LIPID MAPS LipidLIPID MAPS LipidApril 28

www lipidmaps org

Sphingolipids (and precM Camero

www.lipidmaps.org

M. Camero

Schools of Chemistry, BioP tit I tit t f BiPetit Institute for Bioeng

Georgia Institute

Other LIPID MAPS SMass spectr

Al Merrill Jeremy AllegChris HayneSamuel Kelly

domics Workshopdomics Workshop8, 2007

cursor fatty acyl-CoA’s)on Sullardson Sullards

ochemistry and Biology &i i d Bi iineering and Biosciencee of Technology

phingolipid Core members:rometry Cell biologygood Elaine Wang

es Ying Liuy Jia Wei

Outline:A. Brief introduction to the lipid clas

compounds to analyzeB. Sample preparation issues: solv

reproducibilityC. Compound identification: Chara

and MSn (LC for isomers and isoD. Quantitation: MRM, Internal staE. Data analysis/visualization: LIMF. Remaining challenges and oppoG. Discoveries from sphingolipidomH. Comparison of Lipid MAPS met

ss: nomenclature & range of

vents, chromatography, recovery,

acteristic fragmentations; MS/MS obars, etc.)

andards, etc.MS, Website, otherortunitiesmic analysis thus far hods with others in the literature

Backbone variation

A. Brief introduction to the lipid class: nomen

HeBackbone variation

Sphingoid base:

Sphinganine (d18:0)

He

Sphinganine (d18:0)

4 Hydroxysphinganine4-Hydroxysphinganine(phytosphingosine) (t18:0)

D-erythro-sphingosine (d18:1)

Ceramide:

Shown: N-palmitoylsphingosine (d18:1/16:0)Other fatty acids-

GM

typically C16-C260-1 double bondsometimes α-hydroxy

N

nclature & range of compounds to analyze

eadgroup variationeadgroup variation

Phosphosphingolipids:

-OP(O2-)O-choline, Sphingomyelin

Glycosphingolipids:

Glc, Gal, Lac, Sulfatides…>400; seewww.lipidmap.org; www.sphingomap.org

Lactosylceramide (LacCer)

GlcCerGalNAc III

GalGal

GlcII

IV

NeuAcI

II

M1GM3

Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glc1-1CerII3Neu5AcGg4Cer

SphinSphinGGOOMMAPAPP P ((www.sphingomap.org)www.sphingomap.org)

B. Sample preparation issues: solvents, chro

30-100 mm Petri dish

w/cells

+ CH3OH+ CHCl3

+ InternalStds (Avanti)

PBSscrapepellet

Dry,CentrifugeA

(Polar) yRedissolve

in LC mobilephase

g& transfer

(Polar)

+ HOAc CentrifugeB

(Non- HOAc+ CHCl3+ H2O

Centrifuge& Recover

Lower phase

(Nonpolar)

omatography, recovery, reproducibility:

A

Sonicate48 oC ~12 h

0.1 M KOH37 oC ~2 h

B

Centrifuge & transfer to

autoinjector vial

Reverse phase LC-MSn

>80%

Dry,

Centrifuge & transfer to

autoinjector vial

Dry,Redissolve

in LC mobilephase

Normal phase LC MSn

80-90%

C. Compound Identification: Cof sphingof sphing

H

R

H

(CH2)8

O

OR'OHH

(CH2)8

m/z 264

HNH

O

R

R = n-alkyl chain, R' = H, G

Characteristic fragmentations golipidsgolipids

OOH

m/z 184

HNHO O

N+P

HO

OOH

OR"OHH

m/z 266

(CH2)8

H3N+ H

Glu/Gal, Lac, and R" = H, PO3

A (Polar e g So Sa 4 HO Sa SoP So Pcho

Sphingolipid analysis by LC-A (Polar, e.g. So, Sa, 4-HO-Sa, SoP, So-Pcho

B (Nonpolar, e.g. Cer, Cer-P, SM,

InletS t

IonSource

Reversephase

Normal phase HPLC

System SourcephaseHPLC

Species analyzed to date (in most (Dihydro)c

p y (mammalian sphingoid base & FA variants):

SM’s, GlcCer, GalCer, LacCer, Sulfatide (quanlitative analysis of globosides and

Sphingomy(q y ggangliosides)

Cer & Cer-PSo, Sa and 4-hydroxy-SaSo-P, Sa-P and other derivatives (lysoSM,

Glycosph

( ypsychosine, N-methyl-)

Plus metabolites labeled with stable isotope precursors

OHO

OH

OHB

1

23

04

1,5X25

HO

oline Psychosine Cer P etc )

-MS/MSoline, Psychosine, Cer-P, etc.)

GlcCer, GalCer, LacCer, etc.)

Mass Analyzer(Q1 Q2 Q3 or LIT)

Detector(Q1, Q2, Q3 or LIT)

Data System for tit ti b MRM

ceramides

H+OH ORm/z 264 quantitation by MRM

(Multiple Reaction Monitoring) w/

appropriate internal t d d

NH

O

OH OPhosphocholineyelins

m/z 264(266, etc.)

standards

Sullards, Merrill & coworkers STKE (2001),

O O R'

OHZ0

Y0

Z1 45

HO

m/z 184NH

O

hingolipids

Methods (2005),Meth. Enzymol. (in press) &

www.sphingomap.orgO

OH

OH

HN R"

O

Z1Y1

B1C1

B2C2

0

4

2 13

2,5A2 m/z 264(266, etc.)

Summary of conditions used

Reversed PhaseReversed Phase

-- LCBLCB analysisanalysis:: So,So, Sa,Sa, phytophyto So,So, SoSo--11--P,P, SaSa--11--P,P, andand standardsstandards

-- 22..11 xx 5050mmmm SupelcoSupelco DiscoveryDiscovery CC1818,,55 120120 ÅÅ55 µµm,m, 120120 ÅÅ

-- AA:: 7474::2525::11 CHCH33OH/HOH/H22O/HCOOHO/HCOOHBB:: 9999::11 CHCH OH/HCOOHOH/HCOOHBB:: 9999::11 CHCH33OH/HCOOHOH/HCOOH

-- FlowFlow raterate 11 mL/min,mL/min, 00..66 minmin.. 8080::2020A/BA/B 11 88 minmin toto 100100%% BB 00 66 minmin holdholdA/B,A/B, 11..88 minmin.. toto 100100%% B,B, 00..66 minmin.. holdhold100100%% BB

For more information, see Sullards, Merrill &Meth. Enzymol. (in press

d for Liquid Chromatography

Normal PhaseNormal Phase

-- ComplexComplex SL'sSL's:: Cer,Cer, GlcCer,GlcCer, GalCer,GalCer,L CL C SMSM dd t d dt d dLacCer,LacCer, SM,SM, andand standardsstandards

-- 22..11 xx 5050 mmmm SupelcoSupelco NHNH22,, 33 µµm,m, 120120 ÅÅ

-- AA:: 9797::22::11 CHCH33CN/CHCN/CH33OH/CHOH/CH33COOHCOOHBB:: 9999::11 CHCH33OH/CHOH/CH33COOHCOOHbothboth 55mMmM AmmoniumAmmonium AcetateAcetatebothboth 55mMmM AmmoniumAmmonium AcetateAcetate

-- FlowFlow raterate 11..55 mL/min,mL/min, 00..55 minmin.. 100100%% A,A,00..22 minmin.. gradientgradient toto 9090::1010 A/B,A/B, 00..55 minmin..gg ,,hold,hold, 00..44 minmin.. toto 8282::1818 A/B,A/B, 00..66 minmin hold,hold,00..44 minmin.. toto 100100%% BB

& coworkers: STKE (2001), Methods (2005),) & www.sphingomap.org

Sphing3-KetosphinganineSerine + Palmitoyl-CoA

Number of molecules

CH3(CH2)10CH2

Palmitoyl CoA 11<0.1

d18:0;C16:0DHSM

d18:0;C16:0

DHCer-Pd18:0;d18 0

<0.1

37819.5

d18:0;C18:0Cer

d18:0;C16:0DHCer

d18:0;C16:0

d18:0;C16:0

d18:0;C16:0

1-O-AcylDHCer

d18:0;C20:0DHCer

d18:0;C2:0

DHCerd18:0

d18:0-P<0.1

3 913.5

11.7

C 6 0GalDHCer

d18:1;C16:0

SM

GlcDHCer

d18:1;C16:0Cer-P

d18:1;C2:0Cer

All subspecies(only a fe

<0.13.9

<0.1 29.7 211.5

19.5

1023315

d18:1;C18:0Cer

d18:1;C18:0

Cer Pd18:1;C16:0

1-O-AcylCer

d18:1;C20:0Cer

d18:1

d18:1-Pd18:1;C16:0

<0.1d18:1;C16:0

d18:1;C16:0Cer30.3

117

C18:0GlcCer

d18:1 P C16:0GalCer<0.1

SulfatideLacCer <0.1[<1]

C16:0GlcCer30 23.4

ganine (d18:0) Sphinganine 1-phosphate

per RAW cell (million)

H CH2OHHO

NH3 (+)

(d18:0-P)

C

.7 3.9

Ceramide synthases+ Fatty acyl-CoA

d18:0;C22:0DHCer

d18:0;C24:1DHCer

d18:0;C24:0DHCer

d18:0;C26:1DHCer

d18:0;C26:0DHCer

s have been quantified for these chain lengths, tooew are shown; all are at www.lipidmaps.org)

13587 294 6.3 1.50.30.315.37.5.3

d18:1;C22:0Cer

d18:1;C24:1Cer

d18:1;C24:0Cer

d18:1;C26:1Cer

d18:1;C26:0Cer

11 7 23 4 36 9 1 5 0.611.7 23.4 36.9 1.5

Sphing3-KetosphinganineSerine + Palmitoyl-CoA

Acidic SphingolipidsInfusion 0.6 mL/h (MeOH)

Prec 290 scan [MCA 60 Scans (3s/scan)]DP 100 CE 75 IS 4500

CH3(CH2)10CH2

Palmitoyl CoA

Raw cel+KdO2Lipid A

DP = -100 CE = -75 IS = -4500

d18:0;C16:0DHSM

d18:0;C16:0

DHCer-Pd18:0;d18 0 d18:0;

C18:0Cer

d18:0;C16:0DHCer

d18:0;C16:0

d18:0;C16:0

d18:0;C16:0

1-O-AcylDHCer

d18:0;C20:0DHCer

d18:0;C2:0

DHCerd18:0

d18:0-P C 6 0GlcDHCer

d18:1;C16:0

SM

GalDHCer

d18:1;C16:0Cer-P

d18:1;C2:0Cer

Variation Raw cel

d18:1;C18:0Cer

d18:1;C18:0

d18:1;C16:0Cer

Cer Pd18:1;C16:0

1-O-AcylCer

d18:1;C20:0Cer

d18:1

d18:1-Pd18:1;C16:0

d18:1;C16:0C18:0

GlcCerd18:1-P C16:0

GalCerC16:0GlcCer

Sulfatide

ganine (d18:0) Sphinganine 1-phosphate

Next category of compounds being analyzed by “Inside-out”

H CH2OHHO

NH3 (+)

(d18:0-P)

Ceramide synthases

ls2

A

sphingolipidomics:Gangliosidesy

+ Fatty acyl-CoA

d18:0;C22:0DHCer

d18:0;C24:1DHCer

d18:0;C24:0DHCer

d18:0;C26:1DHCer

d18:0;C26:0DHCer

in backbone, acyl chain & headgroup specificitylls

d18:1;C22:0Cer

d18:1;C24:1Cer

d18:1;C24:0Cer

d18:1;C26:1Cer

d18:1;C26:0Cer

GM3 GM2 GM1 GD1a

Work-flow for analysis ofLC-MS/MS MLC MS/MS M

11.. IdentifyIdentify structurestructure specificspecificclassesclasses (e(e..gg..,, SM,SM, GlcCer,GlcCer,(( gg

2.2. Utilize precursor ion and neUtilize precursor ion and neindividual headgroup, sphinindividual headgroup, sphin

3.3. Optimize ionization and disOptimize ionization and disii

combinations.combinations.

species. species. 4.4. Optimize LC as required toOptimize LC as required to

effects and interferences aeffects and interferences aeffects, and interferences aeffects, and interferences aand isomeric species (repeand isomeric species (repe

5.5. Optimize conditions for quaOptimize conditions for quap qp qvs validated internal standavs validated internal standapresent. present.

f new samples using this MethodologyMethodology

dissociationsdissociations uniqueunique toto variousvariousGalCer,GalCer, LacCer,LacCer, etcetc..))))

eutral loss scans to identify eutral loss scans to identify ngoid base, and fatty acid ngoid base, and fatty acid

ssociation conditions for all ssociation conditions for all

o minimize ionization suppression o minimize ionization suppression arisingarising from isobaric isotopicfrom isobaric isotopicarising arising from isobaric, isotopic, from isobaric, isotopic, eat #3 if necessary).eat #3 if necessary).

antitation via ratio of peak areas antitation via ratio of peak areas ppards for all of the species ards for all of the species

Example: Identification of sNeutral Loss or Pr

ance

688.6

tive

Ion

Abu

nda

671.6 709.6

Rel

a

650 700

m

unda

nce

C16:0

Rel

ativ

e Io

n A

buR

650 700

m

C18:0

sphingolipid subspecies via recursor Ion Scans

Q1

750 800 850

m/z

(SM)Pre 184

( )

C24:1

750 800 850

m/z

C22:0C24:1

C2:0

Precursor(25 eV)

ce C24:1

abun

danc

C24:1

300 400 500 600 700

Precursorlativ

e io

n a

Precursor(45 eV)

re

C2:0

300 400 500 600 700

D. Quantitation of Sphingolipids

Comparison of ion abundance for ceramides of varying chain length

when analyzed under singlewhen analyzed under single ionization and dissociation

conditions vs optimized MRMC2:0

C24:1

MRM

300 400 500 600 700m/z

Criteria for selection

1.1. Must have the same chemMust have the same chemthe analyte of interest ideathe analyte of interest ideathe analyte of interest, ideathe analyte of interest, ideaanalogs.analogs.

2.2. Should be practical for “omShould be practical for “omppsubspecies as possible besubspecies as possible bestandard for every analyte standard for every analyte molecules to be synthesizemolecules to be synthesizemolecules to be synthesizemolecules to be synthesizetoo expensive, time consumtoo expensive, time consumimpossible.impossible.

LIPID MAPS Sphingolipidomics cLIPID MAPS Sphingolipidomics cPolar Lipids): 10 uncommon sphiPolar Lipids): 10 uncommon sphip ) pp ) pspike samples prior to extraction (spike samples prior to extraction (

of internal standards

ical and physical properties as ical and physical properties as ally stable isotope labeledally stable isotope labeledally stable isotope labeled ally stable isotope labeled

mic” analysismic” analysis----i.e., cover as many i.e., cover as many yy , y, ycause adding an internal cause adding an internal would require 100s to 1000s of would require 100s to 1000s of

ed added and analyzed which ised added and analyzed which ised, added and analyzed, which is ed, added and analyzed, which is ming and possibly analytically ming and possibly analytically

cocktail (available from Avanti cocktail (available from Avanti ingolipid species that are used to ingolipid species that are used to g p pg p p(Walt Shaw)(Walt Shaw)

LIPID MAPS internal stFor sphingoid bases: odd chain length vFor sphingoid bases: odd chain length vconditions so there is little ionization or dconditions so there is little ionization or dproduct ion masses are slightly shiftedproduct ion masses are slightly shiftedproduct ion masses are slightly shifted.product ion masses are slightly shifted.

d17:1 “sphingosine” and “sphingosind17:1 “sphingosine” and “sphingosind17:0 “sphinganine” and “sphinganid17:0 “sphinganine” and “sphingani

For complex sphingolipids: shorter fatty For complex sphingolipids: shorter fatty l t ith l t f i t t thl t ith l t f i t t th

p g p gp g p g

coco--elute with analytes of interest so thereelute with analytes of interest so theredifferent precursor ion masses but similadifferent precursor ion masses but simila

C12C12 C C25C C25 C C12C C12 CC 11 P CP CC12C12--Cer, C25Cer, C25--Cer, C12Cer, C12--CerCer--11--P, CP, C

Also available:Also available: C12C12 SulfatideSulfatideAlso available: Also available: C12C12--SulfatideSulfatideUnder development:Under development: C12C12--GM1 and otheGM1 and othe

C17:1 sphingosylpC17:1 sphingosylp

tandard cocktail (cont.)variants that elute under similar variants that elute under similar dissociation effects and precursor and dissociation effects and precursor and

ne 1ne 1--phosphate” homologsphosphate” homologsne 1ne 1--phosphate” homologsphosphate” homologs

acid chain length variants (C12:0) that acid chain length variants (C12:0) that i i ti ff t d hi i ti ff t d h

p p gp p g

e are no ionization effects, and have e are no ionization effects, and have ar fragmentation when optimized.ar fragmentation when optimized.

C12C12 Gl C C12Gl C C12 L C C12 SML C C12 SMC12C12--GlcCer, C12GlcCer, C12--LacCer, C12 SMLacCer, C12 SM

er complex glycosphingolipidser complex glycosphingolipidsphosphocholine; Nphosphocholine; N--methylmethyl--sphingoid basessphingoid bases

5.00E+08

6.00E+08Ceramides

5.00E+08

6.00E+08Ceramides

3.00E+08

4.00E+08C12:0C16:0C18:0C24:0C24:1C25:0ea

(cps

)

3.00E+08

4.00E+08C12:0C16:0C18:0C24:0C24:1C25:0ea

(cps

)

1.00E+08

2.00E+08

C25:0

Are

1.00E+08

2.00E+08

C25:0

Are

0.00E+000 100 200 300 400 500 600 700 800 900 10

pmol on column

4.00E+

4.50E+

0.00E+000 100 200 300 400 500 600 700 800 900 10

pmol on column

4.00E+

4.50E+

2.50E+

3.00E+

3.50E+a

(cps

)

2.50E+

3.00E+

3.50E+a

(cps

)

1.00E+

1.50E+

2.00E+

Are

a

1.00E+

1.50E+

2.00E+

Are

a

0.00E+

5.00E+

0.00E+

5.00E+Analyzed by LC MS/MS using an ABI 4000 QTrap

Comparison of MRM for pceramides and

dihydroceramides of varying chain length compared tochain length compared to C12-Cer internal standard

000

+08

+08Dihydroceramides

000

+08

+08Dihydroceramides

+08

+08

+08

C16:0C18:0

+08

+08

+08

C16:0C18:0

+08

+08

+08 C24:0C24:1

+08

+08

+08 C24:0C24:1

+00

+07

0 100 200 300 400 500 600 700 800 900 1000

pmol on column

+00

+07

0 100 200 300 400 500 600 700 800 900 1000

pmol on column

E. Data analysis/visualization:

Example daChanges in Cer

Basic LIPID MAPS Protocol

Add agentPlate cells

Add agent (LPS, Kdo2 Lipid A)

GrowAnalyze

Pre-treatments (if any)

Incubationy

16:018:0

20:022:0

24:124 0N-acyl chainLIPID MAPS 24:0

26:126:0

N acyl chain subspecies

LIPID MAPS

TM

ata from Lipid MAPS experiments:in RAW 267 cells treated with Kdo2 Lipid A ( li id )

60

A)

(see www.lipidmaps.org)

ide/

ug D

NA

30

mol

Cer

ami

12 24

0

(pm

0.5 1 2 4 8 12 24

0 0.5 1 2 4 8 12 24

CORE I: Sp(only C16

Biological Replic3-Ketosphinganine (below LOD)

Serine + Palmitoyl-CoA

Sphinganine

Sphinganine 1-P

DataData ww

Sphingosine

Sphingosine 1-P

C16DHSMphingolipids 6 shown)cates +/- SE

C16DHCer

C16DHGlcCer

Sets Available at:

C16SM

Sets Available at:ww.lipidmaps.org

(Eoin Fahy)

C16Cer

C16GlcCerC16GlcCer

C16Cer-P

KDO2

Control

F. Remaining challenges (an

Discovery of new subspeciesubspecies)--such as

O fObtain standards for glycosp

D t i h t b tt iDetermine how to better visumultiple classes of sp

Develop methods to differenof particular subspeciof particular subspeciturnover.

nd opportunities)

es (and new functions for known s N-methylsphingoid bases

( )phingolipids (and new sphingolipids)

li h i b d iualize changes in abundances in phingolipids over time.

ntiate appearance/disappearance ies via de novo biosynthesis vsies via de novo biosynthesis vs

Example of Lipid MAPS timecoourse data set for sphingolipids(see www.lipidmaps.org)( p p g)

ex SL

gosine

Relational depiction ofComplex

N-acyls

phingo

(Cera

mides)

Relational depiction ofsphingolipid biosynthesis

mplex DHSL

phinganines

ramides

)

18:0

SaP

16:0

Me

Comp

a

N-acyls

ph

(Dihyd

rocera

nine SaP

22:0

24:0

26:1

Sa

26:0 MeSa

N-acylK

etoSa

oSa

Sphingan

in

24:1

24:0

18:0

20:0

16:0

26:1

KSa

Serine +

PalCoA

KetoS

22:0

24:124:0

26:1

KSa

Palmitoyl-CoA

nesHow to display so much information?

18:0

16:0

SoP

HŌenalMe3So

Me2So

Me26 0

Expand as additional

20:0

22:0

24:0

26:1

So

So26:0 additional species are analyzed

24:1HŌanalMe3Sa

Me2Sa

20:0

1

16:0

-Glc

-Gal

-Phosphorylcholine

So

Key

Sphingoid base(KSa, Sa, So, Etc.)

N-acyl-sphingoid base(Fatty acid carbon #:db)

(Sphingomyelin )

-Phosphate(Ceramide 1-P)Me3

Sa

Sphingoid base1-phosphate

N(Men) sphingoid base

Fatty acyl-CoA or fatty aldehyde (HŌanal or enal)

SoP

HÕenal

Ser + PalCoA

3-KetoSa

Sa

GlcDHCer DHCer DHSM

GlcCer Cer SMFenretinide

16

1826

2026:1

SaP

Sa

KSa

22

24:1

24

KSa

D24:1

DHGlcCe

16

1826

SoP

2026:1

SoP

So

22

24:1

24

SM

GlcCer

Zheng et al

DHSM Fold change

Zheng et al., 2006

er0.1 1.0 105.00.5

F. Remaining challenges (an

Discovery of new subspeciesubspecies).

O fObtain standards for glycosp

D t i h t b tt iDetermine how to better visumultiple classes of sp

Develop methods to differenof particular subspeciof particular subspeciturnover.

nd opportunities)

es (and new functions for known

phingolipids.

li h i b d iualize changes in abundances in phingolipids over time.

ntiate appearance/disappearance ies via de novo biosynthesis vsies via de novo biosynthesis vs

13CAnalysis of sphingolipid bi th i i t blbiosynthesis using stable

isotope labeled precursors

RAW 10% time course w/[13C]palmitate

13C D0.1 mM [13C]palmitate

Ser + PalCoA

3 K t S3-KetoSa

Sa

Gl DHC DHC DHSMGlcDHCer DHCer DHSM

GlcCer Cer SM

25

C BASE Ceramide (labeled sphingoid base only)

1015

20

25

ug D

NA

)

C16 C18

C22

r hr 5hr

l 1hr

rol 2

hr

ntro

l 4hr

ontro

l 8hr

Con

trol 1

2hr

Con

trol 2

4hr 0

510

(pm

ol/

DUAL Ceramide (13C in sphingoid base and fatty acid)

C24 C26

0hr

0.5h

r 1hr 2h

r 4hr 8h

r 12h

24h

Con

trol 0

.5

Con

tro

Con

t r

Con C

o C

30

40

50

NA

)

010

20

30

(pm

ol/u

g D

N

C16 C18

C22

C24

C26

0hr

0.5h

r 1hr 2h

r

4hr

8hr 12

hr 24hr

Con

trol 0

.5hr

Con

trol 1

hr

Con

trol 2

hr

Con

trol 4

hr

Con

trol 8

hr

Con

trol 1

2hr

Con

trol 2

4hr 0 (

To understand sphingolipid biosyavailability of the co-sub

100 499 4 NH

SO

499.4

NH

CH3H3COH

O

O

499

261.

MS/MS of C16:0 CoA

50N

NN

N

NH2

OO

PO

PO

33

OH

O

OHO428.0

C16:0-CoA

397.0428.0

(M + H)

NNO

OHOPO

OHHO

136

100 200 300 400 500 600 700 800 900 10000

136.0 261.1 (M + H)1006.4

m/zC16:0-CoA (10 pmol/µL in methanol 10 mM triethylammoparameters: IS = 5400 V, Gs1 = 12 psi, CUR = 10 psi, DP

ynthesis one must also know the bstrate fatty acyl-CoA’s.

Fatty acyl CoA profile of RAW cells

9.4

1

0 3n=7)

Fatty acyl-CoA profile of RAW cells (and for comparison, MCF7 cells)

2624

14541620

2159

0.2

0.3

6 ce

lls (m

ean

+ S

E, n

RAWMCF7

+

6.0404

514

286

6 3 8 18 13187

967

671

236401

871

0

0.1

C14:0 C16:0 C18:1 C18:0 C20:0 C22:0 C24:1 C24:0 C26:0

Molecular Species

pmol

per

10*

6

0

+

onium acetate) was infused at 5 µL/min. 4000QTrapP = 180 V, CE = 52 V, CXP = 14.3 V, CAD = Med

G. Examples of discoveries from th f de novo sphingolipid

Lipid A (has been coshowing increase

thus far

showing increaseBasic LIPID MAPS Protocol

Add agentPlate cells

Add agent (LPS, Kdo2 Lipid A)

GrowAnalyze

Pre-treatments (if any)

Incubationy

16:018:0

20:022:0

24:124 0N-acyl chainLIPID MAPS 24:0

26:126:0

N acyl chain subspecies

LIPID MAPS

TM

sphingolipidomic analysis

biosynthesis is induced by Kdo2orrelated with gene array dataes in SPT1 and SPT2 mRNA)

60

A)

es in SPT1 and SPT2 mRNA)

ide/

ug D

NA

30

mol

Cer

ami

12 24

0

(pm

0.5 1 2 4 8 12 24

0 0.5 1 2 4 8 12 24

Sphing3-KetosphinganineSerine + Palmitoyl-CoA

Number of molecules

CH3(CH2)10CH2

Palmitoyl CoA 11<0.1

d18:0;C16:0DHSM

d18:0;C16:0

DHCer-Pd18:0;d18 0

<0.1

37819.5

DN

A

OH

H

OH

OH

OH

O

d18:0;C18:0Cer

d18:0;C16:0DHCer

d18:0;C16:0

d18:0;C16:0

d18:0;C16:0

1-O-AcylDHCer

d18:0;C20:0DHCer

d18:0;C2:0

DHCerd18:0

d18:0-P<0.1

3 913.5

11.7

l/

CeramideHOH

H

H

OOS

O

O-

SulfatideC 6 0GalDHCer

d18:1;C16:0

SM

GlcDHCer

d18:1;C16:0Cer-P

d18:1;C2:0Cer

All subspecies(only a fe

<0.13.9

<0.1 29.7 211.5

19.5

1023315

Sulfatide (3'-sulfogalactosylceramide)

d18:1;C18:0Cer

d18:1;C18:0

Cer Pd18:1;C16:0

1-O-AcylCer

d18:1;C20:0Cer

d18:1

d18:1-Pd18:1;C16:0

<0.1d18:1;C16:0

d18:1;C16:0Cer30.3

117

C18:0GlcCer

d18:1 P C16:0GalCer<0.1

LacCer <0.1[<1]

C16:0GlcCer30 23.4 Kdo2

Sulfatide

ganine (d18:0) Sphinganine 1-phosphate

per RAW cell (million)

H CH2OHHO

NH3 (+)

(d18:0-P)

C

.7 3.9

Sulfatides in RAW cells treatedCeramide synthases+ Fatty acyl-CoA

Sulfatides in RAW cells treatedwith Kdo2 Lipid A

g D

NA 150

200

+Kdod18:0;C22:0DHCer

d18:0;C24:1DHCer

d18:0;C24:0DHCer

d18:0;C26:1DHCer

d18:0;C26:0DHCerpm

ol/µ

50

100+Kdo2

s have been quantified for these chain lengths, tooew are shown; all are at www.lipidmaps.org)

13587 294 6.3 1.50.30.315.37.5.3Time (h)

00 6 12 18 24

-Kdo2

d18:1;C22:0Cer

d18:1;C24:1Cer

d18:1;C24:0Cer

d18:1;C26:1Cer

d18:1;C26:0Cer

11 7 23 4 36 9 1 5 0.611.7 23.4 36.9 1.5

2 Lipid A385 (exact quantitation still in progress)

G. Examples of additional discovanalysis thus far

Sphingosine-1-phosphate phosphohydrolasgolgi trafficking of ceramide. Giussani PS, Wang E, Kelly S, Merrill AH Jr, Milstien SJul;26(13):5055-69.

Glucosylceramide synthase is an essential rCryptococcus neoformans Rittershaus PCryptococcus neoformans. Rittershaus PJr, Hennig M,Luberto C, Del Poeta M. J Cli

Effects of sphingosine-1-phosphate and cersmooth muscle cells: implications for pKelly S, Wang E, Merrill AH Jr, Kalff JC, vaSep;20(11):1930-2.

Ceramide kinase utilizes ceramide providedLocalization to subcellular compartmenLocalization to subcellular compartmenStahelin RV, Wijesinghe DS, Maceyka M, WChalfant CE. J Lipid Res. 2007 Mar 27; [Ep

Sphingomyelinase Restricts the Lateral DiffFi CM R t SS Ch EH G iffFinnegan CM, Rawat SS, Cho EH, Guiffre Virol. 2007 Mar 7; [Epub ahead of print]

veries from sphingolipidomic

se regulates endoplasmic reticulum-to-P, Maceyka M, Le Stunff H, Mikami A, Lepine S, Spiegel S. Mol Cell Biol. 2006

regulator of pathogenicity of PC Kechichian TB Allegood JC Merrill AHPC, Kechichian TB, Allegood JC, Merrill AH in Invest. 2006 Jun;116(6):1651-9. ramide-1-phosphate on rat intestinal

postoperative ileus. Dragusin M, Wehner S, anEchten-Deckert G. FASEB J. 2006

d by ceramide transport protein. nts of eicosanoid synthesis. Lamour NF,nts of eicosanoid synthesis. Lamour NF, Wang E, Allegood JC, Merrill AH Jr, Cho W, pub ahead of print] fusion of CD4 and Inhibits HIV Fusion.DL L k tt S M ill AH J Bl th l R JDL, Lockett S, Merrill AH Jr, Blumenthal R. J

H. Comparison of these methodt h i i th lit ttechniques in the literature

"Shotgun" techniques: Use the sShotgun techniques: Use the sscans - Great for profiling, nionization suppression, isotointerferences especially with

Nanospray ionization: Greatly imp y ychemical noise: allows detedetailed structural analyses systems can be coupled to Lsystems can be coupled to L

Ultra high resolution mass analyi b i / i i i fisobaric / isotopic interferenctechniques.

s with other sphingolipidomic

same precursor ion / neutral losssame precursor ion / neutral loss ot quantitation, suffer from

opic, isobaric, and isomeric hout hydrolysis and extraction.

mproved sensitivity and reduced p yection of low abundance species, on numerous species, chip-based

LC and fraction collectionLC, and fraction collection.

ysis: allows differentiation of d l i f ices and alternative fragmentation