March, 2012

SNSB

7. CARBOHYDRATES/
GLYCOCONJUGATES
Biological Mass Spectrometry: Applications in the Post-Genome Era

Top fields in Systems Biology

Genomics
Chromati
n
Nucleolus

Nuclear envelope
Nucleus
Plasma membrane

DNAs
Smooth ER
Cytosole
Lysosome
Mitochondrion

Proteins
Centrioles
Centrosome matrix

Rough ER

Carbohydrates

Proteomics

Ribosomes
Golgi apparatus
Microvilli
Secretion being released from
cell by exocytosis

Microfilament
Microtubule
Intermediate filaments

Peroxisome

Glycomics

Cells communicate
to each other by their
carbohydrate
epitopes, covalently
liked either to lipids or
proteins

Definition

Glycoconjugates

Carbohydrates can be attached to another molecule (peptide, protein or

lipid) forming what's called a 'glycoconjugate'.
There are three different types of glycoconjugate classes:

glycoproteins

proteoglycans

glycolipids

Glycoproteins
Glycoproteins=proteins to which carbohydrates are covalently N- or
O-linked. The predominant sugars found in glycoproteins are
glucose, galactose, mannose, fucose, GalNAc, GlcNAc and NeuAc.
GalNAc

O-glycosylation

GlcNAc

N-glycosylation

N-Glycans

Glycoproteins

N-Glycans

Glycoproteins

Glycoproteins

Types of O-Glycans

Glycoproteins

GalNAc
Man

Fuc
GlcNAc

NeuAc

Glycoproteins

Types of O-Glycans

GalNAc
Man

Fuc
GlcNAc
NeuAc

Glycoproteins

GalNAc
Man

Fuc
GlcNAc
NeuAc

Glycoproteins

GalNAc
Man

Fuc
GlcNAc

NeuAc

Glycoproteins

GalNAc
Man

Fuc
GlcNAc

NeuAc

Glycoproteins

GalNAc
Man

Fuc
GlcNAc

NeuAc

Glycoproteins

GalNAc
Man

Fuc
GlcNAc
NeuAc

Strategy for glycoprotein analysis

Glycoproteins

Strategy for O-glycan analysis

Glycoproteins

Strategy for N-glycan analysis

Glycoproteins

Nomenclature for carbohydrate fragmentation*

Y2 Z 2

Y1 Z1
OH

OH

OH

O
HO

Y0

OH

OH

OH

OH

OH

OH

OH

B1 C 1

B2 C 2

B3
reducing end

non reducing end

OH
1,5 X

OH
5
4
HO

O
O

OH
3

bond 4
O
O

OH

bond 5

OH

1
2
OH

0,2

OH

A1

* B. Domon, C.E. Costello, Glycoconjugate J. 5 (1988) 397-409.

bond 0
OH

bond 1
OH

bond 3
OH
bond 2

Fragmentation scheme of linear oligosaccharides

Y4 Z4

Y3 Z3

Y2 Z2

Y1 Z1
1,5X

2,5A

B1 C1

2,4A

B2 C2

0,2A
3

Y4/B3
Z4/C3

B3 C3

B4 C4

ESI MS and tandem MS by CID conditions for detection and

sequencing of glycoconjugates
neutral oligosaccharides

sialylated oligosaccharides

- positive ion mode detection

-pH < 7
- cone voltage: 40-80 V
- capillary voltage: 900-1100V
- collision energy: ~ 40eV

- negative ion mode detection

- pH 7
- cone voltage: 20-50 V
-capillary voltage: 800-1000V
-collision energy: 30-50eV

glycosaminoglycans

glycopeptides

- negative ion mode detection

- pH 7
- cone voltage: up to 20 V
-capillary voltage: 600-800V
-collision energy: 10-30eV

- negative ion mode detection

- pH 7; pH < 7
- cone voltage: 20-50 V
-capillary voltage: 900-1100V
-collision energy: 20-70eV

- negative ion mode detection

- pH 7
glycolipids - cone voltage: 40-150 V
-capillary voltage: 900-1200V
-collision energy: 40-80eV

nanoESI chip-QTOF MS of a complex mixture of sialylated OGalNAc glycosylated peptides

2532.20

100

100

x4

NeuAc2GalGalNAc-Ser
NeuAc2Gal3 GlcNAc2GalNAc-Ser
NeuAc2GalGalNAc-Thr

2525.19

3812.32

2-

NeuAc4Gal3GlcNAc
GalNAc-Ser

NeuAc2GalGalNAc
NeuAc2Gal2GlcNAcGalNAc-Ser
NeuAcGal2GlcNAc -Thr-Pro
NeuAcGalGalNAc-Thr-Pro-H
2O
GalNAc-Thr
2NeuAc2Gal2GlcNAc
Fuc4Gal2GlcNAc3
707.79
GalNAc-Thr
GalNAc -Ser +Na
3NeuAcGalGalNAc-Thr-Pro
1088.40
834.33
NeuAc2GalGalNAc-Thr
Fuc
4 Gal2 GlcNAc3
891.34
834.67
12- 1- GalNAc -Thr +Na
871.36NeuAcGalGalNAc-Ser
21- 2- NeuAc4Gal3GlcNAc
923.87
897.75
NeuAc2GalGalNAc
1065.43
GalNAc-Thr
2834.97
931.36
532.71
NeuAcGalGalNAc-Thr
11- 708.29
898.25
13964.39
Fuc4Gal2GlcNAc3
853.36
839.33
NeuAcGal
714.79
2GlcNAc
NeuAc
854.32
2GalNAc
GalNAc
-Thr2Gal
+2Na
33GalNAc-Thr
872.37
839.67
2931.86
774.32
830.32
NeuAc
22- 2Gal2
901.34
841.99
NeuAc
GalGalNAc
829.65
2
963.40
1864.32
847.32
873.37
GalNAc-H
O
715.29
826.33
942.34
2
-Thr-Pro
901.83
2935.35
956.36
876.33 NeuAc GalGalNAc-Ser

autoMS/MS

819.34

580.75

533.23
820

1087.39

2-

717.62

812.66

7
810

x4

890.35

913.39

879.33

NeuAcGal
GalNAc

2- 830581.26 840
581.77

850

717.94

1-

860
718.79

1-

673.27

916.84

1051.42

autoMS/MS
775.33

870

776.32

880

890

900

2-

1-

910

890.35
834.33 871.36 891.34 923.87 964.39

920

951.35

1088.90
1089.41
1095.40
930 1098.40
940
950
1136.42

1- 1-

960

1184.47
1263.44

0
500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

Solvent: MeOH; ESI tip: 1.50 kV; sampling cone: 30-45 V; acquisition 1 min
average sample consumption: 0.2 pmols

1200

1250

AutoMS/MS of the doubly charged ion at m/z 897.79

100

x2

x2

2-

NeuAc-Gal-GlcNAc-Gal- GlcNAc
x4

GalNAc-Thr

752.38

NeuAc-Gal

Y 5 or Y 2
B1 or B1
1290.10

0,4A

Y 5/B 1
or
Y 2 /B 1

[M-2H] 2-

6/B 2

897.79

0,4A

Y 1/B 1

6/B 1
751.88

10,4A

6/B 3

B5

[M-2H] 2C 3

11424.15

2610.16

518.15

300

673.21
400

500

600

-CO 2
1789.23

1-

292.11

700

1-

Y 5 or Y 2

1214.38

898.24

609.17

291.13

483.18

0,4A

[M-2H] 21-H 2O

1081.34
1080.34

2-

11505.47

1215.40

1082.31
1224.36

2- 889.79
875.76

800

900

1000

1100

1200

1300

1400

Acquisition time 1 min; collision energy: 40 eV; average sample consumption: 0.2 pmols

1500

Determination of glycosylation site by (-) ECD FTICR MS

Y1
3Gal b 1

Z1
O

~ x 10

Y0
3GalNAc

[M+2H]+/
[M+H]+

[M+2H]2+

O
Z0
z 8*

z5

z6
S

z4
S

c4* c5* c6* c 7*

z8*

c9*

c7*

z6

Glycosylation site

Z0

Mucine type of
O-glycosylation

Z1

c9*

c6*

z5
z4

Y0

c4*

Y1
c5*

Proteoglycans

What are proteoglycans?

a special class
glycosylated

of

glycoprotein

heavily

consist of a core protein with one or more

covalently attached glycosaminoglycan chain(s)
the glycosaminoglycan chains are long, linear
carbohydrate polymers negatively charged
under physiological conditions, due to sulphate
and uronic acid groups

Proteoglycans

Proteoglycan structure

Proteoglycans

Proteoglycans with Leucine-Rich Repeats

Decorin
C C
C C

Leucine-Rich Repeat

CC

Cysteine

Biglycan
C C
C C

Chondroitin/
Dermatan Sulphate

CC

Fibromodulin
C C
C C

CC

Tyrosine Sulphate

Lumican
C C
C C

N-Glycan

CC

Keratan Sulphate

Proteoglycans

Tentative Structure of Decorin

DCN

Model of the decorin

The GAG attachment site
on Ser7 is in red. The Nlinked oligosaccharide
attachment sites are in
purple.

Proteoglycans

DECORIN
DECORIN
C C
C C

CC

Leucine-Rich Repeat

Cysteine
Chondroitin/
Dermatan Sulfate
N-Glycan

DERMATAN SULFATE

CHONDROITIN 6-SULFATE

H
COOH
O

H
O

OH

OH

OH

H
H

CH 2 OH

CH2 OSO3 H

H
H

NHCOCH3

SO 3H
O
H

COOH
OH
H
H

OH

O
O

H
H

NHCOCH 3

Proteoglycans
Structure of the repeating disaccharide units
of the major glycosaminoglycans

NHCOCH3

NHCOCH3

Chondroitin 6-sulfate

Dermatan sulfate

Keratan sulfate

NHCOCH3
NHCOCH3

Hyaluronate

Heparin

NHSO3-

Proteoglycans

Depolymerization by lyases
COOH

COOH

CH2 OSO3 H
O
O

OH

O
O

OH

CH2 OSO3 H
O

O OH

OH

OH

OH
OH

NHCOCH3

OH

NHCOCH3

Chondroitin lyases

Condroitinaz

COOH

CH2 OSO3 H
O
O

OH

COOH
O

O OH

OH

CH2 OSO3 H
O

OH

O OH

OH

OH
OH

NHCOCH3

Di-6S
Di-6S

OH

DDDi-6S
Di-6S

NHCOCH3

Proteoglycans

Recognition specificity of chondroitin lyases can be

used as a tool for identification of GlcA- and IdoAcontaining domains in CS-/DS
glycosaminoglycan chains
chondroitinase AC

chondroitinase B

...-GlcA-GalNAc(S)-GlcA-GalNAc(S)-IdoA-GalNAc(S)-...

Proteoglycans

Objectives of Mass Spectrometry

How to...
-obtain high ionization yield in GAG analysis?
-detect long and short chains in mixtures?
-identify sulfation grade?
-detect over- and under-sulfated regions?
-obtain a high coverage on diganostic sequence
ions?

Proteoglycans

Problems associated with the

ionization/sequencing of the GAG chains
lability of the sulfate group
in source loss of sulfate groups resulting in ions
wrongly attributable to non- or undersulfated species
overlapping of isobaric signals

contradictory sequencing conditions required for

correct structural analysis: cleavage of the glycosidic
bond while keeping the sulfate group attached

Proteoglycans

Nanoelectrospray MS and tandem MS conditions

for detection and sequencing of single components
in oligosaccharide mixtures

neutral
- positive ion mode detection
- water/pH < 7
- cone voltage: 40-80 V
- capillary voltage: 900-1100V
- collision energy: ~ 40eV

GAGs
- negative ion mode detection
- water/pH 7
- cone voltage: up to 20 V
-capillary voltage: 600-800V
-collision energy: 10-30eV

Proteoglycans

Strategy for the MS analysis

of GAG chains
a) purification on DEAE AEC
b) -elimination
c) depolymerization of GAG chains by lyases
d) separation of DS by GFC
e) collection of fraction
f) further separation according to the number
of sulfates
g) mass spectrometric analysis

Proteoglycans
Strategy for decorin GAG analysis based on the recognition specificity of
chondroitin lyases and chip ESI multistage MS
Decorin

GAG chain

B lyase

AC lyase

Chip ESI MSn

Proteoglycans
Fully automated (-) nanoESI chip HCT MS1
of decorin CS dissacharide obtained by GAG chain depolymerization
using chondroitin B lyase
Intens.

[M-H]458.11

M=IdoA-GalNAc
M=GlcA-GalNAc(1S)
(1S)

8000

[M-2H+Na]480.22
6000

nS =nSO3, number
of sulfate groups

4000

2000

200

300

400

500

600

700

m/z

Conditions: Solvent: MeOH/H2O/ACN; acquisition time 3 min;

Chip ESI: -0.55 kV; capillary exit: -50 V.

Fully automated (-) nanoESI chip HCT CID MS2

of the singly charged ion at m/z 458.17

B
B11 C C1
1
800

SO
SO
33

Y1300.15

B1

Diagnostic for
sulfation at GalNAc

157.06

400

458.17

4,5 DIdoA-O-GalNAc
4,5-GlcA-O-GalNAc

1000

600

[M-H]-

Y1YZ
1 Z1
1

Intens.

Proteoglycans

Z1-SO3

C1175.08

Y1-SO3
220.12

200

Z1
202.13 230.41

324.15

[M-H-SO3]-

282.14

378.16

0
200

250

300

350

400

450

m/z

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.3-0.6 V

Proteoglycans
Fully automated (-) nanoESI chip HCT MS1
of decorin DS dissacharide obtained by GAG chain depolymerization
using chondroitin AC lyase

Conditions: Solvent: MeOH/H2O/ACN; acquisition time 5 min;

Chip ESI: -0.50 kV; capillary exit: -50 V.

Fully automated (-) nanoESI chip HCT CID MS2

of the singly charged ion at m/z 538.11
-

300

250

200

Proteoglycans

Y1

Z1

C1

150

Diagnostic for
sulfation at GalNAc

100

Diagnostic for
sulfation at GlcA

50

0
500

1000

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.3-0.6 V

nanoESI MS of a depolymerized GAG mixture

Fig.4

IdoA-GalNAc-[GlcA-GalNAc]n
458.02
x4
100

hexa (5S)

611.28

585.03

4-

3-

octa(4S)

octa(4S)

octa(3S)

585.35

hexa(3S)

611.60

2-

552.66
552.91

517.70

3511.38

deca(5S)
546.90

5-

533.82
502.90

687.38

4-

517.38

459.16

3-

dodeca(5S)
deca(4S)

3-

4572.63

619.20

dodeca(4S)

687.87

4-

619.55
647.40
647.91
634.89

deca(3S)

3-

3- 679.37

711.89

664.89

712.92

m/z
460 480 500 520 540 560 580 600 620 640 660 680 700

Proteoglycans

D
DEECCO
OR
RIN
IN

C C
C

C C

L e u c in e -R ic h R e p e a t

C y s te in e
C h o n d r o itin /
D e r m a ta n S u lfa t e

depolymerization

N -G ly c a n

D E R M A TA N S U LFA TE

C H O N D R O IT IN 6 -S U L F A T E

H
COOH
O

H
O

OH

OH

H
H

OH

CH 2 OH

C H2 O S O3 H
O
H
H

H
O

OH

COOH
H
H

H
H

N H C O C H3

SO 3H
O

OH

O
H

H
H

NHCOCH

chondroitin AC-lyase
...- GlcA -GalNAc (S)- GlcA -GalNAc (S)- IdoA - GalNAc (S)-...
CS/DS GAG chain
Robot
Chip

Chip MS analysis
Separation by SEC

Proteoglycans
GFC elution profile of
GAG chain oligosaccharides after cleavage with lyase

collected

Proteoglycans
nanoESI chip MS of the collected octasaccharide fraction
[GlcA-GalNAc]4 (4S)

458.01
100

4-

[GlcA-GalNAc]4 (4S)
6305.00

458.26

[GlcA-GalNAc]4 (4S)

305.16

Octasaccharide completely
desulfated (low abundant,
4- at m/z 379.08)

5-

458.51

366.44
366.24
366.63

305.32

Trace of [GlcA-GalNAc]5
(5S) (low abundant, 6- at
381.52)

[GlcA-GalNAc]4 (3S)

458.76

366.84

459.01

3584.00

0
300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

Proteoglycans
nanoESI chip MS/MS of the ion at 458.02
x10

[M-4H]

5B4(2S) 458.02

100

2-

x4

4-

3584.35

2B8(3S)
Y7(4S)

439.29

379.16

Y3(2S)

C7(3S) B3(0S)
33-

3-

2-

Y6(3S)

611.21

B8(4S)

917.22

2-

917.72

687.21

C8(4S)
Y7(3S)

516.96 553.06

B 8(4S)
Y8(3S)

3418.75

617.34

B7(3S)

Y6(2S)

2-

458.27

3510.96

647.88

Z7 (2S)

Y7(2S)
2-

735.28

696.25

918.22

2-

687. 70

21-

C8(4S)

877.23

2775.34

Z8(2S)
2-

877.75
899.41

2-

837.27

1476.18

0
380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940

m/z

Proteoglycans

CE/UV profile of a fraction collected

from GFC
67

Absorbance

1.471
10-3

8 9 10
11

1
34

-0.368

0.00

Time (min)

6.5

Proteoglycans
100

0.75
39
780

3
0.67
35
670

Introduction of on-line CE/ESI MS for GAGs

CE buffer: 40mM ammonium acetate/ammonia pH 11.8. CE separation voltage 30kV direct
polarity, 6 s injection by pressure. CE column length 100 cm. Nanosprayer potential 600V,
sampling cone potential 12V. MS signal acquisition: 15 min after injection.

0.53
28
673

1.41
71
933

n- number of scans
m/z of the most abundant ion eluted at the moment t

0.26
14
588

t-elution time 15 min after injection

1.32
0.93 67
48 933
841

1.75
88
856

Time
0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Proteoglycans

Spectrum derived from the 7th peak

100

[M-5H]5100

933.12

933.32

933.52

933.72
933.92

934.13
934.33

933.12

4,5 DIdoA-GalNAc
{GlcA-GalNAc}9(11S)
calculated monoisotopic:
m/z 933.126
933.32
933.52

933.72

933.92
727.84

0
575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975 1000 1025 1050 1075

m/z
.

Proteoglycans

on-line CE/nanoESI MS/MS of the eicosasaccharide

bearing 11 sulfate groups
4,5 DIdoA-GalNAc
D 4,5[ HexA -HexNAc(11S)
(S)]11
{GlcA-GalNAc}
9

m/z (1400-1700) u.

100

[M-SO 3 -3H]

3-

1502.23
1528.88

[M-3H]

3-

Y 14 (7S)

1502.5

1529.20

Y 19 (10S)

B19(9S)
C 19 (9S)
%

3-

1502.9 1529.55

Y19(11S)
3-

1434.58

1469.66

1434.90

Z14 (7S)

C 14 (6S)

1605.38

1534.20

1470.1
1495.15
1489.28

1535.25

2-

1605.87

2-

2-

1565.40

2-

Z19(11S)

Y 15 (6S) Y 15 (7S)

1653.22

1596.37

1450

1500

1550

1600

1650

21693.35
1693.84

1700

m/z

Proteoglycans

Fragmentation scheme
of the eicosasaccharide and
localization of the sulfate groups
from MS/MS data

B1

B19

C1

C19

4,5D-IdoA-O-GalNAc- GlcA-GalNAc -GlcA-O-GalNAc-O

S
Y19

Z19

S
Y1 Z1

Polysaccharides
OH
HO
HO

OH
O
HO
HO

OH

OH

OH

OH

HO
OH

OH

HO

OH

OH

OH

HO
OH

n-2

HO
OH

n-2

H
N
3 NH 2

OH

NaBH3CN
H2N

OH
HO
HO

O
o

80 C, 2 days
DMSO
DMF
AcOH

OH
O
HO
HO

O
OH
O
HO
HO

n-2
O
OH
OH

OH

CH2 NH2
HO
HO

O
OH
O
HO
HO

O
OH
O
HO
HO

n-2
OH
OH

H
N
3 NH 2

I. Perdivara, E. Sisu, N. Dinca, K. Tomer, M. Przybylski, A. D. Zamfir, Rapid Commun Mass Spectrom. 2008

(+) ESI FTICR MS of long-chain polysaccharides

OH
O

HO
HO

OH

GlcnHMD [M+H]+

OH
O

O
HO

OH

*GlcnHMD [M+2H]2+

n
OH
OH

O
HO

# GlcnHMD [M+2H]2+ (+14)

H
N
3

OH

NH2

GlcnHMD [M+H]+ (+28)

*
Glc6HMD

< 1 ppm

Side reaction

Glc2HMD
*
Glc5HMD

*
Glc7HMD
Glc3HMD

*
Glc8HMD

Glcn NH

Glc5HMD

Glc6HMD

Glc10HMD

#
#

Glcn NH

H
N
O

*
Glc9HMD

DMF

Glc4HMD

443.2603

471.2553

NH2

*
Glc11HMD
*
Glc12HMD

*
Glc13HMD

Glc7HMD

*
Glc14HMD

500
600
700
800
900
1000
1100
1200
I. Perdivara, E. Sisu, N. Dinca, K. Tomer, M. Przybylski, A. D. Zamfir, Rapid Commun Mass Spectrom. 2008

m/z

HO
HO

(+) ESI FTICR MS of long-chain polysaccharides

OH
O

OH
O
HO

GlcnHMD [M+3H]3+
GlcnHMD [M+2H+Na]3+

O
HO

*Glc HMD [M+2H]2+

n

OH
O
HO
HO

OH

H
N

OH

*
Glc16HMD

Glc25HMD
26
*
Glc15HMD

*
17

NH2

*
Glc19HMD 30

31

36 Glc37HMD

Glc33HMD

34
32

Glc38HMD

Glc39HMD
Glc40HMD

35

29

Glc41HMD

* 28
18
27

Glc42HMD

1400

1600

1800

2000

2200

2400

m/z

I. Perdivara, E. Sisu, N. Dinca, K. Tomer, M. Przybylski, A. D. Zamfir, Rapid Commun Mass Spectrom. 2008

Fully automated (-) nanoESI chip HCT MS1

of polydisperse maltodextrins derivatized with aromatic amines
Intens.
Glc5-AGL

1011.36
Glc3-AGL

Average MW:
1800 Da

687.28
Glc4-AGL

Glc6-AGL

849.32

3000

1173.42

Glc2-AGL

525.25

2000
Glc7-AGL

Glc10-AGL

1335.49

1821.55

Glc11-AGL

1983.47

Glc8-AGL

1000

1497.55
Glc9-AGL

1659.57

600

800

1000

1200

1400

1600

1800

2000

Conditions: Solvent: H2O; concentration 10 pmol/L; acquisition time 2 min;

Chip ESI: -0.60 kV; capillary exit: -20 V.

m/z

Fully automated (-) nanoESI chip HCT MS1

of polydisperse maltodextrins derivatized with aromatic amines
Intens.
x105

Glc7-AGL

1335.67

MS2

OH
O
OH
O
OH
OH

OH
O
OH
OH

O
n

OH
OH
OH

NH

CH2

NH2

OH

Glc6-AGL

1173.56
Glc8-AGL

AGL

1497.74

Area m/z: (800-2400)

4

Glc4-AGL

Average MW:
2800 Da

849.43
Glc5-AGL

1011.50

Glc9-AGL

1659.79
2
Glc10-AGL

1821.84

Glc11-AGL

1983.88
0
800

1000

1200

1400

1600

1800

2000

2200

m/z

Conditions: Solvent: H2O; concentration 10 pmol/L; acquisition time 2 min;

Chip ESI: -0.60 kV; capillary exit: -20 V.

Fully automated (-) nanoESI chip HCT MS1

of polydisperse maltodextrins derivatized with aromatic amines
Area m/z: (2100-3000)
Glc12-AGL

8000

2146.00

OH
O
OH
O
OH
OH

6000

OH
O
OH
OH

OH
OH
OH

NH

CH2

NH2

OH

AGL
4000

Glc13-AGL

2307.00
2000
Glc14-AGL

2469.40

Glc16-AGL

Glc15-AGL

2794.00

2632.00
0

2100

2200

2300

2400

2500

2600

2700

2800

Glc17-AGL

2956.20
2900

m/z

Conditions: Solvent: H2O; concentration 10 pmol/L; acquisition time 2 min;

Chip ESI: -0.60 kV; capillary exit: -20 V.

Fully automated (-) nanoESI chip HCT CID MS2

of the singly charged ion at 1173.46 corresponding to Glc6-AGL
Intens.
1173.46

6000

[M+H]+

MS3
4

5000

B3
487.21

Y1

4000

[M+H]+-H2O

363.17

1155.46

B5

3000

453.18

506.06

2000

Y2

525.23

B4

Y3

Y5

Y4

747.28

1011.37
1071.40
B4

849.33

687.28

649.27
1000

768.26 B3
811.33

471.16
585.19
0

300

400

500

600

700

800

909.36
930.83
900

1000

1100

1200 m/z

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.5-0.8 V

Fully automated (-) nanoESI chip HCT CID MS3

of the singly charged ion detected in MS2 at 363.20
238.20

Intens.

-H2O
-H
O
2
274.00
256.00

201.00
184.90

103.20

[M+H]+-H2O
345.00

[M+H]+
363.20
3

103.20

201.00

256.00

-NH2
2

184.90

238.20

274.00
0

50

100

150

200

250

300

350

400

450

m/z

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.2-0.4 V

Glyco(sphingo)lipids:
...contain one carbohydrate epitope per molecule
...of the same monosaccharide sequence show
conformational diversity according to the
specificity of glycosidic linkages
...their carbohydrate chains conformational
degrees of freedom are restricted according to the
linkage sites (e.g.3- vs. 6-) and steric hindrance
(e.g.3- vs. 4-)

Glycosphingolipids:
...are organized in cell surface lipid
microdomains (rafts) to associate with specific
signaling molecules
...show specificity of expression in different
normal and pathological states according to their
carbohydrate epitopes

Glycosphingolipids:
...are to be analyzed to characterize their
composition regarding the carbohydrate and the
lipid moiety, their way of attachment/sequence,
patterns of branching and the anomericity of the
linkages

...the task for analytical services is to identify

already known and still unknown molecular
species by mapping and de-novo sequencing
...mass spectrometry is the most sensitive and
accurate analytical method for this task
(Methods Enzymol. 193, 1990; Mass Spectrom.
Rev. 1994).

Gangliosides
...are sialylated glycosphingolipids organized in cell surface lipid
microdomains (rafts) to associate with specific signaling molecules
...show specificity of expression in different normal and
pathological states according to their carbohydrate epitopes
....have the highest abundance in central nervous system, being
biomarkers of brain disorders, neurodegeneration and cancer
...are to be analyzed to characterize their composition regarding
the carbohydrate and the lipid moiety, their way of
attachment/sequence, patterns of branching and the anomericity of
the linkages
...the task for analytical services is to identify already known and
still unknown molecular species by mapping and de-novo
sequencing

Gangliosides

GM1a

Neu5Ac3Gal4GlcCer GM3
GalNAc4(Neu5Ac 3)Gal4GlcCer GM2
Gal3GalNAc4(Neu5Ac 3)Gal4GlcCer GM1a
Neu5Ac 3Gal3GalNAc4Gal4GlcCer GM1b
Neu5Ac 8Neu5Ac 3Gal4GlcCer GD3
GalNAc4(Neu5Ac 8Neu5Ac 3)Gal4GlcCer GD2
Neu5Ac 3Gal3GalNAc4(Neu5Ac 3)Gal4GlcCer GD1a
Gal3GalNAc4(Neu5Ac 8Neu5Ac 3)Gal4GlcCer GD1b
Neu5Ac 8Neu5Ac 3Gal3GalNAc4(Neu5Ac 3)Gal4GlcCer GT1a
Neu5Ac 3Gal3GalNAc4(Neu5Ac 8Neu5Ac 3)Gal4GlcCer GT1b
Gal3GalNAc4(Neu5Ac 8Neu5Ac 8Neu5Ac 3)Gal4GlcCer GT1c
Neu5Ac 8Neu5Ac 3Gal3GalNAc4(Neu5Ac 8Neu5c 3)Gal4GlcCer GQ1b

2-8, 2-3-Disialolactosylceramide (GD3)

Gal
Glc
OH
4
1

9
8

O
HO

OH
OH

O
6

OH
NH

2
3

8
5

OH
NH

11

Neu5Ac

11

5
6

Fatty acid

HN

O
OH

Sphingosine

Neu5Ac

OH
10

OH

OH
10

OH

HO
O

O
2

OH

OH

O
HO

OH

Ceramide

Normal brain histology.

Section of the human cerebellum

Neurohistopathological features
of Glioblastoma multiforme/gliosarcoma

nanoESI
chip-QTOF
MS species
of the gliosarcoma
Could
a GM1
(d18:1/18:0)
be present?ganglioside
Calc.M-H=mixture
m/z1544.86
100 x2.5
1179.55

1470.97

GM3
(d18:1/20:0)

GD3
(d18:1/20:0)

1265.45

1526.92

GM2
(d18:1/20:0)

(d18:1/18:0)

1629.80

GD1
(d18:1/18:0)

1756.55

GD2
GD2
(d18:1/22:0) (d18:1/24:0)
1757.53

GM1
(d18:1/20:0)1673.64

1382.15

1835.35

1554.91
1383.16

1181.53

1512.89

1918.26
1758.52

1630.79

1464.10
1263.41

1914.20

1675.66

1462.08

1354.20

GD1
(d18:1/24:1)
GD1
(d18:1/24:0)

1674.65

1235.49

1207.48

GD2
(d18:1/24:1)

GM1
1628.80 (d18:1/24:0)
GD2

GD3
(d18:1/24:0)

GM2
(d18:1/16:0)
1263.44

1553.89

1472.01

GM2
(d18:1/18:0)

1180.56

x12

O-Ac-GD3 O-Ac-GD3
(d18:1/18:0) (d18:1/20:0)

1540.88

GM3
(d18:1/24:0)

x6

GD3
(d18:1/24:1)

GM3
(d18:1/22:0)

GM3
(d18:1/
16:0)

1552.92

GD3
(d18:1/18:0)

GM3
(d18:1/18:0)

1264.44

x3

1919.24

1992.53
1889.28 1920.22

1759.51
1729.58

0
1150

1200

1250

1300

1350

1400

1450

1500

1550

1600

1650

1700

1750

1800

1850

Solvent: MeOH; ESI tip: 1.60 kV; sampling cone: 80 V; acquisition 2 min
average sample consumption: 0.5 pmols

1900

1950

2000

1540.88
100

O-Ac-GD3
(d18:1/20:0)

m/z 1544.86 GM1?

1541.85

?
%

1542.87

1543.84
1539.04
0
1539

1540

1541

1542

1543

1544

1545

1546

1547

1548

290.12

nanoESI chip-QTOF
MS/MS of the singly charged ion at m/z 1544.86

x10

100

B1
B1

GM1 (18/18)

Y0

(GD3
18/20)
(Na)
YB2
0
(GD3 18/20) Y

888.77

Z1

364.25

179.16
202.20

603 (Na)

GD3 (18/20)
NeuAc-NeuAc-Gal-Glc-Cer

726.69

C4

889.77

916

290

603
592

565.77

2,4A
291.15

Y2 (GD3 18/20)

Y1

GalNAc -

888 726/ 564

708

1253 1091

GalGalNAc -

833 995

NeuAc-Gal-GalNAc-Gal-Glc-Cer

564.79

Hex -

364

290

424.38

3/B 1

708.68

562.81 603.01

332.22

727.70

833.52

728.71

890.76

916.81

C5 Y3
995.52

Y4

1091.84

[M-H] 1544.89

1253.79

466.39

0
200

300

400

500

600

700

800

900

1000

1100

1200

1300

Solvent: MeOH; ESI tip: 1.60 kV; collision energy: 45-85 eV;
signal acquisition 50 min. Average sample consumption 15 pmols

1400

1500

290.09
100

x16

B1
B1

x6

x4

1544.93

x10

MS/MS of the singly

charged ion at m/z 1540.88

281.24

[M-H]-

GalNAcB2
Hex-

M= O-Ac GD3 (18/20)

M= GM1 (18/18)

581.20

-H2O
161.03
%

255.22

-CO2
GalGalNAc-

1540.96

1253.84

B2(Na)

220.08

[M-H]-

Y4
701.54

***
Y (Ac)

179.04

-H2O

1249.84

**

Y3

582.19
202.06

419.25
437.29 537.20
290.35

B2 (Ac)

Y2

702.53

-H2O

888.68

623.22

364.13

465.32

729.54

-Ac

1073.71
1207.74
1091.67

851.32

0
200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

m/z

Fully automated (-) nanoESI chip HCT MS1

of An28 native ganglioside mixture from glial islands of anencephalic fetus
Intens.
x10 4

1063.72

MS2
2.0

917.60
1.5

1.0

1671.11
1259.92
1077.73

1237.90 1249.95

1279.88

1519.06

1653.21
1553.07

1179.90

735.53

1918.11

1139.01
0.5

1353.03
1375.03

931.72
836.68

1049.26

1207.01

1756.01
1544.16

1471.03

1757.51

1858.32
1885.08

1572.02

0.0
800

1000

1200

1400

1600

1800

Conditions: Solvent: MeOH; sample concentration 5 pmol/L; acquisition time

10 min; Chip ESI: -0.8 kV; capillary exit: -50 V.

2000

m/z

Fully automated (-) nanoESI chip HCT MS1

of FL27 native ganglioside mixture from normal fetus frontal lobe
Intens.
x10 4

1836.20

917.58
2.5

6000

1858.20
4000

1990.50

2000

2.0

1.5

1700

1750

1800

1850

1900

1950

2000

2050

1383.21
1139.01

1037.60

1.0

931.72

1041.60
1049.18
1063.33

851.60

952.80

0.5

1354.79

1151.71
1165.80
1167.82

1301.82
1279.81
1259.79

1179.74
1181.75

1065.63

1206.77
1077.71
1104.78

1471.03

1221.33
1235.81

735.52

836.71

1444.80

1544.20

0.0
800

1000

1200

1400

1600

1800

Conditions: Solvent: MeOH; sample concentration 5 pmol/L; acquisition time

10 min; Chip ESI: -0.8 kV; capillary exit: -50 V.

m/z

Comparative overview upon gangliosides and

asialo-gangliosides detected in An28 and FL27 mixtures by NanoMate/HCT
GG species

Proposed structure

An28

FL27

GM1

nLM1 and/or LM1 (d18:0/16:0)

nLM1 and/or LM1 (d18:1/18:0)

nLM1 and/or LM1 (d18:0/20:0)

(d18:1/16:0)

(d18:1/18:0)

(d18:1/22:0)
(d18:1/14:0) or (d18:1/h14:0) or HexNAcHex2Cer
(d18:1/22:4)

GM3

GD2

(d18:1/18:0)

(d18:1/18:1)

(d18:1/24:1)

(d18:1/24:0)

(d18:1/20:0)

(d18:0/18:0)

(d18:1/16:0)

(d18:0/16:0)

(d18:1/18:1)

(d18:1/18:0)

(d18:1/24:1)

(18:1/16:0)

+
+

GD3

(d18:1/16:0)

(t18:1/16:0) or (d18:1/h16:0) or
HexNAcHex2Cer(d18:1/24:4)

(d18:1/18:0)

(d18:0/18:0)

(d18:1/20:0)

(d18:1/22:0)

(d18:0/22:0)

(d18:1/24:2)

(d18:1/18:0)

(d18:0/24:0)

(d18:0/20:0)

(d18:1/24:1)

(d18:1/20:0)

(d18:1/24:0)

GT1

(d18:1/24:0)

O-Ac-GM3 (d18:1/20:0) or GM3

(18:1/23:0)

(d18:1/18:0)

O-Ac-GM3 (d18:1/22:1) or GM3

(20:1/23:1)

(d18:1/24:0)

O-Ac-GM3 (d18:1/22:0) (or GM3

(20:1/23:0)

(d18:1/24:1)

GQ1

(d18:1/18:0)

O-Ac-GM3 (d18:0/22:0) (or GM3

(20:0/23:0)

GM4

(d18:1/20:2)

HexNAcHex2Cer (d18:0/14:0) or
(d16:0/16:0)
HexNAcHex2Cer (d18:0/16:0)

Asialo-GG
species

O-Ac-GM3 (d18:1/24:2)

GD1

(d18:1/18:0)

(d18:1/20:0)

(d18:1/23:0)

HexNAcHex2Cer (t18:0/22:0) or
(d18:0/h22:0) or (d18:2/24:4)
HexHexNAcHex2Cer(d18:1/18:0)

(d18:1/24:1)

(d18:0/18:0)

GT3

NanoESI chip HCT CID MS2 of the doubly charged ion at m/z 1063.34
corresponding to GT1 (d18:1/18:0) ganglioside species detected in An28 mixture
Intens.

1063.41

[M-2H]2-- H2O

3000

[M-2H]2-

1054.21
1054.21

Y4

[M-2H]2-- CO2
2000

B2- CO2

MS3

537.32
537,32

1837.01
1837.01

Z421000

B1
290.21

Y4

1544.71
1544.81

1041.21
1041.21

B2

907.32

Y2/B2

/B21
YY44/B

Y42-

Z4

1252.83
1253.89

917.32

Y4+Na

Z4
1818.01

1526.81
1526.82

1859.01
1859.01

888.83
581.31

581.31
0
400

600

800

1000

1200

1400

1600

1800

2000 m/z

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.6-1.0 V

MS2 fragmentation pathway of [M-2H]2- ion at m/z 1063.34

Y4

Z4

NeuAc O Gal O GalNAc O Gal O Glc O Cer

Z4
B1

C1

O
Y4

GT1b (d18:1/18:0)

NeuAc

C2

B2

Y2 /B2
/B1
YY44/B
2

O
B1

NeuAc
Fig3b

NanoESI chip HCT CID MS3 using as a precursor

Y42- ion detected at m/z 917.32 in MS2
Intens.
80

[M-2H]2917.27

MS4

908.27

/C22 or
YZ23/B
Z2/B2

Y0

40

564.62

B1
290.21

20

Z3/C2

1161.50
1161.50

Z3/C2

Y3

1091.32

1254.02

887.63
888.63

982.94
1023.92

B2

1544.87

Z4/B2 or
/C1
YZ2
2/C
1

[M-2H]
-H2O2[M-H22-O-2H]

60

Y4

Z4

Y4/B1or
Y3/B1
1382.92

Y4/CO2

581.32

1500.69

0
200

400

600

800

1000

1200

1400

m/z

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.6-1.0 V

Fig3c

MS3 fragmentation pathway of the ion detected in MS2 at m/z 917.32

Y0

Gal O GalNAc O Gal O Glc O - Cer

Z3/C2 Z3/C2
Z3

C2

Y3

Y2/B2
3/C2 or Z2/B2
Z Z/B
2

NeuAc

B2

Z2Z/C
1 2 or Y2/C1
4/B

C1

/B1 or Y3/B1
Y3Y/B
41

NeuAc

B1

Z4
Y4

NanoESI chip HCT CID MS4 using as a precursor

the Y4- ion detected at m/z 1544.87 in MS3
Intens.

Y2/B1

888.42

1253.81

Y3
15

Z3
1364.56

B4

10

980.32

Y0

Z3-H2O

Y3/B1

564.62

Y2

Y1

Z1

726.22

707.63

1346.50

1090.80

1544.87

[M-H]-CO2 1501.82

1212.61

870.42

[M-H]-H2O 1526.87
1375.83

1024.45

Y2/C1

[M-H]-

1389.01

1179.48

0
600

700

800

900

1000

1100

1200

1300

1400

1500

m/z

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.6-1.0 V

MS4 fragmentation pathway of the ion detected in MS3 at m/z 1544.87

Y3

Z3

Y2

Y1

Z1

Y0

Z0

Gal O GalNAc O Gal O Glc O - Cer

B4
Y2/C1 or Z3 /B2

B2
Y3

O
NeuAc

C1

Y2/B1 or Y3 /B2

B1

Y3/B1

Fig3f

TOP DOWN ANALYSIS OF GANGLIOSIDES BY MULTISTAGE CID

MS2

1077.65
1077.20

Intens.
x105
8

Intens.
5
x10

1063.67

1074.66
1071.20 1074.20

MS1

1063.20

1077.65

1063.20

1049.25

1085.60
1085.20

1085.20
4

1088.20
918.11

1033.36

1088.68

1088.60

926.63

1020.16

1020.16

917.60

1049.25

931.72

939.60

939.60

1098.20

1033.06

931.72

900

925

950

975

1000

1025

1050

1075

926.63
918.11

2
717.80

600

800

1000

1200

1400

1600

m/z

1100

m/z

MS2

Intens.
x10

Y2 (and Y4 /B1)

Y32- (and Y4)

1573.21

932.10

[M-2H]2-- H2O

1068.28
5

Y2/B2

Y3/B1/Y1

916.80
[M-2H]2-- H2O
1055.20

655.40

Y3 (and Y4)

[M-2H]21077.20

1864.21
Z2 (and Y4 /C1)
1555.20

Y3/B2
1120.00

MS3

n.a.
1852.20

B5 - H2O
Y4/B2

n.a.

603.30
B2
581.32

Z3 - H2O
1846.27

1282.06

Y1
754.80

Y3 /CO2
1545.10
B5/B1
1254.00

Z1
736.80

1836.13

Y2
1514.60

B2 /CO2
537.40

1820.20

n.a.
0
400

600

800

1000

1200

1400

1600

1800

2000 m/z

FRAGMENTATION PATTERN
IN MS2
Y4

Y3/B1/Y1

Y2

Y1

Z1

NeuAc O Gal O GalNAc O Gal O Glc O Cer

B5
Z2
Y2/B2
Y2

Y3/B2
B2

Z3
C1

B5/B1
Y4/B1

Y3
B1

Y3/B1

MS3

Y3(and Y4 /B1)

MS4

1573.21

Intens.
Y3/CO2

Y2
1282.06

Y2/B2

Z 3(and Z4/C1)
1555.20

916.80
Z3/B2/CH3CO/CH2O/H2O
3000

[M-H]- - H2O

1011.80
994.66

1528.60

n.a.
B2 - H2O

Y2/C1

562.40
Y1

1338.85
Y2
1514.60

1208.00

754.80

2000

1846.27
2,4X
3

1025.00

n.a.
1820.29

Z2
B2
581.32

[M-H]1864.21

1496.60

Z3/B2/CH2O/H2O
1053.90

n.a.

Z1
736.80

1000

n.a.

1802.20

1481.20

1374.80

n.a.
0
600

800

1000

1200

1400

1600

1800

m/z

FRAGMENTATION PATTERN
IN MS3
Z3

Y2

Z2

Y1

Z1

Gal O GalNAc O Gal O Glc O - Cer

Y2
B2

Y2/C1

Z3
C1
Y3
B1

Y2/B1

MS4

Z3/B1/CH2O/H2O
1054.00

Intens.

MS5

1025.60
300

Y2/B1

Y1

916.80

[M-H]1573.21

[M-H]-/CO2

Y3/C1

754.80

[M-H]-- H2O

1102.00

1555.20

Z0

Z1
736.80

200

Y3/Y1

Y3/B1
1120.00

Y2

1529.20

1282.06
Y2/C1

655.08

2,4X
3

Y2 /CO2

898.00

1163.80

Y0

n.a.

592.66

850.60

Z3 - H2O

C4
997.22

1374.80
Y2
1207.40

100

1512.20

574.60
n.a.

n.a.

1408.80
n.a.

514.60
n.a.

Y3

n.a.

0
200

400

600

800

1000

1200

1400

1600

1800 m/z

FRAGMENTATION PATTERN
IN MS4
Y3

Z3

Y2

Y1

Z1

Y0

Z0

Gal O GalNAc O Gal O Glc O - Cer

C4
Y2/C1

Y3/C1
C1

Y2/B1
Y3/B1

Y2

B1

Z3

Y2

Y1

Y0

Z1

MS5

Gal O GalNAc O Gal O Glc O Cer

Z3/CH2O/H2O
1053.80

Y1
754.80

Intens.

Z3/CH3CO/CH2O/H2O
1011.60

25

Z3
1101.80

Z1
736.80
Y2

20

916.60

15

[M-H]-

Y0

MS6

1282.06

592.66

10

0
200

400

600

800

1000

1200

1400

1600

m/z

MS6
T
308.80

[M-H]-/CH2O
562.60

Intens.
327.60

V
10

283.40

339.20

[M-H]-/CH2O/H2O

[M-H]-

544.60

P
6

592.66

265.80

200

300

400

500

600

700

800 m/z

FRAGMENTATION PATTERN
IN MS6