LIPID MAPS Lipidomics WorkshopApril 28, 2007

Lipid Analysis by Mass SpectrometryIntroduction and ChallengesNeutral and Phospholipids

Robert C. MurphyDepartment of Pharmacology

University of ColoradoHealth Sciences Center

www.lipidmaps.org

Other LIPID MAPS Neutral Lipid Core members:Robert Barkley

Miguel GijonJessica Krank

Outline:A. Electrospray Ionization of Neutral LipidsB. NL- tandem mass spectrometry-TAGs /CEC. Compound identification: Qualitative

Analysis/Challenges/artifactsD. Quantitation: Internal standards, etc.E. Data analysis/visualization: Lipid ProfilerF. PhospholipidsG. LC/MS/MS QuantitationH. Other strategies

Neutral Lipids:Mass spectrometric Challenges

• Formation of gas phase ions– Desorption/Spray Ionization

• Attachment of charging species• Complex mixture – molecular species

– Signal divided by total number of components

– Hundreds of species

Mass Spectrometry

• Electrospray Ionization-Neutral Lipids– Tandem Mass spectrometry

• Product ions • Precursor Ions• Neutral loss

Challenge: Neutral Lipids

Scan m/z 500 to 1000

100

•ESI- no structural information

[M+NH4]+

[M+Na]+

750 770 790 810 830 850 870 890m/z

874.8

52:3-TAG(53:10)

m/z 874.8 16:0/18:1/18:2 TAG

200 300 400 500 600 700 800 900m/z

100577.5

601.5

575.5

874.8

857.8

Product ions (MS/MS)

[M+NH4]+

O

O

HO

O

O O

NH4

-16:0

-18:1

-18:2

[M+H]+

+H-NH3601

(-273)

Scanm/z 874.8

Product ion Scan

Collisional Activation

760 780 800 820 840 860 880 900 920 940 960 980 1000m/z

2.3x106 850.7

876.7

878.7

902.7904.7

924.7

852.7848.7

822.7817.7

812.6

796.7

950.7

ESI/MSIo

n A

bund

ance

(cou

nts/

sec) TAGs

MΦ

Product Ion Scan m/z 824.7TAGs from RAW 264.7 cells

-(16:0)

(48:0)NH4+

(48:0)NH4+

-(15:0)

-(14:0)-(17:0)

500 550 600 650 700 750 800 m/z

100551.5

565.5537.5523.5 579.5

Neutral Lipid extract-(18:0)

List of MS3 identified TAGsFrom 6 different m/z values

TG(48:0) TG(48:1) TG(48:2) TG(49:0) TG(49:1) TG(49:2)

Major 16:0/16:0/16:0 16:0/16:0/16:1 16:0/16:1/16:1 16:0/16:0/17:0 15:0/16:0/18:116:0/16:1/17:016:0/16:0/17:1

15:0/16:1/18:116:0/16:1/17:116:1/16:1/17:0

Minor 14:0/16:0/18:015:0/16:0/17:0

14:0/16:0/18:114:0/16:1/18:015:0/16:1/17:015:0/16:0/17:115:0/15:0/18:1

14:0/16:1/18:114:0/16:0/18:215:0/16:1/17:1

15:0/16:0/18:0 14:0/17:0/18:115:0/16:1/18:0

15:1/16:0/18:1

Trace 15:0/15:0/18:012:0/18:0/18:014:0/17:0/17:0

14:0/17:0/17:114:1/17:0/17:015:1/16:0/17:0

12:0/18:1/18:114:0/17:1/17:114:1/16:0/18:115:0/15:0/18:215:0/15:1/18:115:1/16:0/17:114:1/17:0/17:1

12:0/17:0/18:013:0/18:0/18:014:0/16:0/19:015:0/15:0/19:0

13:0/16:0/20:114:0/16:0/19:114:0/17:1/18:014:1/17:0/18:015:0/15:0/19:115:1/16:0/18:0

13:0/16:0/20:214:0/15:0/20:214:0/17:1/18:114:0/17:0/18:214:1/17:0/18:115:0/16:0/18:215:0/17:1/17:115:1/17:0/17:1

MSMS33 data allowed for the unequivocal identification of 55 TAGsdata allowed for the unequivocal identification of 55 TAGs

m/z 824 m/z 822 m/z 820 m/z 838 m/z 836 m/z 834

Crude Mixture or Purification of Lipids by Class

• Shotgun analysis– Use the power of MS to separate species– Unique product ions after collisional activation

• Separation prior to analysis– LC/MS and LC/MS/MS– Off line separation of classes

• TLC (NP)• HPLC (NP and/or RP)• SPE

CE

TAGsat

TAGunsat

CholesterolDAG

Standards

Origin

FrontUnks

A

BCDE

TAG

CE

TLCSilica gel GHex/EtOEt/HOAc(80/20/1, v/v/v)

850 900 950 1000m/z

4.4e6910.7

908.7884.8

860.8

886.7

882.7

936.8841.8938.8836.8

934.7

819.7815.7

964.6962.7

EMSBand A TAG=55:6

m/z 910.8

A

MΦ

m/z200 300 400 500 600 700 800 900

1.10e6 910.6

893.8563.5

Product ions m/z 910.8

550 600 650

563.5565.6

589.5611.5

637.5561.5 651.5

-22:5

-22:6-20:4

-18:1

-16:0e-18:1e

Unique lossesx10

[M+NH4]+

[M+H]+

Inte

nsit

yO

O

OO

O

NH4

Chemical Formula: C59H108NO5+

Exact Mass: 910.8222Molecular Weight: 911.4920

Bartz R et al. J Lipid Res. 48:837(2007)

Δ 273Neutral loss Scan

7607808008208408608809009209409609801000m/z

2.3x106850.7

876.7

878.7

902.7904.7

924.7

852.7848.7822.7

817.7812.6

796.7

950.7

ESI/MS

Ion

Abu

ndan

ce(c

ount

s/se

c)

TAGsElicited MΦ

577273 signal

700 800 900 1000m/z,

2.3e5In

tens

ity,

cps848.8

874.8

876.9

857.9824.7

900.9822.8 924.7

926.9

All [M+NH4]+ species that contain16:0

10x106

Murine Peritoneal MacrophagesACN/IPA/H2O/CH2Cl2 (45:45:5:5)

IS

Neutral loss spectrum (loss of 273amu)273 = CH3(CH2)14CO2H + NH3

[M+NH4]+

[M+NH4]+

[M+NH4]+

TAGs (9 )

M+NH4]+

675.6

535.5

563.5

591.5

619.6

703.6

sn-319:0

14:0

15:0

16:0

17:0

20:4

20:0

sn-2OH

OH

OH

OH

OH

OH

OH

sn-119:0

14:0

15:0

16:0

17:0

20:0

20:4

DAGs (7)M+NH4]+

19:0 12:0 19:0 857.814:0 16:1 14:0 771.715:0 18:1 15:0 827.816:0 18:0 16:0 857.817:0 17:1 17:0 869.820:4 18:2 20:4 949.720:0 20:1 20:0 995.920:2 18:3 20:2 955.820:5 22:6 20:5 993.7

sn-3sn-2sn-1

O

O

DO

O

O O

DD

D D

D5-glycerol-14:0/16:1/14:0

18- Fatty acyl groups

AvantiPolar Lipids

687.5

Lipid Profiler

Time (hrs)0 5 10 15 20 25 30

Rat

io In

tens

ity/m

g D

NA

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Control

TAG (50:1)18:1

KDO2

KDo2

Time (hrs)0 5 10 15 20 25 30

Rat

io In

tens

ity/u

gD

NA

0.005

0.010

0.015

0.020Control

TAG (50:1) 16:1

DAG-16:0/20:4

Time (hrs)0 5 10 15 20 25

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Control KDo2

Rat

io In

tens

ity/u

gDN

A

A

B

C

RAW 264.7 CellsStimulated with KDO2-Lipid Am/z 850

Precursor ion scanningScan m/z 369

18:1-CE:

200 400 600 800m/z

100 369.3

668.6[M+NH4]+

O

OH4N

Products of m/z 668.6

200 400 600m/z

673.7369.3

668.6

Rela

tive

Abu

ndan

ce

100

Rela

tive

Abu

ndan

ce

[M+NH4]+MS MS/MS

Thioglycolate elicited Peritoneal Macrophage (C57B6)

Precursors of m/z 369.3

500 600 700 800m/z, amu

3.4e5In

tens

ity,

cps

684.9

666.8

668.

9

690.7646.7

567.5 714.8558.6 642.7

614.7

MS/MS

9alCE

14:0CE

18:2CE

[13C18]18:2CE

20:4CE

22:6CE

[2H4]16:0CE

oxIS1

16:0CE

597.7

CE

MΦ

oxIS2

Peritoneal Macrophages(thioglycolate elicited)

(100x106 cells; added 20ug IS)

0

50

100

22:6 20:4 18:1 18:0 18:2 16:0 16:1 14:0

ng/

106ce

lls

Fatty Acid Esterified to Cholesterol

Phospholipids• Classes• Quantitative and Qualitative analysis

– Positive/negative ions – Isotope corrections– Quantity indicating ions and standard curves

• Chromatographic separation– Normal phase (LC)– Reverse phase(LC)

• Artifacts/challenges

X (Polar Head Group)

O

O

R1

OR2

O

O

P

O

OO

X

1,2-diacyl

Glycerophospholipids

NH2

CH

C

CH2

HOO

NH2

CH2CH2

NCH2C

H2

H3C CH3

CH3

Serine (GPSer)

Ethanolamine(GPEtn)

Choline (GPCho)

Inositol (GPIns)HOOH OH

OH

OH

OOO

O PO

OH

O

O NH3+

577-H

200 400 600 800m/z

577

718[M+H]+

Relative

Int

ensity

100

16:0a/18:1 GPEtn

[M+H]+-14116:0a/18:1 GPEtn

[M-H]-

200 400 600 800m/z

281

716

255

452478

Relative

Int

ensity

100

281

OO

O

O PO

O-

O

O NH2

255

CID 16:0a/18:1-GPEtnPositive and negative product ions

200 400 600m/z

100

255

303

480

766.6[M-15] -

Rela

tive

Inte

nsity m/z 766

Negative Ions

200 400 600 800m/z

100 184

782.6Rela

tive

Inte

nsity

[M+H] +

Positive Ions

-ketene

ESI-MS/MS(tandem quadrupole)

OO

O

O

O

POH

OO

NC H3

+H 184C H3C H3

303255 O

OO

O

O

PO

OO

NC H3

C H3

GPCho GPEtn

GPSer GPIns

Diagnostic Product Ions from Positive [M+H]+ Glycerophospholipids (CID)

OHO

OH OH

OH

OH

PHO

O

OH

Neutral loss260 u

OPHO

O

OH

NH2

Neutral loss141 u

OPHO

O

OH

NH2

COOH

Neutral loss185 u

NCH2C

H2

H3C CH3

CH3O

PHO

O

OH

m/z 184

GPCho GPEtnGPSer GPIns

Common Negative Ions of Glycerophospholipids (CID)

R1COO-

R2COO-

[M-H]- -R2COOH[M-H]- -R2C=C=O

[M-H]- -87

R2COO-

[M-15]-

R1COO-

[M-H]- -R2COOH-87

R1COO-

R2COO-

[M-H]- -R2COOH[M-H]- -R2C=C=O

R2COO-

[M-H]- -R2COOH[M-H]- -R2C=C=O

R1COO-

m/z 241

m/z 153m/z 153m/z 153

*

*

*

*

GPGro(12:0/13:0)

GPGro(14:1/17:0)

GPGro(17:0/20:4)

GPGro(21:0/22:6)

LC/MS XICs of GPGro Odd-Carbon Internal StandardsLC/MS Analysis (A. Brown)

I.S.

I.S.

I.S.

I.S.

600 650 700 750 800 850

3.0e6In

tens

ity,

cps

863.76778.88

819.68776.88747.92

795.76783.76

705.84

623.76

724.00

655.76

34:X

GPG

ro

36:X

GPG

ro

38:X

GPG

ro

40:X

GPG

ro

Foam Cell GPGro Region of LC/MS Experiment-EMS: 4.046 to 5.233 min from Sample 1 (Sample003) of Sample003.wiff (Turbo Spray) Max. 2.9e6 cps.

m/z

Avanti Lipid Maps Standards

25:0 GPA 31:1 GPA 37:4 GPA 43:6 GPA25:0 GPCho 31:1 GPCho 37:4 GPCho 43:6 GPCho25:0 GPEtn 31:1 GPEtn 37:4 GPEtn 43:6 GPEtn25:0 GPGro 31:1 GPGro 37:4 GPGro 43:6 GPGro25:0 GPIns 31:1 GPIns 37:4 GPIns 43:6 GPIns25:0 GPSer 31:1 GPSer 37:4 GPSer 43:6 GPSer13:0 Lyso GPA 17:1 Lyso GPA13:0 Lyso GPCho 17:1 Lyso GPCho37:4 GPIns(3)P 37:4 GPIns(4)P 37:4 GPIns(5)P37:4 GPIns(3,4)P2 37:4 GPIns(3,5)P2 37:4 GPInsI(4,5)P237:4 GPIns(3,4,5)P3

XIC from m/z 631.06 to 631.96 (Peak fragmentation indicates 31:1 GPA standard)

Peak IDd

Integration window

LC-MS Computational Analysis

- XIC ASCII data is automatically generated from fullscan data for all m/z values, peaks found, integrated, and aligned across files (samples).

-Visual checks and error checking per m/z are used to confirm automated results.

XIC from m/z 909.06 to 909.96 (LMAPS Raw cell)

LC-MS Analysis (cont’d)

- Peaks areas are normalized to fixed odd-carbon internal standards (when available)- Even carbon titrations are used for estimating quantities in the extract.

Multiple XICs (10 spectra) from m/z 909.06 to 909.96 (LMAPS RAW cell)

- Determine peak areas using automated integration algorithms.- Align integrated values across samples (confirm alignment graphically).- Normalize areas to fixed internal standard areas.- Currently do this for ~130 glycerophospholipid analytes.

GPA

- Peak areas normalized to mean of 4 fixed odd carbon internal standard peak areas within the class (as in KDO2 time-course data set).

- 11 even carbon GPA standard curves generated.

Amount(ng) = slope*(fraction of odd carbon std) + intercept

Standard curves from integrated peak XIC data

O

O

HO

O ON+

P

O

O

O

O

O

HO

O ONH2

P

HO

O

O

H

O

OH

O

O

HO

O ONH2

P

HO

O

O

O

O

HO

O OHP

HO

O

O

O

O

HO

O OP

HO

O

O

OH

HO H

O

O

HO

O OP

HO

O

O

HOOH OH

OH

OH

GPA 101

GPCho 106

GPEtn 136

GPGro 105

GPIns 84

GPSer 68

Glycerophospholipid Parts List (RAW 264.7 cells)

Lipid Class Lipids Identified

Blue light (flame)

Ozonloysis during ESI (Negative ions)

OO

OPO

OOH

O

ON

ESI

O3

O

OH

O

m/z 650.6

m/z 760.7

O

OH

O

HOOCH3

+

m/z 698.6

Thomas, Blanksby et alAnal. Chem. In press2007

Isotopic AbundanceIsotope %

Natural Abun-dance

1H 99.982H 0.01512C 98.913C 1.114N 99.6415N 0.3616O 99.818O 0.219F 100

28Si 92.229Si 4.730Si 3.1

m/z703 704 705 706

100

707 709 710

13C1

13C2

[M+H]+

12C38

702.5728.5

752.5

780.5 806.6

700 720 740 760 780 800 820 840

100

Rel

ativ

e ab

unda

nce

50

860

Positive ion mass spectrum of GPEtn in PMNs

15N1

18O1

C38H77O7NPm/z 702.5437

16:0p/18:1-GPEtn43.7

10.71.92

702

64.0%TotalIon current

Exact mass and Isotopes

O P O O P OOH

O

O

OO

O O

OH

OOOOO

Exact Mass: 1351.96

CardiolipinMitochonrial lipid

1352

1351.96 43.2%Ioncurrent

m/z

C73H141O17P2-

750 1000 1250 1500 1750 2000m/z

100

0

Rela

tive

Int

ensi

ty (%

)

853.1

873.8

1688.9

1709.7

16:0/16:0/18:1-TAG ESI-MS

[16:0/16:0/18:1 TAG + NH4]+

[16:0/16:0/18:1 TAG2 + NH4]+

Na+ adductNa+ adduct

Singly charged dimer

Singly charged monomer

800 1000 1200 1400 1600m/z

100

0

Rela

tive

Int

ensi

ty (%

)

706.6

725.71078.8 1413.0

14:0a/16:0 GPCho ESI-MS

[14:0/16:0PC2 + Ca]2+

[14:0/16:0PC3 + Ca]2+

[14:0/16:0PC2 + H]+

[14:0/16:0PC + H]+

Double charged trimer

O

Singly charged dimer

OO

PO

OOH

O

O

N

+++

++

+

+

++++

+

+

+ +

+

++ +

+ ++

+

+

+

+++

+

+

+

+ ++

+

+

++

+

+

+

+++

+

+

+

+++

+++

+

++

++

+

+

++

+

+

+

+++

+ ++

+

Evaporation

Solvent

CoulombExplosion

Surface tensionexceeded bycharge repulsion

+++

++

++

++++

++

++

+

++

++

+++

+++

+

+

+++

+++

++ +

++

+

++

++

++

++

++

CoulombExplosions

+Direct IonEmission

++

+Evaporationto a gas

Bare ion IntoMass spectrometer

References• Triglyceride analysis

– Qualitative MS/MS and MS3

• McAnoy et al. J Amer Soc Mass Spectrom. 16:1498 (2005)– Quantitation by neutral loss scanning

• Murphy et al. Anal. Biochem. In press 2007• Phospholipid analysis

– Qualitative analysis by ESI Mass Spectrometry• Pulfer and Murphy, Mass Spectrom. Rev. 22:332 (2003)• RCMurphy Mass Spectrometry of Phospholipids (2002).

– Quantitation– Rouzer, R.A., Ivanova, P.T., Byrne, M.O., Milne, S.B., Marnett, L.J., and Brown,

H.A. , (2006) Lipid profiling reveals arachidonate deficiency in RAW264.7 cells: Structural and functional implications., Biochemistry 45: 14795-14808.

– Milne, S.B., Ivanova, P.T., Forrester, J.S., and Brown, H.A., (2006) Lipidomics: Analysis of cellular lipids by ESI-MS. Methods 39: 92-103. Edited by V. Bankaitis. Elsevier Press.

– Callender, H.L., Forrester, J.S., Ivanova, P., Preininger, A., Milne, S., and Brown, H.A., (2007) Quantification of diacylglycerol species from biological extracts by electrospray ionization mass spectrometry. Analytical Chemistry79: 263-272.

– 4. Ivanova, P.T., Milne, S.B., Byrne, M.O., Xiang, Y., and Brown, H.A. (2007) Glycerophospholipid Identification and Quantitation by Electrospray Mass Spectrometry. Methods in Enzymology: Lipidomics and Bioactive Lipids. Voume1. Edited by H. Alex Brown, Elsevier Press. In press.

• Web sites for tools:• www.lipidmaps.org• www2.uchsc.edu/pharmacology/RCMweb1—use Microsoft Internet Explorer

Acknowledgements• Analysis of neutral lipids

– Jessica Krank-Miguel Gijon– Patrick Hutchins

• Analysis of Phospholipids– Alex Brown (Vanderbilt University)

• Lipid Profiler– Eva Duchoslav (Applied Biosystems, Canada)

• Support: National Institutes of HealthLipidMaps GM069338