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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.