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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.