Mass Spectrometry based metabolomics

Metabolomics- A realm of small molecules (<1000 Da)

Jeevan Prasain, PhD

What is metabolomics?• Identification and quantification of the complete set

of metabolites in a biological system• Quantitative global analysis of metabolites from

cells, tissues and fluids• Quantitative measurement of the dynamic metabolite

response of living systems to pathophysiological stimuli or genetic modification

Workflow for metabolome analysis

Sample collection

Treatment orDiseased group

Control group

Urine, plasma, tissueetc.

Sample preparation Internal standard spiking for quantitative analysis Extraction (liquid-liquid, ppt with or without hydrolysis)

Sample analysisLC-MS and LC-MS/MS analysis• Q1 scan using HPLC or UPLC/-TOFMS• +ve/-ve ion mode- accurate mass measurement• MS/MS experiments using a hybrid instrument Q-Trap

Data analysisMultivariat analysis e.g. PCA

Data export

Marker identification

Lipidomics can perhaps best be defined as a comprehensive analysis of lipids on the systems-level scale together with their interacting factors

Genome Transcriptome Proteome Metabolome

What might be happening in a cellSnapshot of the entirephysiology

Lipidomics

Metabolomics in the context of other omics

Lipids are important- as a membrane bilayer- provides hydrophobic environment for protein function- reservoir of energy- signaling molecules

Outlines

• Brief introduction to lipidomics• Analytical methodology: MS/MS

structure elucidation of phospholipids• Phospholipid analysis in lean and

ob/ob mice by mass spectrometry• MS/MS analysis of eicosanoids

Structures of different lipids classes

CH2OHOH

NHR

OCeramide

NH2

N(CH3)3

OHOH

NH2OH

O

HO

OH

HO

OH

OH

Phosphatidylethanolamine (PE)Y=

Phosphatidylcholine (PC)Y=

Phosphatidylglycerol (PG)Y=

Phosphatidylserine (PS)Y=

Phosphatidylinositol (PI)Y=

O

O

O

OOP

OHO

OHO

OP

OOH

OO

O

O

O

Structures of main phospholipids

Sn-1

Sn-2

Cardiolipin (diphosphatidylglycerol)

Sn-3

Choline Phosphocholine Cytidine diphosphocholine

1,2-diacylglycerol

Phosphatidylcholine

Activation of phospholipase A2

Lysophosphatidylcholine (LPC)

+ Free fatty acid

Pro-inflammatoryproperty

Component of oxidized LPL

PhosphatidylethanolamineHydrolysis by phospholipase A2

Lysophosphatidylethanolamine(LPE)

How phospholipids are synthesized?

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.

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

Which ionization mode for which phospholipids?Positive ion mode Negative ion mode

PC PELPC PAPE PILPE PISM PGPS PIPs

PC = phosphatidylcholinePA = phosphatic acidPE = phosphatidylethanolaminePS = phosphatidylserinePG = phosphatidylglycerolPI = phosphatidylinositolPIP = PI monophosphateSM = sphingomyelinLPE = lysoPE

Increasing metabolite coverage using +ve and –ve ion mode

Source: Nordstrom et al. Analytical Chemistry, 2007

No arachidonicAcid in +ve ion mode

Representative Q1 scans of a methanolic extract of human blood serum

Application of shotgun lipidomics: intra-source separation of lipids

Source: Gross and Han, methods in Enzymology, 2007

400 475 550 625 700 775 850 900m/z0

100

%

518.345496.361

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

564.474588.456 794.760816.775

Total scan of metabolites (Q1 SCAN -ve ion mode) for a plasma sample obtained from lean mouse [A]; ob/ob mouse

m/z 184

Phosphatidylethanolamine

Tandem mass spectrometry (MS/MS) of phospholipids

185 Phosphatidylserine

P

O-O

O

NH2O

H OR1

O

OR2

O 141

MS/MS in negative ion mode of phospholipids provide information about the fatty acyl chain

ESI-MS/MS analyses of various lipids

Source: Gross and Han,, 2004

Focused lipidomics

A. Flow injection (ESI-MS/MS)

-Precursor ion scanning at m/z 184-choline-containing phospholipids +ve ion mode

- Neutral scanning of 141, 185, 189, and 277 u used for PE, PS,phosphatidylglycerol (PG), and phosphatidylinositol (PI), respectively

- precursor ion scanning at m/z 153 and 241 in –ve ion mode-glycerol-containing phospholipids and inositol-containing phospholipids, respectively

400 460 520 580 640 700 760 820 880

2.7e6

Intensity, cps

759.1

783.0787.1 807.1761.1

811.1762.1496.9

788.2525.0 835.1704.1 781.1 814.1544.8 733.0 836.1568.8 795.4 805.0767.9702.0546.9494.8 690.0

+Prec (184.00): Exp 1, 2.764 to 30.604 min from Sample 11 (Lean 53 NK) of ZhangSET1.wiff (Turbo Spray), Centroided

400 460 520 580 640 700 760 820 880

3.1e6

Intensity, cps

807.1761.1783.0

759.1

787.0496.9

808.1811.1

525.0522.9 704.1520.8 789.1 835.1781.1757.0 814.2544.7 733.0 805.0568.8 791.3 837.1546.9 702.0494.8 769.3 817.2797.0747.0

+Prec (184.00): Exp 1, 2.764 to 30.603 min from Sample 16 (Ob 27 NK) of ZhangSET1.wiff (Turbo Spray), Centroided

400 460 520 580 640 700 760 820 880

3.1e6

Intensity, cps

807.1761.1783.0

759.1

787.0496.9

808.1811.1

525.0522.9 704.1520.8 789.1 835.1781.1757.0 814.2544.7 733.0 805.0568.8 791.3 837.1546.9 702.0494.8 769.3 817.2797.0747.0

+Prec (184.00): Exp 1, 2.764 to 30.603 min from Sample 16 (Ob 27 NK) of ZhangSET1.wiff (Turbo Spray), Centroided

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

526.5454.5483.5 792.8

751.8636.8 729.1 762.8703.0 738.9548.5 661.7690.8 797.0528.7430.1 476.5 606.6508.4 810.8600.5583.7452.4 619.5 732.2 892.5

+NL (141.00): Exp 2, 2.278 to 33.485 min from Sample 11 (Lean 53 NK) of ZhangSET1.wiff (Turbo Spray), Centroided

400 460 520 580 640 700 760 820 880 900

3400

Intensity, cps

769.0

764.9769.9482.5

454.5 740.9480.5 502.5 716.8478.5483.5 526.5 792.9778.9762.7

794.9738.8659.3528.6430.0 600.4 752.8524.4 687.4636.8548.3 795.9452.4 715.9 786.7 836.9426.1 898.8663.6581.7 812.9 857.9

+NL (141.00): Exp 2, 2.278 to 33.485 min from Sample 15 (Ob 26 NK) of ZhangSET1.wiff (Turbo Spray), Centroided

[M+H]+, m/z 452 = 16:1 lysoEtn[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 738 = 36a:5 GPEtn[M+H]+, m/z 740 = 36a:4 GPEtn[M+H]+, m/z 762 = 38e:0 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[M+H]+, m/z 792 = 40a:6 GPEtn[M+H]+, m/z 794 = 40a:5 GPEtn

MSMS fragmentation of m/z 496 obtained from plasma of ob/ob mousesupplemented with kudzu

TOF MSMS 494.35ES+

75 150 225 300 375 450 525 m/z0

100

%

184.080

104.113

86.104 478.348

HOP

HOO

Om/z 125

125 220 280 340 400 460 520m/z0

100

%

478.348

313.285

258.118

239.247 311.266283.283

419.277

417.264 476.331496.357

-59

-18

MS/MS of m/z 480 [M-15]- from a ob/ob no kudzu supplemented plasma sample TOF MSMS 480.40ES-

220 280 340 400 460m/z0

100

%

255.267

224.102242.116

256.275

+

m/z 480, [M-15]-

C16:0

O

O

OH

O POH

OO

N m/z 524LysoPC

O

O

O

O POH

OO

N

O

m/z 524

O

O POH

OO

N

O

O m/z 524

Platelet activating factor

LysoPC

sn-1

sn-2

Several isomeric compounds exits and unambiguous identification is a challenge

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 distinguish positional isomersof lithiated phospholipids

A 2D ESI mass spectrometric finger print for TG molecules

Source: Han and Gross, 2004

Eicosanoids, meaning 20 derived from a 20-carbon acid,

arachidonic acid

-Important lipid mediators and elicit potent effects in various biological systems mediated through specific protein receptors

MS/MS analysis of eicosanoids

Structural representation PG based on ring features

R = aliphatic chain

Intensity, cps

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

255.0173.0229.2

273.2137.1 181.2

281.2219.043.2 113.0

124.8 299.2

335.159.3 138.8

ESI-MS/MS of the [M-H]- from PGF2α

m/z 353 using a quadrupole mass spectrometer

-44 Da

O-

O

OH

HO

HO

Source: Murphy et al. Analytical Biochemistry, 2005

What information does deuterium labeling at C-2 and C-3 ofPGF2 provide us for structure elucidation of PG?

Fragmentation scheme of PGF2α

[M-H]- m/z 353

Ions m/z 309, 291, 273 and 193 are indicative of F2-ring

MS/MS fragmentation of PGE2 and PGD2 m/z 351.00

m/z, amu

20 80 140 200 260 320

1.9e6

Intensity, cps

189.0

271.2

108.9112.9106.9 135.0 162.9 217.1174.8 203.9121.0 158.967.0 191.0186.9 269.395.1 333.3206.1 315.0123.0 235.2170.9 228.8137.2 253.3119.0 184.981.1 157.958.9 176.964.7 296.7273.1

20 80 140 200 260 320

2.1e6

Intensity, cps

189.1

271.1

203.0120.959.1 157.8 186.9 242.8135.0106.9 215.1173.681.2 94.9 191.0 289.1269.3229.0146.8 297.2 315.1131.6

PGD2, Rt 12.4 min

PGE2, Rt 12.07 min

Deuterated PG standards are used for quantitativeanalysis of PGs in a extract

Source: Cao et al. Analytical Biochemistry, 2008

Library search for eicosanoid http://www.lipidmaps.org/

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 +ve ion 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.