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3/10/2017
1
The use of mass spectrometry in lipidomics
Jeevan Prasainjprasain@uab.edu6-2612
Outlines
• Brief introduction to lipidomics
• Analytical methodology: MS/MS structure elucidation of phospholipids
• Phospholipid analysis in lean and ob/ob mice by mass spectrometry
• LC-MS/MS quantification of ceramides
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Lipidomics- A comprehensive analysis of lipid molecules in response to cellular
stress and challenges
Structures of different lipids classes
CH2OH
OH
NHR
OCeramide
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NH2
N(CH3)3
OHOH
NH2
OH
O
HO
OH
HO
OH
OH
Phosphatidylethanolamine (PE)Y=
Phosphatidylcholine (PC)Y=
Phosphatidylglycerol (PG)Y=
Phosphatidylserine (PS)
Y=
Phosphatidylinositol (PI)Y=
Structures of main phospholipids
Sn-1
Sn-2
Cardiolipin (diphosphatidylglycerol)
Sn-3
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 and added 1.0 ml water and 1.25 ml chloroform additionally 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 phase and evaporate to dryness and stored at -20 oC untill analysis.
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Shotgun lipidomics: intrasource separation of lipids for quantitative lipidomics
The ionization efficiency of an analyte greatly depends on the electrical propensity of an individual analyte in its own microenvironment to lose or gain a charge
Source: Gross and Han,, 2004
Profiling phospholipids and sphingosinesin a complex mixture using MS/MS
PENeutral Loss scan 141 PC & SM
Precursor ion scan 184
PSNeutral Loss scan 185
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How to profile sphingolipidsin a complex mixture using MS/MS?
m/z 264 is a characteristic ion for all compounds containing a sphingosine backbone
Phosphatidylcholine loses a methyl group to form a negatively charged, pseudomolecular ion
Phospholipids may undergo demethylation and then theloss 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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400 475 550 625 700 775 850 900m/z0
100
%
518.345
496.361
494.352
758.599
542.347544.367
546.380
566.351568.366
590.351756.584591.359 703.605
786.632
806.604810.628
811.636812.644
835.632
869
Total scan of metabolites (Q1 SCAN + ion mode) for a plasma sample obtained from lean mouse [A]; ob/ob mouse
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
[A]
[B] 1.52e3
Total metabolomics
20.3
400 475 550 625 700 775 850 900m/z0
100
%
480.425
478.419
504.432
508.464
558.466885.817820.780794.751588.418 646.397 732.383674.430 830.818 886.779
400 475 550 625 700 775 850 900m/z0
100
%
52.5480.415
504.431506.445540.459508.463
564.474588.456794.760816.775
Total scan of metabolites (Q1 SCAN -ve ion mode) for a plasma sample obtained from lean mouse [A]; ob/ob mouse
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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
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 m/z
703.7
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ESI-MS/MS analyses of various lipids
Source: Gross and Han,, 2004
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
C22:0Calc. 622.6133
C24:1Calc. 648.6289
C20C16
-H2O
Precursor ion scan m/z 264 in +ve ion mode is specific to identify ceramides in a sample
m/z 264
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Intensity, cps
400 460 520 580 640 700 760 820 880
6239769.0
744.9
778.8764.9
745.8478.3482.5 750.8 779.8740.9716.9502.6
526.5454.5483.5
792.8751.8636.8 729.1 762.8703.0 738.9548.5 661.7 690.8 797.0528.7430.1 476.5 606.6508.4 810.8600.5583.7452.4 619.5 732.2 892.5
[M+H]+, m/z 454 = 16:0 lysoEtn[M+H]+, m/z 478 = 18:2a lysoEtn[M+H]+, m/z 482 = 18:0 lysoEtn[M+H]+, m/z 502 = 20:4a lysoEtn[M+H]+, m/z 526 = ?
[M+H]+, m/z 716 = 34a:2 GPEtn[M+H]+, m/z 744 = 36a:2 GPEtn[M+H]+, m/z 764 = 38a:6 GPEtn[M+H]+, m/z 768 = 38a:4 GPEtn[M+H]+, m/z 778 = 40p:5/40e:6 GPEtn
400 460 520 580 640 700 760 820 880
2.7e6759.1
783.0
787.1 807.1761.1
811.1762.1
496.9
788.2525.0835.1704.1 781.1 814.1544.8 733.0 836.1
568.8795.4 805.0767.9702.0
546.9494.8 690.0
+Prec (184.00)
[M+H]+, m/z 496 = 16:0 lysoPC[M+H]+, m/z 546 = 20:3 lysoPC
[M+H]+, m/z 758 = 34:2 PC[M+H]+, m/z 782 = 36:4 PC[M+H]+, m/z 806 = 38:6
Profiling of phospholipids using precursor ion m/z 184 and neutral loss scan 141 for PC, SM and PE
MSMS fragmentation of m/z 496 obtained from a plasma sample in positive ion mode
75 150 225 300 375 450 525m/z0
100
%
184.080
104.113
86.104478.348
HOP
HO
O
Om/z 125
125
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MS/MS of m/z 480 [M-15]- from a plasma sample
220 280 340 400 460m/z0
100
%
255.267
224.102
242.116
256.275
+
m/z 480, [M-15]-
C16:0
Several isomeric compounds-Identification by high resolution mass spectrometry
O
O
OH
O P
OH
OO
Nm/z 524calc. m/z 524.3711
O
O
O
O P
OH
OO
N
O
O
O P
OH
OO
N
O
O
m/z 524calc. m/z 524.3347
m/z 524calc. m/z 524.3711
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Source: Hsu et al. J. Am Soc. Mass Spectrom, 1998
Lithiated adducts of phosphocholine provide more
structural information in their MS/MS spectra
Source: Hsu et al. J. Am Soc. Mass Spectrom, 1998
Relative abundances of product ion can be used to distinguishpositional isomers of lithiated phospholipids
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Library search for eicosanoid http://www.lipidmaps.org/
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Product ion spectra of deprotonated arachidonic acid [AA] and its oxidation product 5-hydroxy-eicosatetraenoic
acids [5-HETE]
30 60 90 120 150 180 210 240 270 300
8.0e4 59.04
303.28
83.08177.17 231.24 259.22163.1470.96 205.23135.06 285.28
44 Da
18 Da
20 60 100 140 180 220 260 300 340
9.0e4
Inten
sity, cps
115.059.0
203.2
167.0 257.2 261.0177.3 319.2
m/z 115.0401
AA
COO-
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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1.0e2
(C24 650.5 / 264.0): "Quadratic" Regression ("1 / x" weighting): y = -7.9e-006 x^2 + 0.0349 x + 0.035 (r = 0.9995)
1.0e-1 1.0e0 1.0e1Analyte Conc. ng/ml/ IS Conc.
1.0e-1
1.0e0
An
alyteA
rea / IS A
rea
A linear response for Cer C24:0 was observed over a range of 0.1-100 ng/ml with correlation coefficient greater than 0.99
Sample Name Analyte Peak Name Calculated Concentration (ng/mL) Accuracy (%)Ceramide Standard 100 ng/ml C24 650.5 / 264.0 100 100Ceramide Standard 50 ng/ml C24 650.5 / 264.0 49.8 99.6Ceramide Standard 10 ng/ml C24 650.5 / 264.0 10.5 105Ceramide Standard 1 ng/ml C24 650.5 / 264.0 0.634 63.4
Ceramide Standard 0.1 ng/ml C24 650.5 / 264.0 0.132 132
[A]-[C] represent base line plasma ceramide C20 (594/264) from three animals[D] after 4 months treatment with cranberry (1 g/kg b. w. and high fat diet[E] & [F] after 4 months treatment with high fat diet only
Time, min
0.5 1.5 2.5 3.5 4.50100
2586
Intensity, cps
1.97
0.18 2.81
[B]
0.5 1.5 2.5 3.5 4.50
3400
Intensity, cps
1.96
0.17
[D]
0.5 1.5 2.5 3.5 4.50
5407
Intensity, cps
1.97
2.11
[E]
0.5 1.5 2.5 3.5 4.50
4986
Intensity, cps
1.97
1.81
[F]
0.5 1.5 2.5 3.5 4.50200
3129
Intensity, cps
1.98
[C]
0.5 1.5 2.5 3.5 4.50
3800
Intensity, cps
1.97
0.26 1.74
[A]
Time, min Time, min
Cranberry + HF4 months
HF only4 months
HF only4 months
Cranberry fruit powder treatment reduced the HF induced increased levels of Ceramide C20 in rats
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Conclusions
• Shotgun lipidomics approaches are high throughput and applicable to perform profiling as well as quantitative analysis of various lipids in biological samples.
• Tandem mass spectrometry analysis of phospholipids in +veion mode characterizes phospholipid polar head groups, whereas –ve ion mode provide fatty acid chain structural information
• Identification of phospholipids at a molecular level present a great challenge due to their structural diversity and dynamic metabolism.
• A rapid five minute liquid chromatography tandem mass spectrometry (LC-MS/MS) method operating in multiple reaction ion monitoring mode (MRM) was developed for identification and simultaneous quantification of six ceramides.
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