Identification and Structure Determination of Higher Order Glycosphingolipids via LC-MS/MS

M. Cameron Sullards, Ph. D.

Georgia Institute of Technology: School of Chemistry and Biochemistry and School of Biology, Atlanta, GA 30332-0363

Gene

mRNA

Protein

Post-translationalmodification(s)

Metabolites

Cel

lula

r fu

ncti

ons

Membrane lipids and regulation of cell structure/function

Membrane (lipid)functions:• Energy• Structure• Transport• Signaling

Implications for disease:• Energy excess /deficit• Aberrant cell structure/function• Transport defects• Incorrect signaling• Developmental abnormalities

Ago

nist

s &

oth

er e

xtra

cell

ular

sig

nals

,in

clud

ing

nutr

ient

s &

xen

obio

tics

LIPID MAPS

LIPID MAPSLipid Metabolites And Pathways Strategy

GENOMICS

PROTEOMICS

METABOLOMICS

“LIPIDOMICS”

Lipid Metabolites and Pathways Strategy

LIPID MAPS GOALS(1) To separate and detect all of the lipids in a specific

cell and to discover and characterize any novel lipids that may be present.

(2) To quantitate each of the lipid metabolites present and toquantitate the changes in their levels and location during cellular function.

(3) To define the biochemical pathways for each lipid and develop lipid maps which define the interaction networks.

LIPID MAPS CORES

Bioinformatics

Neutral Lipids Other Lipids/Structural Lipidomics

SterolsFatty Acids/Eicosanoids

GlycerophospholipidsSphingolipids/Gangliosides

Lipid Synthesis/Characterization Michael VanNieuwenhze - UCSD

Walter Shaw - Avanti Polar LipidsSteven White - UC Irvine

Shankar Subramaniam - UCSD

Robert C. Murphy - Colorado Christian Raetz - Duke

David W. Russell - UTSW

Alfred H. Merrill - Georgia Tech H. Alex Brown - Vanderbilt

Macrophage BiologyChristopher Glass - UCSD

Edward A. Dennis - UCSD

LC/Mass SpecRobert C. Murphy - Colorado

PI Edward A. Dennis - UCSD

LIPID MAPS

Sphingolipids are the most structurally complex and diverse lipids of eukaryotes

H O

O

O

O H

OHO

OO

O HO

OH

H

O

H O

OO H

H O

OHH

1

OO H

H O

O H

O

H N AcOH

H O

H O H

H

Cer

Lactosylceram id e (LacCer)

34

3

1

2

11

4

1'

N-Acetyl-galactosamine

Galactose Galactose Glucose

AcNH

HO2C

N-Acetyl-Neuraminic acid

GM1

GM2

GM3

Glu cosylceram id e (GlcCer)

OHOH

NH

D-erythro-sphingosine

Ceramide (N-acylsphingosine)

O

(CH3)3NCH2CH2O-P(O2H)-O-Cer

Sphingomyelin

J. L. W. Thudichum1884

SphinSphinGGOOMMAPAP© (Download available at (Download available at www.sphingomap.org))

MS/MS Methodology

Identify structure specific dissociations unique to various Identify structure specific dissociations unique to various classes SL’s (ie. Cer, GlcCer, LacCer, Gb3, and Gb4)classes SL’s (ie. Cer, GlcCer, LacCer, Gb3, and Gb4)

Optimize ionization and dissociation conditions for all SL’s Optimize ionization and dissociation conditions for all SL’s

Utilize precursor ion & neutral loss scans to Utilize precursor ion & neutral loss scans to identify identify individual headgroup, base, and fatty acid combinationsindividual headgroup, base, and fatty acid combinationsof endogenous SL’sof endogenous SL’s

Quantify SL’s using internal standards and LC-MS/MSQuantify SL’s using internal standards and LC-MS/MS

O

O

OH

OH

OH

O

OO

OH

OH

OH

OHO

OH

OH

OH OH

NH

O

OHO

OH

OH

NH

O

O

OH

OH

OH

O

OO

OH

OH

OH

OHO

O

OH

OH OH

NH

O

O

Globoside Gb3

Globoside Gb4

+Q1: 60 MCA scans from Sample 10 (Gb3) of mcs 0178.wiff (Turbo Spray) Max. 9.1e6 cps.

1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 1200m/z, amu

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

Inte

nsity

, cp

s

1152.9

1136.9

1150.91108.8

1158.81109.9

1130.9 1175.0

(M1 + H)+

*

*

*

Gb3(M2 + H)+ (M3 + H)+

* = (M + Na)+

4000 Q TRAPTM System – Ion Path

Q0 Q1 Q2 Q3

LIT

Curtain PlateOrifice

Skimmer

LINAC Exit Lens

+MS2 (1108.80) CE (50): 36 MCA scans from Sample 12 (Gb3) of mcs 0178.wiff (Turbo Spray) Max. 7.3e5 cps.

100 200 300 400 500 600 700 800 900 1000 1100m/z, amu

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

Inte

nsity

, cp

s

604.8

264.5

586.81091.0

928.9767.0

282.31108.8574.6252.1 831.1135.1

340u = C22:0Glc

GalGal

Quad FragmentsQuad Scan

d18:1

(M1 + H)+

O

O

OH

OH

OH

O

OO

OH

OH

OH

OHO

OH

OH

OH OH

NH

O

Y1 Y0

Y2

N"

+EPI (1108.80) CE (50): 119 MCA scans from Sample 14 (Gb3) of mcs 0178.wiff (Turbo Spray) Max. 8.5e7 cps.

100 200 300 400 500 600 700 800 900 1000 1100m/z, amu

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

6.0e7

7.0e7

8.0e7

8.5e7

Inte

ns

ity, c

ps

604.6

264.2

586.6 1090.8

928.7766.6

282.2 574.6 1108.8

Quad FragmentsLIT Scan

d18:1

340u = C22:0Glc

GalGal

(M1 + H)+

+EPI (1108.80) CE (50): 119 MCA scans from Sample 15 (Gb3) of mcs 0178.wiff (Turbo Spray) Max. 8.0e8 cps.

100 200 300 400 500 600 700 800 900 1000 1100m/z, amu

1.0e8

2.0e8

3.0e8

4.0e8

5.0e8

6.0e8

7.0e8

8.0e8

Inte

ns

ity, c

ps

604.6

586.7

264.21090.8

928.7766.7

574.7 1108.7282.2 659.4 830.8252.2 340.3

Quad FragmentsLIT Scan

w/ Q0 Trapping

d18:1

340u = C22:0 GlcGal

Gal

(M1 + H)+

4000 Q TRAPTM System – Ion Path

Q0 Q1 Q2 Q3

LIT

Curtain PlateOrifice

Skimmer

LINAC Exit Lens

+MS3 (1108.70),(604.70): 93 MCA scans from Sample 24 (GB3) of mcs 0176.wiff (Turbo Spray) Max. 2.7e8 cps.

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z, amu

2.0e7

4.0e7

6.0e7

8.0e7

1.0e8

1.2e8

1.4e8

1.6e8

1.8e8

2.0e8

2.2e8

2.4e8

2.6e82.7e8

Inte

ns

ity, c

ps

264.2

604.6

340.3252.2

574.6282.2364.3

MS/MS/MS1108.7604.6

+EPI (1136.90) CE (50): 119 MCA scans from Sample 19 (Gb3) of mcs 0178.wiff (Turbo Spray) Max. 8.8e8 cps.

100 200 300 400 500 600 700 800 900 1000 1100m/z, amu

1.0e8

2.0e8

3.0e8

4.0e8

5.0e8

6.0e8

7.0e8

8.0e8

8.8e8

Inte

ns

ity, c

ps

632.7

614.81118.8

264.2

956.8794.7

602.8 1137.3282.2659.4768.5252.2 825.8368.3

d18:1

368u = C24:0

Gal

Gal

Glc

(M2 + H)+

+EPI (1152.90) CE (50): 117 MCA scans from Sample 25 (Gb3) of mcs 0178.wiff (Turbo Spray) Max. 7.4e8 cps.

100 200 300 400 500 600 700 800 900 1000 1100m/z, amu

5.0e7

1.0e8

1.5e8

2.0e8

2.5e8

3.0e8

3.5e8

4.0e8

4.5e8

5.0e8

5.5e8

6.0e8

6.5e8

7.0e87.4e8

Inte

ns

ity, c

ps

648.7

630.8

1134.8

618.8972.8 1152.8810.7

264.2 666.7

792.7384.3282.2

d18:1

384u = h24:0 GlcGal

Gal

(M3 + H)+

Q1 Q2Q3

Precursor Ion Scan

scan m/z fixed m/z

+Prec (264.20): 179 MCA scans from Sample 11 (Gb3) of mcs 0178.wiff (Turbo Spray) Max. 3.5e6 cps.

1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 1200m/z, amu

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

Inte

ns

ity, c

ps

1137.1

1153.1

1109.2

1151.1

1024.91053.0

1081.1

1125.1

1135.1

C16:0

h24:1

h24:0

C18:0C20:0

C22:0

h22:0

C24:1

C24:1

d18:1

+Q1: 30 MCA scans from Sample 2 (Gb4) of mcs 0179.wiff (Turbo Spray) Max. 9.4e6 cps.

1200 1220 1240 1260 1280 1300 1320 1340 1360 1380 1400m/z, amu

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e69.4e6

Inte

ns

ity, c

ps

1356.0

1362.0 1379.0

1340.0

1358.01354.01333.9

1377.01373.0

1311.9

1328.9 1359.9 1375.01349.9

Gb4

(M1 + H)+

(M1 + H)+

(M1 + H)+

*

*

*

* = (M + Na)+

OHO

OH

OH

NH

O

O

OH

OH

OH

O

OO

OH

OH

OH

OHO

O

OH

OH OH

NH

O

O

Y1 Y0

Y2

N"

Y3

GalNAc

+EPI (1311.80) CE (60): 35 MCA scans from Sample 8 (Gb4) of mcs 0177.wiff (Turbo Spray) Max. 1.0e8 cps.

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z, amu

1.00e7

2.00e7

3.00e7

4.00e7

5.00e7

6.00e7

7.00e7

8.00e7

9.00e7

1.00e8

Inte

ns

ity, c

ps

604.6

586.6

1090.7928.7264.2

766.6

1293.8574.6204.1282.2144.0 946.6

(M1 + H)+

GalNAc

GalGal

Glc

340u = C22:0

d18:1

+EPI (1340.00) CE (65): 105 MCA scans from Sample 4 (Gb4 c24:0) of mcs 0178.wiff (Turbo Spray) Max. 5.4e8 cps.

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z, amu

5.0e7

1.0e8

1.5e8

2.0e8

2.5e8

3.0e8

3.5e8

4.0e8

4.5e8

5.0e8

5.4e8

Inte

ns

ity, c

ps

632.6

264.2614.7

956.71118.8794.7

602.7204.1282.2

1321.9144.1 368.3 813.7168.1

(M2 + H)+

GalNAcGal

GalGlc368u = C24:0

d18:1

+EPI (1356.00) CE (65): 104 MCA scans from Sample 8 (Gb4 h24:0) of mcs 0178.wiff (Turbo Spray) Max. 5.7e8 cps.

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z, amu

5.0e7

1.0e8

1.5e8

2.0e8

2.5e8

3.0e8

3.5e8

4.0e8

4.5e8

5.0e8

5.5e85.7e8

Inte

ns

ity, c

ps

648.6

618.7264.2

810.7 972.7204.11134.8

666.7 1356.4144.1 252.2 282.2 384.3126.0 792.7

(M3 + H)+

GalNAcGal

GalGlc

d18:1

384u = h24:0

+Prec (264.20): 90 MCA scans from Sample 3 (Gb4) of mcs 0179.wiff (Turbo Spray) Max. 1.6e6 cps.

1200 1220 1240 1260 1280 1300 1320 1340 1360 1380 1400m/z, amu

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

Inte

ns

ity, c

ps

1340.0

1356.1

1312.0

1341.1

1354.11358.2

1313.1 1329.0

1373.21338.0

1228.01256.1 1284.1

C16:0C18:0 C20:0

C22:0

C24:0

h24:0

h24:1

d18:1

LC ESI MS/MSSample In

(Autosampler)

InletSystem

IonSource

Mass Analyzer(Q1, Q2, Q3) Detector

DataSystem

Dataout

HPLC

Rel

ativ

e Io

n A

bund

ance

0 2 4 6 8 10Time (min)

Cer

SM

LacCer

GlcCer

O

HN

O

OH

HOO

HO

OH

OH

N

0

132

45

XIC of +MRM (9 pairs): 1125.0/264.3 amu from Sample 9 (Gb 3/4 mix) of mcs 0184.wiff (Turbo Spray) Max. 2.6e4 cps.

24 25 26 27 28 29 30 31 32 33 34 35Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

Inte

ns

ity, c

ps

Gb3d18:1

C16:0C18:0

C20:0

h24:1

h22:0

C24:1

C22:0

h24:0C24:0

XIC of +MRM (9 pairs): 1340.0/264.3 amu from Sample 6 (Gb 3/4 mix) of mcs 0184.wiff (Turbo Spray) Max. 9.1e4 cps.

23 24 25 26 27 28 29 30 31 32 33 34 35Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

Inte

ns

ity, c

ps

Gb4

C20:0

C18:0

C16:0h24:1

C24:1

C22:0

h22:0

h24:0

C24:0

d18:1

ConclusionsConclusions Enhanced product ion scans in conjunction w/ Q0 trapping yield

more highly abundant fragment ions enabling detailed structural analysis

Precursor ion scans reveal critical information regarding low abundance globosides with high sensitivity in crude lipid extracts

MS/MS/MS may be used for determination of specific headgroup, long chain base, and fatty acid combinations in globosides

LC in conjunction w/ MS/MS is a powerful tool for separation and identification of complex mixtures of globosides

AcknowledgementsAcknowledgements

Prof. Alfred H. Merrill, Jr.Prof. Alfred H. Merrill, Jr.Meeyoung ParkMeeyoung ParkAnu KoppikarAnu Koppikarhttp://www.sphingomap.org http://www.sphingomap.org MatreyaMatreyaNIH/NIGMS/Lipid MAPSNIH/NIGMS/Lipid MAPShttp://www.lipidmaps.orghttp://www.lipidmaps.org