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Lipidomics
Lipidomics- A comprehensive analysis of lipid molecules in response to cellular
pathophysiology
Outlines
• Brief introduction to lipidomics
• Analytical methodology: MS/MS structure elucidation of phospholipids
• Phospholipid analysis by MS/MS
• MS/MS analysis of eicosanoids
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GlycerolipidsCholesteryl esters
Why measure lipids?
Lipidomics can perhaps best be defined as a comprehensive analysis of lipids on the systems-level scale together with their
interacting factors
Lipids are important- as a membrane bilayer- provides hydrophobic environment for protein function- reservoir of energy- signaling molecules
Membrane lipidsStorage lipids
PhospholipidsSphingolipidsSterols
Landmarks in the analysis of lipids
• Early 1800s Fractional distillation• 1908-1950s Liquid (adsorption) and paper chromatography• 1930-1950 Fractional crystallization• 1940-1960 Urea-fatty acid and metal-fatty acid complexes• 1952 AT James and AJP Martin invent gas-liquid
chromatography for volatile fatty acids (C1-C12)• 1958 Fatty acid methyl esters and polar liquid phases extend
analysis to very long chain fatty acids C34 andunsaturated fatty acids
• 1963 EC Horning applies capillary gas chromatography• 1980s Thermospray, electrospray and APCI ionization
interfaces link LC (HPLC) with mass spectrometry• 2000s Improvements in mass accuracy and uPLC
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Structures of major phospholipids
Cardiolipin (diphosphatidylglycerol)
How to profile phospholipidsin a complex mixture using MS/MS?
PENeutral Loss scan 141
PC & SMPrecursor ion scan 184
PSNeutral Loss scan 185
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How to profile sphingolipidsin a complex mixture using MS/MS?
m/z 264 and 282 are characteristic ions for compounds containing a sphingosine backbone
OH
H2N
OH
[M+H-H2O]+m/z 282
Tandem mass spectrometry has the ability to characterize the fatty acyl chain in -ve ion mode
Phospholipids may undergo demethylation and then the loss of the fatty acyl groups from glycerophosphocholinebackbone.
PO-
O
O
N+OO
O
R1O
R2O
PO-
O
O
NO
OO
R1O
R2O [M-15]-
CID
CH3 O
O
CH3 OCH3
O
+
O
R2O
O
R1O +
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Increasing metabolite coverage using +ve and –ve ion mode
Source: Nordstrom et al. Analytical Chemistry, 2008
Representative Q1 scans of a methanolic extract of human blood serum
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How to extract lipids?Extraction of lipids by Bligh/Dyer method
• To a homogenized sample (1 ml containing internal standards) add methanol (2.5 ml) and chloroform (1.25 ml), sonicate by 4-5 bursts; extra 1.0 ml water and 1.25 ml chloroform added and vigorously shaken.
• Centrifuge (1,000 x g) for 2 min and separate the chloroform layer (bottom layer) and repeat the process twice.
• Combine the chloroform soluble phases and evaporate to dryness and store at -20oC until analysis.
Survey scan of metabolites (+ ion mode) for a plasma sample from lean mouse [A]; ob/ob mouse [B]. Plasma
lipidomes of obese mice are higher than lean littermates (1.5e3 vs. 863)
400 475 550 625 700 775 850 m/z0
100
%
518.318
496.335
494.326
760.570758.553544.339
546.352566.322
568.337590.321
732.558602.288
782.552
806.556810.592
811.599812.608
835.594
869
400 475 550 625 700 775 850 900m/z0
100
%
518.345496.361
494.352
758.599zz
542.347544.367
546.380
566.351568.366
590.351756.584591.359 703.605
786.632
806.604810.628
811.636812.644
835.632
[A]
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MSMS fragmentation of m/z 496 obtained from plasma of obese mouse identified to be LPC 16:0
75 150 225 300 375 450 525 m/z0
100
%
184.080
104.113
86.104 478.348
HOP
HO
O
Om/z 125
125
-H2O
50 200 350 500 650
6.5e6 184.0
703.7
685.8
MS/MS of sphingomyelin standard (2S,3R,4E)-2-acylaminooctadec-4-ene-3-hydroxy-1-Phosphocholine
Even = Odd # NitrogenOdd = Even # Nitrogen
m/z 703.7 is reported as m/z 703, instead of m/z 704odd number m/z = sphingomyelin
Even number m/z = phosphatidylcholine
How to differentiate PC and SM?
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MS/MS of m/z 746.90: PE Std
141 Da
Neutral loss of 141 is a characteristic for detecting PE
Targeted lipidomics- Precursor ion spectra (PRE m/z184) from LNK, ObK and ObNK hepatocytes.
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A 2D ESI mass spectrometric finger print for TG molecules
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300 360 420 480 540 600 660 700
8.7e4
Intensity, cps
649.0
623.1
651.0
605.0
631.1621.0594.8 636.8
647.1538.8394.7407.6 620.3602.7 633.2576.7
C32
C22:1
C20C16
-H2O
Precursor ion scan m/z 264 in +ve ion mode is specific to identify ceramides in a sample
C20 m/z 594/264
C4, m/z 370/264
C6, m/z 398/264
C8, m/z 426/264
C18, m/z 566/264
C17, m/z 552/264IS
Time, min
0.5 1.5 2.5 3.5 4.50
700
Intensity, cps
0.5 1.5 2.5 3.5 4.50
269
Intensity, cps
0.5 2.0 3.50
299
Intensity, cps
0.5 2.0 3.50
1500
Intensity, cps
1.84
0.5 2.0 3.50
2336
Intensity, cps
0.5 2.0 3.50.0
5.0e4Intensity, cps
1.74
0.5 2.0 3.50
2998
Intensity, cps
2.28
C24 m/z 650/264
MRM chromatograms showing simultaneous determination of ceramides (C4-C24)
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Eicosanoids, meaning 20derived from a 20-carbon acid,
arachidonic acid
-Important lipid mediators and elicit potent effects in various biological systems mediated through specific protein receptors
15(R)-Prostaglandin F2α
Prostaglandin F2α
8-iso Prostaglandin F2α
8-iso-15(R)-Prostaglandin F2α
min
PGs and their diastereoisomer isoprostanes can be distinguished based on retention time in LC-MS
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m/z, amu40 100 160 220 280 340
3.8e6193.1
309.2164.9
171.2 291.1208.9
247.2191.1111.2
353.4263.1
255.0173.0
229.2273.2
137.1 181.2281.2
219.043.2 113.0
124.8 299.2
335.159.3 138.8
MS/MS of the [M-H]- from PGF2a m/z 353 using a quadrupole mass spectrometer
-44 Da
Fragmentation scheme of PGF2 [M-H]- m/z 353
Ions m/z 309, 291, 273 and 193 are indicative of F2-ring
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The first loss of water, m/z 189 and m/z 233 arecharacteristics of PGE2/PGD2
MS/MS fragmentation of PGE2 [M-H]- m/z 351
Conclusions• Tandem mass spectrometry analysis of phospholipids in
+ve ion mode characterizes phospholipid polar head groups, whereas –ve ion mode provide fatty acid chain structural information.
• Shotgun lipidomics can be used for rapid and reproducible global analysis of lipids in biological samples.
• Identification of metabolites (lipids or any other metabolites) at a molecular level present a great challenge due to their structural diversity (isobars and isomers) and dynamic metabolism.