivity?
A. Lipid class -specific scanning (MS/MS)
B. On-line chromatographic separation (LC-MS)
Lipid class -specific scanning
Phospholipid class consist of species with the same
polar head-group but different fatty acids
O
CH2
O
O
CH
O
H2C
O
O
P
O
X
O
Phospholipid class
Phosphatidylcholines
Phosphatidylinositols
Phosphatidylethanolamines
Phosphatidylserines
Specific scan
Precursors of +184
Precursors of -241
Neutral-loss of 141
Neutral-loss of
87
Precursor ion scanning
 Requires a characteristic, charged product ion
PC => Diglyceride + phosphocholine (+184)
Scanning
Fragmentation
Static (+184)
MS1
Collision cell
MS2
(Helium or Argon)
Precursors of +184 => PC + SM
SM-16:0
-Alkaline hydrolysis can be used to remove PCs
Neutral-loss scanning
..when the characteristic fragment is uncharged
PE => Diglyceride (+) + phosphoethanolamine (141)
Mass interval = 141
Scanning
MS1
Fragmentation
Collision cell
(Helium or Argon)
Scanning
MS2
Neutral-loss of 141 (= PE)
700
750
800
MS-scan
Intens.
PE 18:0/18:2
PS 18:0/18:1
PE 18:0/20:4
6000
PE 18:0/22:5
4000
PE 18:0/20:4
PE 16:0/18:1
LBPA 18:1/18:1
2000
0
PI 18:0/20:3
PI
PA 18:0/18:2
700
725
750
775
800
825
850
!8:0/18:2
875
m/z
MS analysis of Sphingolipids
OH
OH
HO
COOH
O
O
AcHN
O
HO
OH
OH
OH
HO
COOH
AcHN
OHOH
HO
O H
H
CH 2
H
HC N
O
HO
O H
OH
H
OH
H
HO
H
HO
CH
H
H
OH
H
O
H
H
O
CH
H
C
H
N
O
O
H
HC
H
N
O
O
C
OH
H
CH2
CH2
CH2
HC
O
O
O
O
O
O
O
H
H
H
O
H
OH
H
H
OH
C OH
H
OH
H
CH 2
H
HC N O
H3SO3-
H
OH
C OH
H
OH
H
O
O
OH
H
H3SO3-
O
O
HC
O
O
CH
C
H
N
OH
O
O
CH
CH
Sphingosine
Ceramide
Lactosylceramide Ganglioside
Sulfatide
Ceramide and Neutral Glycosphingolipids
- Precursors of sphingosine (m/z +264)
Glucosylceramides
Ceramides
24:1
Sulfatides
- Precursors of Sulfate (m/z -97)
Liquid chromatography-MS (LC-MS)
Advantages
- Increased sensitivity due to diminished
suppression of minor species by
- Major species
- Impurities
Disadvantages
– Takes more time (not UPLC)
– Data analysis more complex (?)
LC-MS analysis of mouse brain lipids
Time
Hermansson et al. (2005) Anal Chem.77:2166-75
Data analysis => software
A. Processing of the data
=> Identification
=> Concentrations
B. Bioinformatics
=> Biomarkers?
=>Biological significance?
Quantification not simple
Signal intensity depends on:
•
•
•
•
•
•
Lipid head-group
Acyl chain length
Acyl chain unsaturation
Ions present (adduct formation)
Detergent and other impurities (suppression)
Solvent composition and instrument settings
=> Internal standards necessary!
LIMSA
Excel add-on for Quantitative Analysis of MS data
(Haimi et al. .2006. Anal Chem. 78:8324-31)
 LIMSA does:
 Peak picking and fitting
 Peak overlap correction
 Peak assignment
(database of >3000 lipids)
 Quantification with
internal standards
 Batch analysis
MS-imaging of Lipids by MALDI
PI 38:4
Sulfatide 24:1
Hydroxy-Sulfatide 24:1
Analysis of Lipid Metabolism by MS
 Adds another, dynamic dimension to lipidomics
 Labeled lipids can be selectively detected!
D9-PC
D4-PE
D4-PS
D6-PI
=> +193 (Unlabeled PC => +184)
=> 145 (Unlabeled PE => 141)
=> 90 (Unlabeled PS => 87)
=> -247 (Unlabeled PI => -241)
Precursors
 Water soluble precursors (D9-choline etc)
 Exogenous lipids
Phospholipid Remodeling: Exchange of acyl
PO4
Glycerol
Fatty acid
OH
Alcohol
Alcohol
PO4
Glycerol
Acyl
transferase
Fatty acid
Fatty acid
Fatty acid
Glycerol
PLA2
Fatty acid
PO4
Alcohol
chains
Analysis of phospholipid remodeling using
soluble precursor is problematic
D4-ethanolamine => cells => D4-PE species
18:1/18:1
16:0/18:1
100
Kinetics
18:0/18:1
16:0/17:1
0h
16:0/16:1
50
1h
100
5h
100
24h
Relative abundance (%)
Relative intensity (%)
100
16:0/16:1
16:0/17:1
16:0/18:1
16:1/18:1
18:0/18:1
18:1/18:1
18:1/18:2
18:0/20:4
18:1/20:4
18:0/22:6
25
0
100
0
unlabeled
680
700
720
740
m/z
760
780
800
5
10
15
Chase time (h)
20
25
Our approach: Use intact exogenous phospholipids
with a deuterium-labeled head-group
PROTOCOL
• Synthesize a phospholipid with a deuterium-labeled
head group
• Make vesicles containing the labeled phospholipid
• Incubate cells with these vesicles and β-cyclodextrin
(carrier)
• Extract and analyze lipids using MS/MS scans showing
the labeled (or unlabeled) lipid only
• Determine the pathways and kinetics of remodeling
Unnatural 14:0/14:0-PE is remodeled very rapidly
14:0/14:0-D4-PE
KINETICS
100%
14:0/14:0
0h
100%
14:0/18:1
14:0/20:4
50
3h
18:0/18:1
Relative Intensity
16:0/14:0
14:1/14:1
16:0/14:1
18:0/14:1
18:1/14:1
16:0/18:1
14:1/20:4
18:0/18:1
18:1/18:1
18:0/20:4
18:1/20:4
100%
18:1/18:1
18:0/20:4
16:0/18:1
18:1/20:4
7h
25
100
24h
0
100%
0
Endogenous
700
750
m/z
800
850
5
10
15
20
25
”Natural” 18:1/18:1-PE is hardly remodeled
18:1/18:1
100%
0h
100%
Relative abundance
1h
100%
3h
100%
5h
100%
24 h
100%
endogenous
700
750
800
Positional isomers are remodeled with
very different kinetics
14:0/18:1
100%
18:1/14:0
14:0/18:1
100%
18:1/14:0
18:1/18:1
18:1/20:4
18:1/18:1
18:1/18:2
16:1/16:1
0h
0h
18:1/22:6
18:1/16:1
Relative abundance
100%
700
750
800
18:1/18:1
100%
700
750
800
3h
3h
18:1/18:2
100%
700
750
800
700
700
750
750
800
7h
100%
700
750
endogenous
700
800
7h
16:1/18:1 16:0/18:1
100%
100%
750
m/z
800
800
endogenous
700
750
m/z
800
Pathways of 14:0/14:0-PE remodeling
14:0/14:0
14:0/ 18:2
14:0/ 16:1
18:1 /18:2
18:1 /16:1
14:0/ 18:1
14:0/ 20:4
14:0/ 22:6
18:1 /20:4
18:1 /22:6
16:1 /18:2
16:0 /18:2
16:0 /18:1
16:1 /18:1
18:1 /18:1
18:0 /20:4
Kainu et al. (2008) J Biol Chem. 283:3676-87
Studies with >50 phospholipid species
(and PLA inhibitors) indicate that
=> Multiple acyl chain specific PLAs are involved
in remodeling of phospholipids in mammalian cells
BUT which PLAs? ..and what determines their
specificity?
..and which acyltransferases are involved?
Conclusions
 MS-based lipidomics is highly usefull in
– Biology
– Medicine
– Food industry
....but needs to be integrated with other “omics”
and functional assays
 Heavy isotope –labeling adds an important extra
dimension to lipidomics
Contributors
• Martin Hermansson
• Ville Kainu
• Perttu Haimi