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
Implications for
disease:
• Energy excess
/deficit
• Aberrant cell
structure/function
• Transport defects
• Incorrect signaling
• Developmental
abnormalities
Gene
mRNA
Protein
Post-translational
modification(s)
Metabolites
Cellular functions
Membrane (lipid)
functions:
• Energy
• Structure
• Transport
• Signaling
Agonists & other extracellular signals,
including nutrients & xenobiotics
Membrane lipids and regulation of cell structure/function
LIPID MAPS
LIPID MAPS
Lipid 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 to
quantitate 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
Macrophage Biology
PI
Edward A. Dennis - UCSD
Christopher Glass - UCSD
LC/Mass Spec
Robert C. Murphy - Colorado
Bioinformatics
Lipid Synthesis/Characterization
Michael VanNieuwenhze - UCSD
Walter Shaw - Avanti Polar Lipids
Steven White - UC Irvine
Shankar Subramaniam - UCSD
LIPID MAPS
Fatty Acids/Eicosanoids
Sterols
David W. Russell - UTSW
Edward A. Dennis - UCSD
Sphingolipids/Gangliosides
Alfred H. Merrill - Georgia Tech
Neutral Lipids
Robert C. Murphy - Colorado
Glycerophospholipids
H. Alex Brown - Vanderbilt
Other Lipids/Structural Lipidomics
Christian Raetz - Duke
Sphingolipids are the most structurally complex and diverse lipids of
eukaryotes
Glu cosylceram id e (GlcCer)
N-Acetylgalactosamine
Lactosylcera m id e (La cCer)
Galactose
Galactose
OH
HO
OH
HO
H
HO
O
O
AcNH
1 O
3
OH
OH
1 O
4
O
HO2C
H
2
O
1 O
H
OH
4
HO
O
1 O
OH
H
NH
1'
Cer
O
Ceramide (N-acylsphingosine)
O
HO
HO
OH OH
Glucose
OH
3
D-erythro-sphingosine
OH
OH
H N Ac
N-AcetylNeuraminic acid
GM3
GM2
GM1
(CH3)3NCH2CH2O-P(O2H)-O-Cer
Sphingomyelin
J. L. W. Thudichum
1884
SphinGOMAP©
(Download available at www.sphingomap.org)
MS/MS Methodology

Identify structure specific dissociations unique to various
classes SL’s (ie. Cer, GlcCer, LacCer, Gb3, and Gb4)

Utilize precursor ion & neutral loss scans to identify
individual headgroup, base, and fatty acid combinations
of endogenous SL’s

Optimize ionization and dissociation conditions for all SL’s

Quantify SL’s using internal standards and LC-MS/MS
OH
OH
Globoside Gb3
O
HO
OH
OH
OH O
O
O
HO
OH
OH
O
HO
O
OH
HN
O
OH
OH
O
HO
NH
OH
Globoside Gb4
OH
O
O
OH O
OH
OH
O
O
O
HO
OH
OH
O
HO
O
OH
HN
O
+Q1: 60 MCA scans from Sample 10 (Gb3) of mcs 0178.wiff (Turbo Spray)
Max. 9.1e6 cps.
(M2 + H)+
9.0e6
8.0e6
Gb3
7.0e6
* = (M + Na)+
(M3 + H)+
1136.9
(M1 + H)+
1150.9
1108.8
In te n sity , c p s
1152.9
6.0e6
*
5.0e6
1158.8
*
1109.9
*
1130.9
1175.0
4.0e6
3.0e6
2.0e6
1.0e6
1000
1020
1040
1060
1080
1100
m/z, amu
1120
1140
1160
1180
1200
4000 Q TRAP System – Ion Path
TM
Skimmer
Q0
Q1
Q2
Q3
LIT
Orifice
Curtain Plate
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.
604.8
7.0e5
6.5e5
6.0e5
(M1 + H)+
Quad Fragments
Quad Scan
5.5e5
In te n sity , c p s
5.0e5
4.5e5
4.0e5
d18:1
3.5e5
264.5
3.0e5
340u = C22:0
Glc
2.5e5
Gal
Gal
2.0e5
586.8
1091.0
1.5e5
928.9
1.0e5
5.0e4
135.1 252.1
100
200
767.0
282.3
300
574.6
400
500
600
m/z, amu
1108.8
831.1
700
800
900
1000
1100
Y2
OH
Y1
OH
O
HO
OH O
OH
OH
OH
OH
O
O
HO
Y0
O
HO
O
OH
HN
O
N"
+EPI (1108.80) CE (50): 119 MCA scans from Sample 14 (Gb3) of mcs 0178.wiff (Turbo Spray)
604.6
8.5e7
8.0e7
7.0e7
Max. 8.5e7 cps.
Quad Fragments
LIT Scan
(M1 + H)+
In te n sity , c p s
6.0e7
5.0e7
4.0e7
d18:1
3.0e7
264.2
Glc
Gal
340u = C22:0
2.0e7
Gal
586.6
1090.8
1.0e7
766.6
282.2
100
200
300
928.7
574.6
400
500
600
m/z, amu
1108.8
700
800
900
1000
1100
+EPI (1108.80) CE (50): 119 MCA scans from Sample 15 (Gb3) of mcs 0178.wiff (Turbo Spray)
604.6
8.0e8
(M1 + H)+
Quad Fragments
LIT Scan
w/ Q0 Trapping
7.0e8
6.0e8
In te n sity , c p s
Max. 8.0e8 cps.
5.0e8
4.0e8
Gal
d18:1
586.7
3.0e8
264.2
Gal
340u = C22:0
Glc
2.0e8
1090.8
928.7
766.7
1.0e8
252.2
100
200
282.2
300
574.7
659.4
340.3
400
500
600
m/z, amu
700
1108.7
830.8
800
900
1000
1100
4000 Q TRAP System – Ion Path
TM
Skimmer
Q0
Q1
Q2
Q3
LIT
Orifice
Curtain Plate
LINAC
Exit Lens
+MS3 (1108.70),(604.70): 93 MCA scans from Sample 24 (GB3) of mcs 0176.wiff (Turbo Spray)
2.7e8
2.6e8
Max. 2.7e8 cps.
264.2
MS/MS/MS
604.6
2.4e8
1108.7
2.2e8
In te n sity , c p s
2.0e8
1.8e8
1.6e8
1.4e8
1.2e8
1.0e8
8.0e7
6.0e7
604.6
4.0e7
2.0e7
252.2
340.3
282.2
364.3
574.6
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z, amu
+EPI (1136.90) CE (50): 119 MCA scans from Sample 19 (Gb3) of mcs 0178.wiff (Turbo Spray)
Max. 8.8e8 cps.
632.7
8.8e8
(M2 + H)+
8.0e8
7.0e8
In te n sity , c p s
6.0e8
368u = C24:0
5.0e8
4.0e8
d18:1
614.8
Glc
Gal
264.2
3.0e8
1118.8
956.8
2.0e8
794.7
1.0e8
602.8
282.2
252.2
100
200
400
1137.3
659.4 768.5
368.3
300
Gal
500
600
m/z, amu
700
800
825.8
900
1000
1100
+EPI (1152.90) CE (50): 117 MCA scans from Sample 25 (Gb3) of mcs 0178.wiff (Turbo Spray)
Max. 7.4e8 cps.
648.7
7.4e8
7.0e8
(M3 + H)+
6.5e8
6.0e8
5.5e8
630.8
In te n sity , c p s
5.0e8
4.5e8
4.0e8
3.5e8
3.0e8
384u = h24:0
2.5e8
Gal
Glc
Gal
2.0e8
1.5e8
618.8
d18:1
1.0e8
264.2
200
972.8
810.7
1152.8
666.7
5.0e7
100
1134.8
282.2
384.3
300
400
792.7
500
600
700
m/z, amu
800
900
1000
1100
Q1
scan m/z
Precursor Ion Scan
Q2
Q3
fixed m/z
+Prec (264.20): 179 MCA scans from Sample 11 (Gb3) of mcs 0178.wiff (Turbo Spray)
3.5e6
Max. 3.5e6 cps.
C24:1
1137.1
h24:0
d18:1
1153.1
C22:0
3.0e6
1109.2
h24:1
In te n sity , c p s
2.5e6
C24:1
2.0e6
1151.1
1135.1
1.5e6
h22:0
1.0e6
C16:0
5.0e5
1024.9
C18:0
C20:0
1125.1
1081.1
1053.0
1000
1020
1040
1060
1080
1100
m/z, amu
1120
1140
1160
1180
1200
+Q1: 30 MCA scans from Sample 2 (Gb4) of mcs 0179.wiff (Turbo Spray)
9.4e6
9.0e6
8.0e6
Max. 9.4e6 cps.
(M1 + H)+
Gb4
1356.0
* = (M + Na)+
(M1 + H)+
7.0e6
In te n s ity , c p s
*
*
1362.0
1379.0
1340.0
6.0e6
(M1 + H)+
5.0e6
1358.0
*
1333.9
1354.0
1377.0
1373.0
4.0e6
1311.9
1328.9
1349.9
3.0e6
1359.9 1375.0
2.0e6
1.0e6
1200
1220
1240
1260
1280
1300
m/z, amu
1320
1340
1360
1380
1400
Y3
OH
OH
O
HO
OH
Y2
Y1
OH
O
O
NH
OH O
OH
OH
O
O
HO
GalNAc
OH
OH
O
Y0
O
HO
O
OH
HN
O
N"
+EPI (1311.80) CE (60): 35 MCA scans from Sample 8 (Gb4) of mcs 0177.wiff (Turbo Spray)
Max. 1.0e8 cps.
604.6
1.00e8
(M1 + H)+
9.00e7
8.00e7
In te n s ity , c p s
7.00e7
6.00e7
5.00e7
340u = C22:0
4.00e7
3.00e7
586.6
d18:1
2.00e7
Gal
Gal
GalNAc
Glc
1090.7
928.7
264.2
766.6
1.00e7
144.0
100
204.1
200
300
1293.8
574.6
282.2
400
500
600
946.6
700
800
m/z, amu
900
1000
1100
1200
1300
+EPI (1340.00) CE (65): 105 MCA scans from Sample 4 (Gb4 c24:0) of mcs 0178.wiff (Turbo Spray)
Max. 5.4e8 cps.
632.6
5.4e8
(M2 + H)+
5.0e8
4.5e8
In te n s ity , c p s
4.0e8
3.5e8
3.0e8
d18:1
2.5e8
264.2
368u = C24:0
2.0e8
614.7
Glc
Gal
Gal
1.5e8
1.0e8
956.7
794.7
144.1 168.1
100
200
1118.8
602.7
204.1
5.0e7
GalNAc
282.2
368.3
300
400
1321.9
813.7
500
600
700
800
m/z, amu
900
1000
1100
1200
1300
+EPI (1356.00) CE (65): 104 MCA scans from Sample 8 (Gb4 h24:0) of mcs 0178.wiff (Turbo Spray)
Max. 5.7e8 cps.
648.6
5.7e8
5.5e8
(M3 + H)+
5.0e8
4.5e8
In te n s ity , c p s
4.0e8
3.5e8
3.0e8
384u = h24:0
2.5e8
Glc
2.0e8
d18:1
1.5e8
264.2
Gal
Gal
618.7
1.0e8
810.7
204.1
GalNAc
972.7
1134.8
5.0e7
144.1
126.0
100
252.2 282.2
200
300
666.7
384.3
400
500
600
1356.4
792.7
700
800
m/z, amu
900
1000
1100
1200
1300
+Prec (264.20): 90 MCA scans from Sample 3 (Gb4) of mcs 0179.wiff (Turbo Spray)
1.5e6
1.4e6
C24:0
Max. 1.6e6 cps.
1340.0
d18:1
h24:0
1356.1
1.3e6
1.2e6
C22:0
In te n s ity , c p s
1.1e6
1312.0
1.0e6
9.0e5
h24:1
1341.1
8.0e5
7.0e5
1354.1
1358.2
6.0e5
1313.1 1329.0
5.0e5
4.0e5
C16:0
3.0e5
2.0e5
1373.2
1338.0
C18:0
C20:0
1256.1
1284.1
1228.0
1.0e5
1200
1220
1240
1260
1280
1300
m/z, amu
1320
1340
1360
1380
1400
HO
LC ESI MS/MS
5
4
OH
HO
Sample In
(Autosampler)
3
N
O
OH
0
1
2
OH
O
HN
O
Mass Analyzer
(Q1, Q2, Q3)
Ion
Source
Inlet
System
HPLC
Detector
Relative Ion Abundance
SM
Data
System
LacCer
Cer
Data
out
GlcCer
0
2
4
6
Time (min)
8
10
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.
C24:0
9.0e4
Gb3
8.0e4
d18:1
h24:0
C22:0
7.0e4
In te n s ity , c p s
6.0e4
h24:1
5.0e4
h22:0
4.0e4
C24:1
3.0e4
2.0e4
1.0e4
0.0
C16:0
24
25
C20:0
C18:0
26
27
28
29
30
Time, min
31
32
33
34
35
XIC of +MRM (9 pairs): 1340.0/264.3 amu from Sample 6 (Gb 3/4 mix) of mcs 0184.wiff (Turbo Spray)
1.00e5
Gb4
9.00e4
d18:1
C22:0
C24:0
h24:0
8.00e4
In te n s ity , c p s
7.00e4
Max. 9.1e4 cps.
C24:1
C16:0
h24:1
6.00e4
C18:0
h22:0
5.00e4
C20:0
4.00e4
3.00e4
2.00e4
1.00e4
0.00
23
24
25
26
27
28
29
Time, min
30
31
32
33
34
35
Conclusions

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

LC in conjunction w/ MS/MS is a powerful tool for separation and
identification of complex mixtures of globosides

MS/MS/MS may be used for determination of specific
headgroup, long chain base, and fatty acid combinations in
globosides
Acknowledgements
Prof. Alfred H. Merrill, Jr.
Meeyoung Park
Anu Koppikar
http://www.sphingomap.org
Matreya
NIH/NIGMS/Lipid MAPS
http://www.lipidmaps.org