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MassSpectrometryBasedLipidomics

ElisabeteMaciel,ElianaAlves,

PedroDomingues,RosárioDomingues

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Genomics Proteomics Lipidomics

Lipidomics

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Lipidomics

-  Thefullcharacterizationoflipidmolecularspeciesandoftheirbiologicalroleswithrespecttoexpressionofproteinsinvolvedinlipidmetabolismandfunction,includinggeneregulation

(AOCSLipidsLibrary)

-Analysisoflipidprofileanditsrelationtocellphysiologyandpathophysiology

Lipidomics

Lipidisolation Lipidanalysis

Studyofmetabolicpathawys

interactionsLipid-proteins

Ø Profilingcellularlipidome

Ø Membranelipiddomains&dynamics

Ø Regulatory(e.g.signaling)functionsoflipids

Ø  Integrationofomics&interactionofcellularcomplement&machinerytoformcells/organism

Lipidomics

3

Whylipidsaresoimportant?

EnergyMetabolism/Reserves

Membranes

CellularRegulationsignalingmessengers,hormones,…

Cell membranes

Dysfunctionoflipidsignaling&metabolismplaysacentralroleinhealth&diseases

Lipids-CellMembrane

MembraneAssymetry

MembranedomainsLipidrafts

4

Lipidprofilingincell,tissuesandbiofluids

% of total acyl lipid content

GlycerolipidsChloroplast

thylakoid

innermitochondrial

membraneplasmamembrane

MGDG 51% 0 0DGDG 26 0 0SQDG 7 0 0PC 3 27 32PS 0 25 0PG 9 0 0PE 0 29 46PI 1 0 19CL 0 20 0

Eachtypeofcell,tissueandbodyfluidhaveacharacteristiclipidprofilewithadefinedlipidcompositions.

Identificationofallcellularlipids-

ProfileofPhospholipidclasses

Cardiomyocytescellline

BreastcancercellLines

Phospholipid classes quantification

Main classes of phospholipids

Rel

ativ

e ab

unda

nce

(%)

SM PC PIPS PE PA CL

0

10

20

30

40

50CTLStress**

** *

Moussebrain

LPC SM PC PI

PG PE CL-10

0

10

20

30

40

50 CONT

T1DM*

*

PL Classes

Rela

tive

Perc

enta

ge (%

)

MitochondriafromheartMitochondriafrommuscle

5

ProfileofFattyAcids

EffectsofPLfattyacidcompositioninmembraneproperties

6

Whatarethebigchallengesinlipidomics?

Structuralcomplexityoflipids

7

Phospholipids

Alsocalledglycerophospholipids

Phospholipids/GlycerolipidsMolecularSpecies

Phospholipids/GlycerolipidsMolecularSpecies

16:0

18:1

P choline

Glyc

ero

l

18:2

18:3

P ethanol- amine

Glyc

ero

l

PC,PE,PS,NPE,PG,CL,PI,PIP,PIP2,PIP3,LysoPLs……

8

PhospholipidshapeandmembranesPL&fattyacidcomposition&membraneproperties

PhospholipidBiosynthesis

DefineandregulateSpecificLipidomesignatures

9

DeviationsintheLipidome

Disease– Alterationinmetabolicpathways– OxidativemodificationofsomelipidsOthers:– Diet–sourceofdifferentlipids

Importance:NewbiomarkersNewtherapeuticstrategiesNewbiotechnologicalapplications

Lipidomicanalyticalstrategiestoovercomethecomplexityofthelipidome

10

Lipidomicapproach

Lipossomes TissuesCells

LipidExtraction

Fractionation/SeparationbyChromatography:TLC,HPLCor

SPE

MassSpectromatry(MS)AnalysisUntargetanalysisTargeretanalysis

Lipidomicsworkflow

Tissue and cells

Bligh and Dyer Folch MTBE

LC- MS

LipidomicsbasedonMassSpectrometry(MS)

analysis

11

LipidExtractionChemical extraction using organic solvents:

q Folch method (CHCl3:CH3OH 2:1)

q Bligh and Dyer (CHCl3:CH3OH 1:2)

q others

12

LipidExtraction

AnalyzedMatrices Extrationprotocols

CajkaandFiehn,TrensinAnalyticalchemistry,2014

LipidExtraction

Selective extraction of phospholipids from plasma using Hybrid SPE

13

Fractionationoflipidextracts

SolidphaseextractionToseparateneutralfrompolarlipidsNeutrallipids(TG)fromPL

Chromatographicmethods

TLC(Thinlayerchromatography)HPLC(Highperformanceliquidchromatography)

Toseparatelipidclasses/molecularspecies

A

Separationofphospholipidclasses

TLC HPLCl

Phospholipid classes can be separated based on their polarity by:

14

TLC–ThinLayerChromatography

CL-Cardiolipin

PA-Phosphatidic Acid

PE-Phosphatidylethanolamine

PS-Phosphatidylserine

PI-Phosphatidylinositol

PC-Phosphatidylcholine

SM-Spingomyelin

LPC-Lyso Phosphatidylcholine

Different elution systems- different TLC profiles

2D-TLC

Two Diferent Solvent Systems

Prof Valerian Kagan lab

15

HPLC-MS

CajkaandFiehn,TrensinAnalyticalchemistry,2014

on-lineHPLC-MS

MSAnalysis

off-lineTLC-MS

LipidExtraction

MSAnalysis

m/z700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850

%

0

100 744.5

742.5

716.5700.5

701.5

714.5

703.5

728.5717.5

718.5729.5

740.5

772.5745.5770.5

746.5758.5

766.5

773.5844.4

828.4774.5788.5

802.5 814.4 818.4842.4 845.4

849.8

A

HPO-

O

O

NH3+OR1 O

O

OR2

O

PE

B PI

CPS

DPG

A PE

B PI

m/z760 770 780 790 800 810 820 830 840 850

%

0

100 788.6

760.6761.6 786.6

774.6762.6 776.6

789.6

802.6790.6

791.6 819.6804.6 810.6 834.6826.6 836.6 842.6 848.6

C

H NH3+

OO-H

PO-

O

O

O

O

OR1OR2

O

PS

DPG

A PE

m/z820 825 830 835 840 845 850 855 860 865 870 875 880 885 890 895 900

%

0

100 861.6

835.6833.6

821.6 836.6 859.6849.6847.6

863.7

885.6864.7

883.6865.7

875.7 877.6

886.6

890.7 899.6

BHO

OH

OHOH

OH

PO

O-O

R2

O

R1

O

O HO O

PI

CPS

DPG

A PE

B PI

m/z760 770 780 790 800 810 820 830 840 850

%

0

100 788.6

760.6761.6 786.6

774.6762.6 776.6

789.6

802.6790.6

791.6 819.6804.6 810.6 834.6826.6 836.6 842.6 848.6

CPS

m/z690 700 710 720 730 740 750 760 770 780 790 800 810

%

0

100 773.6

713.6695.5691.5699.6

711.5745.6727.6723.5

737.6747.6 771.6

759.6

774.6

775.6

776.6802.7800.6787.6 804.7

D

HOH

OHPO-

O

O

O

O

OR1OR2

O

PG

16

MSDATAANALYSIS:

Ions formed during ionization of lipids

HighResolutionMassSpectrometry(HRMS)

Cold Spring Harb Perspect Biol. 2011 Sep; 3(9): a004614. doi:† 10.1101/cshperspect.a004614

o  Highmassaccuracy:

o  molecularweightcalculation

o  elementalcompositionandmolecularformuladetermination

o  molecularstructure

o  Molecular ions of isobaric species (samem/z value but different

molecularformulaandstructure)couldbedistinguished

17

TandemMassSpectrometry(MS/MS)dataanalysis

Fragmentation: §  Selection of ion of interest in MS §  Formation of fragment ions in MS/MS §  Structural information

MS Spectrum

MS/MS Spectrum

The interpretation of the MS/MS spectrum is like solving a puzzle

↓ Allows us to obtain structural information about the initial

compound.

Tandemmassspectromety(MS/MS)Glycerophospholipids

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Glycerophospholipidsorphospholipids(PL)

O OP

OPolar Head group

O

O

OH

Fatty acyl

Fatty acyl

R1

R2

R1 and R2 are used to designate undefined alkyl groups at the sn-1 and sn-2 positions, respectively

N+

NH2

H

OHOH

OHOH

H

H

H

OH

NH2O

OH

OHOH

Choline

Ethanolamine

Inositol

Serine

Glycerol

-H Hydrogen

Informationneededtobe

confirm:

-Polarheadgroup

-Fattyacids

Fragmenationdependson:

-Typeofpercursorion

-Colisionenergy

-others

m/z100 200 300 400 500 600 700 800

%

0

100

%

0

100 x36184.1

478.4450.4 760.7504.5549.6

x82 599.5

147.0184.1 478.3441.2

247.3

504.4

505.2

723.5782.6

O

O

O

POH

O

O

N+ CH3

CH3

CH3

CH3

O

CH3

O

[MH-R1COOH]+

m/z 504

CH2O

O

POH

O

O

N+ CH3

CH3

CH3

R1

O

[MH-R2COOH]+

m/z 478

O

OP

OH

O

O

N+ CH3

CH3

CH3

R2

O

OHP

OH

O

O

N+ CH3

CH3

CH3

m/z 184

[MH]+

m/z 760

-R1COOH-R2COOH [M+H]+

Phosphatidylcholine–MS/MS[M+H]+

MS/MSspectrum-  m/z184-  Lossoffattyacids

PC16:0/18:1R

R

+

19

•  PositiveMode[M+H]+

•  NegativeMode[M-H]–

100 200 300 400 500 600 700m/z0

100

%

x36 577.6381.3

360.4

308.3 454.4 718.6-R2CO

-141Da

Phosphatidylethanolamine–PE

[M+H]+

H+Na+H+

Characteristiclossof141Da

LossofRCOOHandRC=O

(R1=CO+andR2=CO+).

ESI-MS/MS[M+H]+

R

R

PE16:0/18:1

R1COO-

R2COO- [M-H]-

[M-H]-

Phosphatidylethanolamine–MS/MS

MS/MSspectruminnegativemode•  R1COO-ion•  R2COO-ion

R2COO-

R1COO-

-O

ESI-MS/MS[M-H]-

20

03-Mar-201009:49:39

.

m/z500 550 600 650 700 750

%

0

100RO-RK PLPS MS760 76 (0.802) Sm (SG, 5x3.00); Cm (15:113)

1.07e3575.4

760.4576.4

[M+H]+

-185Da

ESI-MS/MS[M+H]+POPS

Phosphatidylserines–PS

R

R

MS/MS[M+H]+Characteristiclossof185Da

RK-PLPS neg #134-260 RT: 0.70-1.46 AV: 127 NL: 3.94E5T: ITMS - c ESI Full ms2 758.40@17.00 [ 205.00-770.00]

300 400 500 600 700m/z

0

20

40

60

80

100

Rela

tive

Abu

ndan

ce

671.3

391.1

415.1255.1

758.2433.2 496.2 672.0

x10 -87DaESI-MS/MS[M-H]-POPSESI-MS/MSof[M-H]-

•  Lossof87Da•  R1COO-ion•  R2COO-ion

R2COO-

R1COO-

-87Da

-185Da

m/z100 200 300 400 500 600 700

%

0

100R-D-TLC-DC-CONT-PG-2904MS699 64 (0.674) Cm (58:140)

95281.3

253.2

153.0 171.1

699.5

389.3

282.3567.4417.3

418.3567.3

699.0

700.0

700.2

[M-H]-

EspectrodeESI-MS/MSinnegativemode

Phosphatidylglycerol-PG

v

-

m/z171

H

R2COO-

R1COO-

R

R

R2COO-

R1COO-

21

R R

-

m/z241

ESI-MS/MSofM-H]-16:0/18:1-PI

Phosphatidylinositol-PI

m/z100 200 300 400 500 600 700 800

%

0

100R-GPIL-IBMC-1MS835 82 (1.266) TOF MSMS 835.50ES-

82.8281.2241.0

223.0

391.2

835.5553.3392.2 571.3

R R

H+

OH

OH

OH

OH

O PO

O

O-

R2COO-

R1COO-

[M-H]-

R2COO-

R1COO-

PhospholipidclassesandMS/MS

Informationaboutfattyacylcomposition–  Positivemode

•  LossofRCOOHeR=C=O

–  Negativemode•  FormationofcarboxylateanionsRCOO-

22

RT: 0.0 - 30.0 SM: 9B

0 5 10 15 20 25 30Time (min)

0

20

40

60

80

100

Relat

ive A

bund

ance

19.2

24.15.63.1

7.7 14.811.8

NL: 3.30E7Base Peak m/z= 480.00-525.00+700.00-1500.00 F: FTMS + p ESI Full ms [200.0000-1600.0000] MS oxHaCaT_2h_1

HT2 #5478-6045 RT: 15.75-17.33 AV: 26 NL: 8.26E7T: FTMS + p ESI Full ms [200.00-1600.00]

700 750 800 850m/z

0

20

40

60

80

100

Relat

ive A

bund

ance

782.6760.6

784.6

780.5 786.6756.6

787.6778.5 806.6 868.7840.6754.5732.5703.5

TICPositiveionmode

PE

PC

LPC

Time

10 15 20 25Time (min)

0

20

40

60

80

100

Relat

ive A

bund

ance

RT: 16.3AA: 9968214328

NL: 1.16E8m/z= 782.56-782.57 F: FTMS + p ESI Full ms [200.00-1600.00] MS ICIS HT2RIC

MS m/z: 782.6

SM

10 20 30Time (min)

0

20

40

60

80

100

Rela

tive

Abun

danc

e

740 760 780 800 820m/z

0

20

40

60

80

100

Rela

tive

Abun

danc

e

TIC

MS

200 400 600m/z

0

20

40

60

80

100

Rela

tive

Abun

danc

e MS/MS

PG/PI

PE PC

LPC PS

PC

Bioinformatictools:MzmineCompoundDiscoverLipidizerLipidBlastOthers

UntargetLipidomicsanalysis

Shotgun Lipidomics

TargetLipidomicsanalysis

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QuantificationbyLCMS

InternalStandard

BeforeMSAnalysis

•  Different lipid species have different quantitative responses

•  One internal standard for each one phospholipid class

Normalization of each molecular species to the internal standard of the corresponding class

possiblelossesthatcanoccurduring

extraction

di-C14 di-C15di-C17

BeforeExtraction

Internal Standard Phospholipid

Molecular Species

PL Species

Internal Standard

Extracted Ion Chromatogram

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